Isolated hypercalciuria with mutation in CLCN5: relevance to idiopathic hypercalciuria

Isolated hypercalciuria with mutation in CLCN5: Relevance to idiopathic hypercalciuria. BACKGROUND: Idiopathic hypercalciuria (IH) is the most common risk factor for kidney stones and often has a genetic component. Dent's disease (X-linked nephrolithiasis) is associated with mutations in the CLCN5 chloride channel gene, and low molecular weight (LMW) proteinuria was universally observed in affected males. We sought to identify mutations in CLCN5 or abnormalities in LMW protein excretion in a large group of patients with IH and in a rat model of genetic hypercalciuria. METHODS: One hundred and seven patients with IH (82 adults and 25 children) and one asymptomatic hypercalciuric man with a known inactivating mutation in CLCN5 were studied. Secondary causes of hypercalciuria were excluded in all. The excretion of retinol-binding protein and beta2-microglobulin was measured by immunoassay in 101 patients with IH. Mutation analysis of the CLCN5 gene was performed in 32 patients with IH and in the genetic hypercalciuric stone-forming (GHS) rat strain. RESULTS: LMW protein excretion was normal in 92 patients with IH, and only slight abnormalities were found in the other nine, none of whom had a mutation in CLCN5. One 27-year-old man who had a CLCN5 mutation was found to have isolated hypercalciuria without LMW proteinuria, renal failure, or other evidence of renal disease. Mutation analysis was normal in 32 patients with IH. The CLCN5 sequence was normal in the GHS rat. CONCLUSIONS: Inactivation of CLCN5 can be found in the setting of hypercalciuria without other features of X-linked nephrolithiasis. However, mutations in CLCN5 do not represent a common cause of IH.     

Increased mucosal inflammatory cytokines in children with Helicobacter pylori-associated gastritis

The concentrations of tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and IL-1-β in tissue homogenates of gastric mucosal biopsy specimens, and in gastric juice samples from Helicobacter pylori-positive and -negative children, were determined. The study population comprised 30 children with recurrent abdominal pain attending upper gastrointestinal endoscopy. Of these patients 18 were infected with H. pylori. Cytokine concentrations in gastric biopsy homogenate supernatants and in gastric juice were measured by enzyme-linked immunosorbent assay (ELISA). TNF-α levels in gastric juice and in gastric biopsy homogenate supernatants in patients with H. pylori-positive gastritis were found to be significantly higher than those in children without H. pylori infection. IL-6 levels were also higher in H. pylori -infected subjects, but the difference in IL-6 concentrations measured in gastric juice and biopsy homogenate supernatants did not reach statistical significance. IL-1-β concentrations in both specimens showed no significant difference between the two groups of children. It was suggested that increased levels of inflammatory cytokines, especially TNF-α and IL-6 generated locally within the gastric mucosa might be implicated in the pathogenesis of H. pylori-associated gastritis in childhood.     

人ERRα基因小分子干扰RNA慢病毒载体构建及鉴定

目的构建并鉴定靶向人ERRα基因的小分子干扰RNA的慢病毒载体。方法针对ERRαmRNA设计了4条si RNA,并构建pGCSIL-GFP-siERRα慢病毒质粒,PCR扩增阳性克隆并测序鉴定。用pGCSIL-GFP-siERRα、pHelp-er1.0和pHelper2.0质粒共转染293T细胞包装产生慢病毒,测定病毒滴度。将慢病毒干扰RNA及含有ERRα过表达载体共转染293T细胞,Western-blot检测ERRα表达,观察蛋白表达抑制效果。结果 PCR和测序结果与设计的干扰序列一致,病毒滴度达2×109TU/ml。转染细胞中ERRα蛋白表达显著降低。结论成功构建高表达、高效率的人ERRα基因小分子干扰RNA慢病毒载体,为进一步研究ERRα在细胞核内转导中的作用机制和靶向ERRα治疗奠定基础。     

GNAS1 mutational analysis in pseudohypoparathyroidism

OBJECTIVE

Mutations of the GNAS1 gene, which is located on chromosome 20q13.11 and encodes the α-subunit of the stimulatory GTP-binding protein, have been identified in patients with pseudohypoparathyroidism type Ia (PHPIa) and pseudopseudohypoparathyroidism (PPHP). We have undertaken studies to determine the prevalence of GNAS1 mutations and to explore methods for their more rapid detection.

METHODS

Thirteen unrelated families (8 with PHPIa and PPHP patients, and 5 with PPHP patients only) were investigated for GNAS1 mutations in the 1050 base-pair (bp) region spanning exons 2–13 by single-stranded conformational polymorphism (SSCP) and DNA sequence analysis.

