Publications by authors named "Sian E Piret"

21 Publications

  • Page 1 of 1

Proximal Tubular Transcription Factors in Acute Kidney Injury: Recent Advances.

Nephron 2020 9;144(12):613-615. Epub 2020 Jul 9.

Division of Nephrology, Department of Medicine, Stony Brook University, Stony Brook, New York, USA.

The proximal tubule (PT) is a major target in acute kidney injury (AKI), leading to profound changes in PT cell biology. Amongst the genes with early and robust changes in expression are many transcription factors (TFs), which themselves account for other transcriptomic changes. Potentially important TFs are being revealed in large sequencing datasets; however, to understand whether these TFs account for adaptive or maladaptive changes requires further mechanistic studies, which may reveal novel therapeutic targets. This mini review will highlight the identification and biology of 3 novel TFs in AKI: Sox9, Foxm1, and Foxo3.
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http://dx.doi.org/10.1159/000508856DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7708397PMC
July 2020

Genetic background influences tumour development in heterozygous Men1 knockout mice.

Endocr Connect 2020 May;9(5):426-437

Academic Endocrine Unit, Radcliffe Department of Medicine, University of Oxford, Oxford Centre for Diabetes, Endocrinology and Metabolism (OCDEM), Churchill Hospital, Headington, Oxford, UK.

Multiple endocrine neoplasia type 1 (MEN1), an autosomal dominant disorder caused by MEN1 germline mutations, is characterised by parathyroid, pancreatic and pituitary tumours. MEN1 mutations also cause familial isolated primary hyperparathyroidism (FIHP), a milder condition causing hyperparathyroidism only. Identical mutations can cause either MEN1 or FIHP in different families, thereby implicating a role for genetic modifiers in altering phenotypic expression of tumours. We therefore investigated the effects of genetic background and potential for genetic modifiers on tumour development in adult Men1+/- mice, which develop tumours of the parathyroids, pancreatic islets, anterior pituitary, adrenal cortex and gonads, that had been backcrossed to generate C57BL/6 and 129S6/SvEv congenic strains. A total of 275 Men1+/- mice, aged 5-26 months were macroscopically studied, and this revealed that genetic background significantly influenced the development of pituitary, adrenal and ovarian tumours, which occurred in mice over 12 months of age and more frequently in C57BL/6 females, 129S6/SvEv males and 129S6/SvEv females, respectively. Moreover, pituitary and adrenal tumours developed earlier, in C57BL/6 males and 129S6/SvEv females, respectively, and pancreatic and testicular tumours developed earlier in 129S6/SvEv males. Furthermore, glucagon-positive staining pancreatic tumours occurred more frequently in 129S6/SvEv Men1+/- mice. Whole genome sequence analysis of 129S6/SvEv and C57BL/6 Men1+/- mice revealed >54,000 different variants in >300 genes. These included, Coq7, Dmpk, Ccne2, Kras, Wnt2b, Il3ra and Tnfrsf10a, and qRT-PCR analysis revealed that Kras was significantly higher in pituitaries of male 129S6/SvEv mice. Thus, our results demonstrate that Kras and other genes could represent possible genetic modifiers of Men1.
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http://dx.doi.org/10.1530/EC-20-0103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7274560PMC
May 2020

Mice with a Brd4 Mutation Represent a New Model of Nephrocalcinosis.

J Bone Miner Res 2019 07 4;34(7):1324-1335. Epub 2019 Mar 4.

Academic Endocrine Unit, Oxford Centre for Diabetes, Endocrinology, and Metabolism, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.

