Increased Transactivation Associated with SOX3 Polyalanine Tract Deletion in a Patient with Hypopituitarism

Backgound and Aims: Correct gene dosage of SOX3 is critical for the development of the hypothalamo-pituitary axis. Both overdosage of SOX3, as a result of gene duplication, and loss of function resulting from expansion of the first polyalanine (PA) tract are associated with variable degrees of hypopituitarism, with or without mental retardation. The aim of this study was to further investigate the contribution of SOX3 in the etiology of hypopituitarism and the mechanisms involved in the phenotypic variability. Methods: We screened 154 patients with congenital hypopituitarism and an undescended posterior pituitary for mutations in SOX3 and variability in the length of the first PA tract. In addition, 300 patients with variable septooptic dysplasia were screened for variability of the PA tract. Results: We report a novel 18-base pair deletion (p.A243_A248del6, del6PA) in a female patient with hypopituitarism resulting in a 2-fold increase in transcriptional activation in vitro, compared with wild-type SOX3. We also identified a previously reported seven-alanine expansion (p.A240_A241ins7, +7PA) in two male siblings with isolated GH deficiency and a distinct phenotype, in addition to the nonsynonymous variant p.R5Q in an unrelated individual; this appears to have no functional effect on the protein. In contrast to +7PA, del6PA maintained its ability to repress beta-catenin mediated transcription in vitro. Conclusion: This is the first study to report that PA tract deletions associated with hypopituitarism have functional consequences in vitro, possibly due to increased activation of SOX3 target genes. In addition, we have expanded the phenotypic spectrum associated with PA tract expansion (+7PA) mutations to include panhypopituitarism or isolated GH deficiency, with or without mental retardation. (J Clin Endocrinol Metab 96: E685-E690, 2011)

Lactic acid stimulates interleukin-23 production by peripheral blood mononuclear cells exposed to bacterial lipopolysaccharide

Lactic acid is the predominant acid present in the vagina. We evaluated the consequences of lactic acid, at physiological levels present in the vagina, on cytokine responses of peripheral blood mononuclear cells (PBMCs) obtained from 10 individuals in the presence or absence of bacterial lipopolysaccharide. Preincubation of PBMCs in 15 mM lactic acid before the addition of lipopolysaccharide resulted in a 246% mean increase in interleukin-23 (IL-23) secretion over that released in the presence of lipopolysaccharide alone (P=0.0068). The lipopolysaccharide-induced production of tumor necrosis factor-alpha, IL-6, IL-10 and IL-12 was unaffected by lactic acid. IL-23 stimulation was not observed if the lactic acid was neutralized before its addition to the culture medium or if hydrochloric acid was substituted for lactic acid. In the absence of lipopolysaccharide, lactic acid did not stimulate the production of IL-23 or any of the other cytokines. The increase in IL-23 production was proportional to the lactic acid concentration over a 15-60 mM range. We conclude that at body sites characterized by lactic acid accumulation, such as in the human vagina, exposure to gram-negative bacteria results in selective IL-23 production, leading to a subsequent preferential stimulation of the Th17 T lymphocyte pathway.

Fibroblast Growth Factor 23 in Hemodialysis Patients: Effects of Phosphate Binder, Calcitriol and Calcium Concentration in the Dialysate

Background: Fibroblast growth factor 23 (FGF23) concentrations increase early in chronic kidney disease (CKD), and the influence of current CKD-mineral and bone disorder (MBD) therapies on serum FGF23 levels is still under investigation. Methods: In this post-hoc analysis of a randomized clinical trial, phosphate binders and calcitriol were washed out of 72 hemodialysis patients who were then submitted to bone biopsy, coronary tomography and biochemical measures, including FGF23. They were randomized to receive sevelamer or calcium acetate for 1 year and the prescription of calcitriol and the calcium concentration in the dialysate were adjusted according to serum calcium, phosphate and PTH and bone biopsy diagnosis. Results: At baseline, bone biopsy showed that 58.3% had low-turnover bone disease, whereas 38.9% had high-turnover bone disease, with no significant differences between them with regard to FGF23. Median baseline FGF23 serum levels were elevated and correlated positively with serum phosphate. After 1 year, serum FGF23 decreased significantly. Repeated measures ANOVA analysis showed that the use of a 3.5-mEq/l calcium concentration in the dialysate, as well as the administration of calcitriol and a calcium-based phosphate binder were associated with higher final serum FGF23 levels. Conclusions: Taken together, our results confirm that the current CKD-MBD therapies have an effect on serum levels of FGF23. Since FGF23 is emerging as a potential treatment target, our findings should be taken into account in the decision on how to manage CKD-MBD therapy. Copyright (C) 2010 S. Karger AG, Basel

