NT5E mutations and arterial calcifications.

BACKGROUND: Arterial calcifications are associated with increased cardiovascular risk, but the genetic basis of this association is unclear. METHODS: We performed clinical, radiographic, and genetic studies in three families with symptomatic arterial calcifications. Single-nucleotide-polymorphism analysis, targeted gene sequencing, quantitative polymerase-chain-reaction assays, Western blotting, enzyme measurements, transduction rescue experiments, and in vitro calcification assays were performed. RESULTS: We identified nine persons with calcifications of the lower-extremity arteries and hand and foot joint capsules: all five siblings in one family, three siblings in another, and one patient in a third family. Serum calcium, phosphate, and vitamin D levels were normal. Affected members of Family 1 shared a single 22.4-Mb region of homozygosity on chromosome 6 and had a homozygous nonsense mutation (c.662C→A, p.S221X) in NT5E, encoding CD73, which converts AMP to adenosine. Affected members of Family 2 had a homozygous missense mutation (c.1073G→A, p.C358Y) in NT5E. The proband of Family 3 was a compound heterozygote for c.662C→A and c.1609dupA (p.V537fsX7). All mutations found in the three families result in nonfunctional CD73. Cultured fibroblasts from affected members of Family 1 showed markedly reduced expression of NT5E messenger RNA, CD73 protein, and enzyme activity, as well as increased alkaline phosphatase levels and accumulated calcium phosphate crystals. Genetic rescue experiments normalized the CD73 and alkaline phosphatase activity in patients' cells, and adenosine treatment reduced the levels of alkaline phosphatase and calcification. CONCLUSIONS: We identified mutations in NT5E in members of three families with symptomatic arterial and joint calcifications. This gene encodes CD73, which converts AMP to adenosine, supporting a role for this metabolic pathway in inhibiting ectopic tissue calcification. (Funded by the National Human Genome Research Institute and the National Heart, Lung, and Blood Institute of the National Institutes of Health.).

Comparison of fracture risk calculators in elderly fallers: a hospital-based cross-sectional study.

OBJECTIVE: Elderly patients presenting with falls are known to carry an extremely high risk of future fragility fractures. Current osteoporosis guidelines recommend using fracture risk calculators such as FRAX, QFracture or Garvan to guide management. However, they differ considerably in their inputs and may therefore provide contrasting risk estimations in certain individuals. In this study, we compare these risk calculators in a high-risk cohort of elderly patients admitted to hospital with falls. DESIGN: Hospital-based cross-sectional study. SETTING: Secondary care, London, UK. PARTICIPANTS: Data from 120 consecutive elderly patients who had falls presenting to a single hospital over 4 months were collected. 10-year major and hip fracture risks were calculated using FRAX, QFracture and Garvan. 1-year major and hip fracture risks from QFracture were assessed against prospective incidence of fracture. RESULTS: Median 10-year major fracture risk was: FRAX 19.5%, QFracture 26.0%, Garvan 32.5%. Median 10-year hip fracture risk was: FRAX 9.6%, QFracture 21.1%, Garvan 6.5%. Correlation between FRAX and QFracture was r=0.672 for major, r=0.676 for hip fracture (both p<0.0001); FRAX and Garvan r=0.778 (p<0.0001) for major, r=0.128 (p=0.206) for hip fracture; QFracture and Garvan r=0.658 (p<0.0001) for major, r=0.318 (p<0.001) for hip fracture. QFracture 1-year predicted major and hip fracture rates were 1.8% and 1.2%, respectively, compared with actual rates of 2.1% and 0%, respectively. CONCLUSIONS: Although strong correlations between calculators were observed in the study cohort, there were differences of up to 13% between estimated risks. QFracture captured several elderly-specific inputs not considered by other calculators and so projected higher fracture risk than the other calculators. QFracture provided 1-year fracture risks that were comparable with the prospective observed fracture incidence in the cohort. This study has important clinical implications for the use of fracture risk calculators to guide treatment decisions, particularly in the high-risk cohort of elderly patients admitted to hospital following falls.

Altered electroretinograms in patients with KCNJ10 mutations and EAST syndrome.