Nephrolithiasis (NL) and nephrocalcinosis (NC), which comprise renal calcification of the collecting system and parenchyma, respectively, have a multifactorial etiology with environmental and genetic determinants and affect ∼10% of adults by age 70 years. Studies of families with hereditary NL and NC have identified >30 causative genes that have increased our understanding of extracellular calcium homeostasis and renal tubular transport of calcium. However, these account for <20% of the likely genes that are involved, and to identify novel genes for renal calcification disorders, we investigated 1745 12-month-old progeny from a male mouse that had been treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU) for radiological renal opacities. This identified a male mouse with renal calcification that was inherited as an autosomal dominant trait with >80% penetrance in 152 progeny. The calcification consisted of calcium phosphate deposits in the renal papillae and was associated with the presence of the urinary macromolecules osteopontin and Tamm-Horsfall protein, which are features found in Randall's plaques of patients with NC. Genome-wide mapping located the disease locus to a ∼30 Mbp region on chromosome 17A3.3-B3 and whole-exome sequence analysis identified a heterozygous mutation, resulting in a missense substitution (Met149Thr, M149T), in the bromodomain-containing protein 4 (BRD4). The mutant heterozygous (Brd4 ) mice, when compared with wild-type (Brd4 ) mice, were normocalcemic and normophosphatemic, with normal urinary excretions of calcium and phosphate, and had normal bone turnover markers. BRD4 plays a critical role in histone modification and gene transcription, and cDNA expression profiling, using kidneys from Brd4 and Brd4 mice, revealed differential expression of genes involved in vitamin D metabolism, cell differentiation, and apoptosis. Kidneys from Brd4 mice also had increased apoptosis at sites of calcification within the renal papillae. Thus, our studies have established a mouse model, due to a Brd4 Met149Thr mutation, for inherited NC. © 2019 American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbmr.3695DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6658219PMC
July 2019

An N-Ethyl-N-Nitrosourea (ENU)-Induced Tyr265Stop Mutation of the DNA Polymerase Accessory Subunit Gamma 2 (Polg2) Is Associated With Renal Calcification in Mice.

J Bone Miner Res 2019 03 14;34(3):497-507. Epub 2018 Dec 14.

Academic Endocrine Unit, Oxford Centre for Diabetes, Endocrinology and Metabolism, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.

Renal calcification (RCALC) resulting in nephrolithiasis and nephrocalcinosis, which affects ∼10% of adults by 70 years of age, involves environmental and genetic etiologies. Thus, nephrolithiasis and nephrocalcinosis occurs as an inherited disorder in ∼65% of patients, and may be associated with endocrine and metabolic disorders including: primary hyperparathyroidism, hypercalciuria, renal tubular acidosis, cystinuria, and hyperoxaluria. Investigations of families with nephrolithiasis and nephrocalcinosis have identified some causative genes, but further progress is limited as large families are unavailable for genetic studies. We therefore embarked on establishing mouse models for hereditary nephrolithiasis and nephrocalcinosis by performing abdominal X-rays to identify renal opacities in N-ethyl-N-nitrosourea (ENU)-mutagenized mice. This identified a mouse with RCALC inherited as an autosomal dominant trait, designated RCALC type 2 (RCALC2). Genomewide mapping located the Rcalc2 locus to a ∼16-Mbp region on chromosome 11D-E2 and whole-exome sequence analysis identified a heterozygous mutation in the DNA polymerase gamma-2, accessory subunit (Polg2) resulting in a nonsense mutation, Tyr265Stop (Y265X), which co-segregated with RCALC2. Kidneys of mutant mice (Polg2 ) had lower POLG2 mRNA and protein expression, compared to wild-type littermates (Polg2 ). The Polg2 and Polg2 mice had similar plasma concentrations of sodium, potassium, calcium, phosphate, chloride, urea, creatinine, glucose, and alkaline phosphatase activity; and similar urinary fractional excretion of calcium, phosphate, oxalate, and protein. Polg2 encodes the minor subunit of the mitochondrial DNA (mtDNA) polymerase and the mtDNA content in Polg2 kidneys was reduced compared to Polg2 mice, and cDNA expression profiling revealed differential expression of 26 genes involved in several biological processes including mitochondrial DNA function, apoptosis, and ubiquitination, the complement pathway, and inflammatory pathways. In addition, plasma of Polg2 mice, compared to Polg2 littermates had higher levels of reactive oxygen species. Thus, our studies have identified a mutant mouse model for inherited renal calcification associated with a Polg2 nonsense mutation. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/jbmr.3624DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446808PMC
March 2019

An -Ethyl--Nitrosourea (ENU) Mutagenized Mouse Model for Autosomal Dominant Nonsyndromic Kyphoscoliosis Due to Vertebral Fusion.

JBMR Plus 2018 May 8;2(3):154-163. Epub 2018 Mar 8.

Academic Endocrine Unit Radcliffe Department of Medicine University of Oxford Oxford Centre for Diabetes, Endocrinology and Metabolism Churchill Hospital Headington UK.