Insertion/deletion polymorphism of the angiotensin converting enzyme gene and loss of the insertion allele in aldosterone-producing adenoma

The genetic mechanisms responsible for the formation of adrenocortical adenomas which autonomously produce aldosterone are largely unknown, The adrenal renin-angiotensin system has been implicated in the pathophysiology of these tumours, Angiotensin-converting enzyme (ACE) catalyses the generation of angiotensin II, and the insertion/deletion (I/D) polymorphism of the ACE gene regulates up to 50% of plasma and cellular ACE variability in humans. We therefore examined the genotypic and allelic frequency distributions of the ACE gene I/D polymorphism in 55 patients with aldosterone-producing adenoma, APA, (angiotensin-unresponsive APA n = 28, angiotensin-responsive APA n = 27), and 80 control subjects with no family history of hypertension, We also compared the ACE gene I/D polymorphism allelic pattern in matched tumour and peripheral blood DNA in the 55 patients with APA, The frequency of the D allele was 0.518 and 0.512 and the I allele was 0.482 and 0.488 in the APA and control subjects respectively, Genotypic and allelic frequency analysis found no significant differences between the groups, Examination of the matched tumour and peripheral blood DNA samples revealed the loss of the insertion allele in four of the 25 patients who were heterozygous for the ACE I/D genotype. The I/D polymorphism of the ACE gene does not appear to contribute to the biochemical and phenotypic characteristics of APA, however, the deletion of the insertion allele of the ACE gene I/D polymorphism in 16% of aldosterone-producing adenomas may represent the loss of a tumour suppressor gene/s or other genes on chromosome 17q which may contribute to tumorigenesis in APA.

FGF-23 as a Predictor of Renal Outcome in Diabetic Nephropathy

Background and objectives Fibroblast growth factor 23 (FGF-23) has emerged as a new factor in mineral metabolism in chronic kidney disease (CKD). An important regulator of phosphorus homeostasis, FGF-23 has been shown to independently predict CKD progression in nondiabetic renal disease. We analyzed the relation between FGF-23 and renal outcome in diabetic nephropathy (DN). Design, setting, participants, & measurements DN patients participating in a clinical trial (enalapril+placebo versus enalapril+losartan) had baseline data collected and were followed until June 2009 or until the primary outcome was reached. Four patients were lost to follow-up. The composite primary outcome was defined as death, doubling of serum creatinine, and/or dialysis need. Results At baseline, serum FGF-23 showed a significant association with serum creatinine, intact parathyroid hormone, proteirturia, urinary fractional excretion of phosphate, male sex, and race. Interestingly, FGF-23 was not related to calcium, phosphorus, 25OH-vitamin D, or 24-hour urinary phosphorus. Mean follow-up time was 30.7 +/- 10 months. Cox regression showed that FGF-23 was an independent predictor of the primary outcome, even after adjustment for creatinine clearance and intact parathyroid hormone (10 pg/ml FGF-23 increase = hazard ratio, 1.09; 95% CI, 1.01 to 1.16, P = 0.02). Finally, Kaplan-Meier analysis showed a significantly higher risk of the primary outcome in patients with FGF-23 values of >70 pg/ml. Conclusions FGF-23 is a significant independent predictor of renal outcome in patients with macroalbuminuric DN. Further studies should clarify whether this relation is causal and whether FGF-23 should be a new therapeutic target for CKD prevention. Clin J Am Soc Nephrol 6: 241-247, 2011. doi: 10.2215/CJN.04250510