The K+ channel expressed by the KCNJ10 gene (Kir4.1) has previously demonstrated importance in retinal function in animal experiments. Recently, mutations in KCNJ10 were recognised as pathogenic in man, causing a constellation of symptoms, including epilepsy, ataxia, sensorineural deafness and a renal tubulopathy designated as EAST syndrome. We have studied the impact of KCNJ10 mutations on the human electroretinogram (ERG) in four unrelated patients with EAST syndrome. Corneal ganzfeld ERGs were elicited in response to flash stimuli of strengths of 0.001­10 phot cd s/m2 presented scotopically, and 0.3­10 phot cd s/m2 presented photopically. ERG waveforms from light-adapted retinae of all patients showed reduced amplitudes of the photopic negative response (PhNR) (P < 0.001). The photopic ERGs showed a delay in b-wave time to peak, but the photopic hill, i.e. the relative variation of time to peak and amplitude with luminance flash strength, was preserved. Scotopic ERGs to flash strengths 0.01 to 0.1 phot cd s/m2 showed a delay of up to 20 ms before the onset of the b-wave in two patients compared to controls. Stimulus­response functions were fitted by Michaelis­Menten equations and showed significantly lower retinal sensitivity in two patients than in controls (P < 0.001). Our study for the first time in the human ERG shows changes in association with KCNJ10 mutations affecting a Muller cell K+ channel. These data illustrate the role of KCNJ10 function in the physiology of proximal and possibly also the distal human retina.

KCNJ10 mutations disrupt function in patients with EAST syndrome.

BACKGROUND/AIMS: Mutations in the inwardly-rectifying K+ channel KCNJ10/Kir4.1 cause an autosomal recessive disorder characterized by epilepsy, ataxia, sensorineural deafness and tubulopathy (EAST syndrome). KCNJ10 is expressed in the kidney distal convoluted tubule, cochlear stria vascularis and brain glial cells. Patients clinically diagnosed with EAST syndrome were genotyped to identify and study mutations in KCNJ10. METHODS: Patient DNA was sequenced and new mutations identified. Mutant and wild-type KCNJ10 constructs were cloned and heterologously expressed in Xenopus oocytes. Whole-cell K+ currents were measured by two-electrode voltage clamping. RESULTS: Three new mutations in KCNJ10 (p.R65C, p.F75L and p.V259fs259X) were identified, and mutation p.R297C, previously only seen in a compound heterozygous patient, was found in a homozygous state. Wild-type human KCNJ10-expressing oocytes showed strongly inwardly-rectified currents, which by comparison were significantly reduced in all the mutants (p < 0.001). Specific inhibition of KCNJ10 currents by Ba2+ demonstrated residual function in all mutant channels (p < 0.05) but V259X. CONCLUSION: This study confirms that EAST syndrome can be caused by many different mutations in KCNJ10 that significantly reduce K+ conductance. EAST syndrome should be considered in any patient with a renal Gitelman-like phenotype with additional neurological signs and symptoms like ataxia, epilepsy or sensorineural deafness.

Genetic and Pharmacological Targeting of Transcriptional Repression in Resistance to Thyroid Hormone Alpha.

Background: Thyroid hormones act in bone and cartilage via thyroid hormone receptor alpha (TRα). In the absence of triiodothyronine (T3), TRα interacts with co-repressors, including nuclear receptor co-repressor-1 (NCoR1), which recruit histone deacetylases (HDACs) and mediate transcriptional repression. Dominant-negative mutations of TRα cause resistance to thyroid hormone alpha (RTHα; OMIM 614450), characterized by excessive repression of T3 target genes leading to delayed skeletal development, growth retardation, and bone dysplasia. Treatment with thyroxine has been of limited benefit, even in mildly affected individuals, and there is a need for new therapeutic strategies. It was hypothesized that (i) the skeletal manifestations of RTHα are mediated by the persistent TRα/NCoR1/HDAC repressor complex containing mutant TRα, and (ii) treatment with the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) would ameliorate these manifestations. Methods: The skeletal phenotypes of (i) Thra1PV/+ mice, a well characterized model of RTHα; (ii) Ncor1ΔID/ΔID mice, which express an NCoR1 mutant that fails to interact with TRα; and (iii) Thra1PV/+Ncor1ΔID/ΔID double-mutant adult mice were determined. Wild-type, Thra1PV/+, Ncor1ΔID/ΔID, and Thra1PV/+Ncor1ΔID/ΔID double-mutant mice were also treated with SAHA to determine whether HDAC inhibition results in amelioration of skeletal abnormalities. Results:Thra1PV/+ mice had a severe skeletal dysplasia, characterized by short stature, abnormal bone morphology, and increased bone mineral content. Despite normal bone length, Ncor1ΔID/ΔID mice displayed increased cortical bone mass, mineralization, and strength. Thra1PV/+Ncor1ΔID/ΔID double-mutant mice displayed only a small improvement of skeletal abnormalities compared to Thra1PV/+ mice. Treatment with SAHA to inhibit histone deacetylation had no beneficial or detrimental effects on bone structure, mineralization, or strength in wild-type or mutant mice. Conclusions: These studies indicate treatment with SAHA is unlikely to improve the skeletal manifestations of RTHα. Nevertheless, the findings (i) confirm that TRα1 has a critical role in the regulation of skeletal development and adult bone mass, (ii) suggest a physiological role for alternative co-repressors that interact with TR in skeletal cells, and (iii) demonstrate a novel role for NCoR1 in the regulation of adult bone mass and strength.