RESULTS

GNAS1 mutations were detected in 4 of the 8 families with PHPIa patients. These consisted of: two novel de novo missense mutations (Pro115Ser and Glu259Val) in two families and an identical 4 bp deletion of codons 189 and 190 resulting in a frameshift in two unrelated families. These results expand the spectrum of GNAS1 mutations associated with this disorder and confirm the presence of a mutational hot-spot involving codons 189 and 190. SSCP analysis was found to be a specific and sensitive method that detected all 4 mutations. GNAS1 mutations were not detected in any of the PPHP only families.

CONCLUSIONS

The pseudohypoparathyroid disorders appear to represent a heterogeneous group with GNAS1 mutations forming the molecular aetiology in approximately 50% of pseudohypoparathyroidism type Ia families. Such mutations can be reliably identified by single-stranded conformational polymorphism and this will help to supplement the clinical evaluation of some patients and their families, particularly as the disease may not be fully penetrant.

     

Kidney Stones: A Fetal Origins Hypothesis

Kidney stones are common, with a multifactorial etiology involving dietary, environmental, and genetic factors. In addition, patients with nephrolithiasis are at greater risk of hypertension, diabetes mellitus, metabolic syndrome, and osteoporosis, although the basis for this is not fully understood. All of these renal stone–associated conditions have also been linked with adverse early‐life events, including low–birth weight, and it has been suggested that this developmental effect is due to excess exposure to maternal glucocorticoids in utero. This is proposed to result in long‐term increased hypothalamic‐pituitary‐axis activation; there are mechanisms through which this effect could also promote urinary lithogenic potential. We therefore hypothesize that the association between renal stone disease and hypertension, diabetes mellitus, metabolic syndrome, and osteoporosis may be related by a common pathway of programming in early life, which, if validated, would implicate the developmental origins hypothesis in the etiology of nephrolithiasis. © 2013 American Society for Bone and Mineral Research.     

Symmetrically reduced stiffness and increased extensibility in compression and tension at the mineralized fibrillar level in rachitic bone

In metabolic bone diseases, the alterations in fibrillar level bone-material quality affecting macroscopic mechanical competence are not well-understood quantitatively. Here, we quantify the fibrillar level deformation in cantilever bending in a mouse model for hereditary rickets (Hpr). Microfocus in-situ synchrotron small-angle X-ray scattering (SAXS) combined with cantilever bending was used to resolve nanoscale fibril strain in tensile- and compressive tissue regions separately, with quantitative backscattered scanning electron microscopy used to measure microscale mineralization. Tissue-level flexural moduli for Hpr mice were significantly (p < 0.01) smaller compared to wild-type (~ 5 to 10-fold reduction). At the fibrillar level, the fibril moduli within the tensile and compressive zones were significantly (p < 0.05) lower by ~ 3- to 5-fold in Hpr mice compared to wild-type mice. Hpr mice have a lower mineral content (24.2 ± 2.1 Ca wt.% versus 27.4 ± 3.3 Ca wt.%) and its distribution was more heterogeneous compared to wild-type animals. However, the average effective fibril modulus did not differ significantly (p > 0.05) over ages (4, 7 and 10 weeks) between tensile and compressive zones. Our results indicate that incompletely mineralized fibrils in Hpr mice have greater deformability and lower moduli in both compression and tension, and those compressive and tensile zones have similar moduli at the fibrillar level.     

Hypoparathyroidism,deafness, and renal dysplasia syndrome: 20 Years after the identification of the first GATA3 mutations

The hypoparathyroidism, deafness, and renal dysplasia (HDR) syndrome is an autosomal dominant disorder caused by heterozygous mutations of the GATA3 gene. In the last 20 years, since the identification of the genetic cause of the HDR syndrome, GATA3 mutations have been reported in 124 families (177 patients). The clinical aspects and molecular genetics of the HDR syndrome are reviewed here together with the reported mutations and phenotypes. Reported mutations consist of 40% frameshift deletions or insertions, 23% missense mutations, 14% nonsense mutations, 6% splice‐site mutations, 1% in‐frame deletions or insertions, 15% whole‐gene deletions, and 1% whole‐gene duplication. Missense mutations were found to cluster in the regions encoding the two GATA3 zinc‐finger domains. Patients showed great clinical variability and the penetrance of each HDR defect increased with age. The most frequently observed abnormality was deafness (93%), followed by hypoparathyroidism (87%) and renal defects (61%). The mean age of diagnosis of HDR was 15.3, 7.5, and 14.0 years, respectively. However, patients with whole‐gene deletions and protein‐truncating mutations were diagnosed earlier than patients with missense mutations.