Spectrum and Prevalence of FP/TMEM127 Gene Mutations in Pheochromocytomas and Paragangliomas

Context Pheochromocytomas and paragangliomas are genetically heterogeneous neural crest-derived neoplasms. We recently identified germline mutations of the novel transmembrane-encoding gene FP/TMEM127 in familial and sporadic pheochromocytomas consistent with a tumor suppressor effect. Objectives To examine the prevalence and spectrum of FP/TMEM127 mutations in pheochromocytomas and paragangliomas and to test the effect of mutations in vitro. Design, Setting, and Participants We sequenced the FP/TMEM127 gene in 990 individuals with pheochromocytomas and/or paragangliomas, including 898 previously unreported cases without mutations in other susceptibility genes from 8 independent worldwide referral centers between January 2009 and June 2010. A multiplex polymerase chain reaction-based method was developed to screen for large gene deletions in 545 of these samples. Confocal microscopy of 5 transfected mutant proteins was used to determine their subcellular localization. Main Outcome Measures The frequency and type of FP/TMEM127 mutation or deletion was assessed and correlated with clinical variables; the subcellular localization of 5 overexpressed mutants was compared with wild-type FP/TMEM127 protein. Results We identified 19 potentially pathogenic FP/TMEM127 germline mutations in 20 independent families, but no large deletions were detected. All mutation carriers had adrenal tumors, including 7 bilateral (P=2.7 x 10(-4)) and/or with familial disease (5 of 20 samples; P=.005). The median age at disease onset in the FP/TMEM127 mutation group was similar to that of patients without a mutation (41.5 vs 45 years, respectively; P=.54). The most common presentation was that of a single benign adrenal tumor in patients older than 40 years. Malignancy was seen in 1 mutation carrier (5%). Expression of 5 novel FP/TMEM127 mutations in cell lines revealed diffuse localization of the mutant proteins in contrast with the discrete multiorganelle distribution of wild-type TMEM127. Conclusions Germline mutations of FP/TMEM127 were associated with pheochromocytoma but not paraganglioma and occured in an age group frequently excluded from genetic screening algorithms. Disease-associated mutations disrupt intracellular distribution of the FP/TMEM127 protein. JAMA. 2010;304(23):2611-2619 www.jama.com

Highlights from the II international symposium of thrombosis and anticoagulation (ISTA), October 22-23, 2009, So Paulo, Brazil

sanofi-aventis

Genetic heterogeneity in familial acute myelogenous leukemia: Evidence for a second locus at chromosome 16q21-23.2

The identification of genes responsible for the rare cases of familial leukemia may afford insight into the mechanism underlying the more common sporadic occurrences. Here we test a single family with 11 relevant meioses transmitting autosomal dominant acute myelogenous leukemia (AML) and myelodysplasia for linkage to three potential candidate loci. In a different family with inherited AML, linkage to chromosome 21q22.1-22.2 was recently reported; we exclude linkage to 21q22.1-22.2, demonstrating that familial AML is a heterogeneous disease. After reviewing familial leukemia and observing anticipation in the form of a declining age of onset with each generation, we had proposed 9p21-22 and 16q22 as additional candidate loci. Whereas linkage to 9p21-22 can be excluded, the finding of a maximum two-point LOD score of 2.82 with the microsatellite marker D16S522 at a recombination fraction theta = 0 provides evidence supporting linkage to 16q22. Haplotype analysis reveals a 23.5-cM (17.9-Mb) commonly inherited region among all affected family members extending from D16S451 to D1GS289, In order to extract maximum linkage information with missing individuals, incomplete informativeness with individual markers in this interval, and possible deviance from strict autosomal dominant inheritance, we performed nonparametric linkage analysis (NPL) and found a maximum NPL statistic corresponding to a P-value of .00098, close to the maximum conditional probability of linkage expected for a pedigree with this structure. Mutational analysis in this region specifically excludes expansion of the AT-rich minisatellite repeat FRA16B fragile site and the CAG trinucleotide repeat in the E2F-4 transcription factor. The ''repeat expansion detection'' method, capable of detecting dynamic mutation associated with anticipation, more generally excludes large CAG repeat expansion as a cause of leukemia in this family.