Assessment and Management of Anti-Insulin Autoantibodies in Varying Presentations of Insulin Autoimmune Syndrome.

Context: Insulin autoimmune syndrome (IAS), spontaneous hyperinsulinemic hypoglycemia due to insulin-binding autoantibodies, may be difficult to distinguish from tumoral or other forms of hyperinsulinemic hypoglycemia, including surreptitious insulin administration. No standardized treatment regimen exists. Objectives: To evaluate an analytic approach to IAS and responses to different treatments. Design and Setting: Observational study in the UK Severe Insulin Resistance Service. Patients: Six patients with hyperinsulinemic hypoglycemia and detectable circulating anti-insulin antibody (IA). Main Outcome Measures: Glycemia, plasma insulin, and C-peptide concentrations by immunoassay or mass spectrometry (MS). Immunoreactive insulin was determined in the context of polyethylene glycol (PEG) precipitation and gel filtration chromatography (GFC). IA quantification using ELISA and RIA, and IA were further characterized using radioligand binding studies. Results: All patients were diagnosed with IAS (five IgG, one IgA) based on a high insulin/C-peptide ratio, low insulin recovery after PEG precipitation, and GFC evidence of antibody-bound insulin. Neither ELISA nor RIA result proved diagnostic for every case. MS provided a more robust quantification of insulin in the context of IA. One patient was managed conservatively, four were treated with diazoxide without sustained benefit, and four were treated with immunosuppression with highly variable responses. IA affinity did not appear to influence presentation or prognosis. Conclusions: IAS should be considered in patients with hyperinsulinemic hypoglycemia and a high insulin/C-peptide ratio. Low insulin recovery on PEG precipitation supports the presence of insulin-binding antibodies, with GFC providing definitive confirmation. Immunomodulatory therapy should be customized according to individual needs and clinical response.

Rapid phenotyping of knockout mice to identify genetic determinants of bone strength.

The genetic determinants of osteoporosis remain poorly understood, and there is a large unmet need for new treatments in our ageing society. Thus, new approaches for gene discovery in skeletal disease are required to complement the current genome-wide association studies in human populations. The International Knockout Mouse Consortium (IKMC) and the International Mouse Phenotyping Consortium (IMPC) provide such an opportunity. The IKMC generates knockout mice representing each of the known protein-coding genes in C57BL/6 mice and, as part of the IMPC initiative, the Origins of Bone and Cartilage Disease project identifies mutants with significant outlier skeletal phenotypes. This initiative will add value to data from large human cohorts and provide a new understanding of bone and cartilage pathophysiology, ultimately leading to the identification of novel drug targets for the treatment of skeletal disease.

Renal biopsy in the very elderly.

BACKGROUND AND OBJECTIVES: Data regarding renal biopsy in the very elderly (>or=age 80 yr) are extremely limited. The aim of this study was to examine the causes of renal disease and their clinical presentations in very elderly patients who underwent native renal biopsy. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: All native renal biopsies (n = 235 including 106 men, 129 women) performed in patients aged >or=80 yr over a 3.67-yr period were retrospectively identified. Results were compared with a control group of 264 patients aged 60 to 61 who were biopsied over the same period. RESULTS: The indications for biopsy were acute kidney injury (AKI) in 46.4%, chronic-progressive kidney injury in 23.8%, nephrotic syndrome (NS) in 13.2%, NS with AKI in 9.4%, and isolated proteinuria in 5.5%. Pauci-immune GN was the most frequent diagnosis (19%), followed by focal segmental glomerulosclerosis secondary to hypertension (7.6%), hypertensive nephrosclerosis (7.1%), IgA nephropathy (7.1%) and membranous nephropathy (7.1%). Comparison with the control group showed pauci-immune GN to be more frequent (P < 0.001) and diabetic glomerulosclerosis (P < 0.001) and membranous nephropathy (P < 0.05) less frequent in the very elderly. Diagnostic information had the potential to modify treatment in 67% of biopsies from the very elderly, particularly in those with AKI or NS. CONCLUSIONS: Renal biopsy in very elderly patients is a valuable diagnostic tool that should be offered in clinical settings with maximal potential benefit. Advanced age per se should no longer be considered a contraindication to renal biopsy.