Investigating 22q11.2 Deletion and Other Chromosomal Aberrations in Fetuses With Heart Defects Detected by Prenatal Echocardiography

Congenital heart disease (CHD) is the most common birth defect and the leading cause of mortality in the first year of life. In fetuses with a heart defect, chromosomal abnormalities are very frequent. Besides aneuploidy, 22q11.2 deletion is one of the most recognizable chromosomal abnormalities causing CHD. The frequency of this abnormality varies in nonselected populations. This study aimed to investigate the incidence of the 22q11.2 deletion and other chromosomal alterations in a Brazilian sample of fetuses with structural cardiac anomalies detected by fetal echocardiography. In a prospective study, 68 fetuses with a heart defect were evaluated. Prenatal detection of cardiac abnormalities led to identification of aneuploidy or structural chromosomal anomaly in 35.3% of these cases. None of the fetuses with apparently normal karyotypes had a 22q11.2 deletion. The heart defects most frequently associated with chromosomal abnormalities were atrioventricular septal defect (AVSD), ventricular septal defect (VSD), and tetralogy of Fallot. Autosomal trisomies 18 and 21 were the most common chromosomal abnormalities. The study results support the strong association of chromosome alterations and cardiac malformation, especially in AVSD and VSD, for which a chromosome investigation is indicated. In fetuses with an isolated conotruncal cardiopathy, fluorescence in situ hybridization (FISH) to investigate a 22q11.2 deletion is not indicated.

Immunogenicity of 23-valent pneumococcal polysaccharide vaccine in HIV-infected pregnant women and kinetics of passively acquired antibodies in young infants

Whether gestational immunization of HIV-infected mothers with the 23-valent pneumococcal polysaccharide vaccine (PPV) confers maternal and infant early life, passive protection is not known. We evaluated safety, immunogenicity and placental transfer of antibodies in 44 HIV-infected women. Pneumococcal IgG antibodies against serotypes 1, 3, 5, 613, 9V, and 14 were measured in mothers (pre-vaccination and at delivery), and infants (at birth, 1, 2, 3, and 6 months). PPV was safe and immunogenic in mothers. Newborns received 46-72% of maternal antibody titers. Overall, infants had antibody levels lower than protective by 2 months of age. Alternative pneumococcal vaccination of HIV-infected pregnant women should be explored with the aim of prolonging passive protection in their infants. (C) 2009 Elsevier Ltd. All rights reserved.

Cytogenetic and Molecular Evaluation and 20-Year Follow-Up of a Patient With Ring Chromosome 14

We present a 20-year follow-up on a patient with a ring chromosome 14. The ring chromosome was studied by fluorescence in-situ hybridization (FISH), multiplex-ligation probe amplification (MLPA), and genome wide SNP array, and no deletions of chromosome 14 were detected, although the telomeric repeat sequence was absent from the ring chromosome. The patient had skeletal abnormalities, and susceptibility to infections, as well as seizures and retinal pigmentation, which are commonly found in individuals with a ring 14. Our patient corroborates the idea that even when no genes are lost during ring formation, a complete ring chromosome can produce phenotypic alterations, which presumably result from ring instability or gene silencing due to the new chromosomal architecture. (C) 2010 Wiley-Liss, Inc.

Nonsense Mutations in FGF8 Gene Causing Different Degrees of Human Gonadotropin-Releasing Deficiency

Context: FGFR1 mutations cause isolated hypogonadotropic hypogonadism (IHH) with or without olfactory abnormalities, Kallmann syndrome, and normosmic IHH respectively. Recently, missense mutations in FGF8, a key ligand for fibroblast growth factor receptor (FGFR) 1 in the ontogenesis of GnRH, were identified in IHH patients, thus establishing FGF8 as a novel locus for human GnRH deficiency. Objective: Our objective was to analyze the clinical, hormonal, and molecular findings of two familial IHH patients due to FGF8 gene mutations. Methods and Patients: The entire coding region of the FGF8 gene was amplified and sequenced in two well-phenotyped IHH probands and their relatives. Results: Two unique heterozygous nonsense mutations in FGF8(p.R127X and p.R129X) were identified in two unrelated IHH probands, which were absent in 150 control individuals. These two mutations, mapped to the core domain of FGF8, impact all four human FGF8 isoforms, and lead to the deletion of a large portion of the protein, generating nonfunctional FGF8 ligands. The p.R127X mutation was identified in an 18-yr-old Kallmann syndrome female. Her four affected siblings with normosmic IHH or delayed puberty also carried the p.R127X mutation. Additional developmental anomalies, including cleft lip and palate and neurosensorial deafness, were also present in this family. The p.R129X mutation was identified in a 30-yr-old man with familial normosmic IHH and severe GnRH deficiency. Conclusions: We identified the first nonsense mutations in the FGF8 gene in familial IHH with variable degrees of GnRH deficiency and olfactory phenotypes, confirming that loss-of-function mutations in FGF8 cause human GnRH deficiency. (J Clin Endocrinol Metab 95: 3491-3496, 2010)

MLPA analysis in 30 Sotos syndrome patients revealed one total NSD1 deletion and two partial deletions not previously reported

Sotos syndrome (MIM #117550) is an autosomal dominant condition characterized by pre and postnatal overgrowth, macrocephaly and typical facial gestalt with frontal bossing, hypertelorism, antimongoloid slant of the palpebral fissures, prominent jaw and high and narrow palate. This syndrome is also frequently associated with brain, cardiovascular, and urinary anomalies and is occasionally accompanied by malignant lesions such as Wilms turnout and hepatocarcinoma. The syndrome is known to be caused by mutations or deletions of the NSD1 gene. To detect both 5q35 microdeletions and partial NSD1 gene deletions we screened 30 Brazilian patients with clinical diagnosis of Sotos syndrome by multiplex ligation dependent probe amplification. We identified one patient with a total deletion of NSD1 and neighbouring FGFR4, other with missing NSD1 exons 13-14 and another with a deletion involving FGFR4 and spanning up to NSD1 exon 17. All deletions were de novo. The two NSD1 partial deletions have not been previously reported. The clinical features of the three patients included a typical facial gestalt with frontal bossing, prominent jaw and high anterior hairline; macrocephaly, dolichocephaly, large hands; neonatal hypotonia and jaundice. All presented normal growth at birth but postnatal overgrowth. Two patients with NSD1 and FGFR4 gene deletions presented congenital heart anomalies. (C) 2009 Elsevier Masson SAS. All rights reserved.

A Duplex Allele-Specific Amplification PCR to Detect SMN1 Deletion

Spinal muscular atrophy (SMA), the leading genetic cause of death in childhood, is an autosomal recessive neuromuscular disorder characterized by progressive muscle weakness, associated with deletions of the survival motor neuron (SMN) gene identified and mapped to chromosome 5q13. SMN is present in two highly homologous copies (SMN1 and SMN2). In the general population, normal individuals (noncarriers) have at least one telomeric (SMN1) copy, and 5% of them have no copies of SMN2. Approximately 95% of SMA patients carry homologous deletions of SMN1 exon(s) 7 (and 8). SMN1 and SMN2 exons 7 and 8 differ only by 1 bp each, and SMA diagnosis might be performed by single-strand conformational polymorphism, PCR amplification followed by restriction fragment length polymorphism (RFLP), multiple ligation-dependent probe amplification, or realtime PCR of SMNs exons 7 and 8. We developed a simpler and cost-effective method to detect SMN1 exon 7 deletion based on allele-specific amplification PCR.