Kyphosis and scoliosis are common spinal disorders that occur as part of complex syndromes or as nonsyndromic, idiopathic diseases. Familial and twin studies implicate genetic involvement, although the causative genes for idiopathic kyphoscoliosis remain to be identified. To facilitate these studies, we investigated progeny of mice treated with the chemical mutagen -ethyl--nitrosourea (ENU) and assessed them for morphological and radiographic abnormalities. This identified a mouse with kyphoscoliosis due to fused lumbar vertebrae, which was inherited as an autosomal dominant trait; the phenotype was designated as hereditary vertebral fusion (HVF) and the locus as . Micro-computed tomography (μCT) analysis confirmed the occurrence of nonsyndromic kyphoscoliosis due to fusion of lumbar vertebrae in HVF mice, consistent with a pattern of blocked vertebrae due to failure of segmentation. μCT scans also showed the lumbar vertebral column of HVF mice to have generalized disc narrowing, displacement with compression of the neural spine, and distorted transverse processes. Histology of lumbar vertebrae revealed HVF mice to have irregularly shaped vertebral bodies and displacement of intervertebral discs and ossification centers. Genetic mapping using a panel of single nucleotide polymorphic (SNP) loci arranged in chromosome sets and DNA samples from 23 HVF (eight males and 15 females) mice, localized to chromosome 4A3 and within a 5-megabase (Mb) region containing nine protein coding genes, two processed transcripts, three microRNAs, five small nuclear RNAs, three large intergenic noncoding RNAs, and 24 pseudogenes. However, genome sequence analysis in this interval did not identify any abnormalities in the coding exons, or exon-intron boundaries of any of these genes. Thus, our studies have established a mouse model for a monogenic form of nonsyndromic kyphoscoliosis due to fusion of lumbar vertebrae, and further identification of the underlying genetic defect will help elucidate the molecular mechanisms involved in kyphoscoliosis. © 2018 The Authors. is published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbm4.10033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6124210PMC
May 2018

Podocyte-Specific Loss of Krüppel-Like Factor 6 Increases Mitochondrial Injury in Diabetic Kidney Disease.

Diabetes 2018 11 16;67(11):2420-2433. Epub 2018 Aug 16.

Division of Nephrology, Department of Medicine, Stony Brook University, Stony Brook, NY

Mitochondrial injury is uniformly observed in several murine models as well as in individuals with diabetic kidney disease (DKD). Although emerging evidence has highlighted the role of key transcriptional regulators in mitochondrial biogenesis, little is known about the regulation of mitochondrial cytochrome c oxidase assembly in the podocyte under diabetic conditions. We recently reported a critical role of the zinc finger Krüppel-like factor 6 (KLF6) in maintaining mitochondrial function and preventing apoptosis in a proteinuric murine model. In this study, we report that podocyte-specific knockdown of increased the susceptibility to streptozotocin-induced DKD in the resistant C57BL/6 mouse strain. We observed that the loss of in podocytes reduced the expression of with resultant increased mitochondrial injury, leading to activation of the intrinsic apoptotic pathway under diabetic conditions. Conversely, mitochondrial injury and apoptosis were significantly attenuated with overexpression of in cultured human podocytes under hyperglycemic conditions. Finally, we observed a significant reduction in glomerular and podocyte-specific expression of KLF6 in human kidney biopsies with progression of DKD. Collectively, these data suggest that podocyte-specific KLF6 is critical to preventing mitochondrial injury and apoptosis under diabetic conditions.
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http://dx.doi.org/10.2337/db17-0958DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6198342PMC
November 2018

Cinacalcet corrects hypercalcemia in mice with an inactivating Gα11 mutation.

JCI Insight 2017 10 19;2(20). Epub 2017 Oct 19.

Academic Endocrine Unit, Radcliffe Department of Medicine, University of Oxford, Oxford, United Kingdom.

Loss-of-function mutations of GNA11, which encodes G-protein subunit α11 (Gα11), a signaling partner for the calcium-sensing receptor (CaSR), result in familial hypocalciuric hypercalcemia type 2 (FHH2). FHH2 is characterized by hypercalcemia, inappropriately normal or raised parathyroid hormone (PTH) concentrations, and normal or low urinary calcium excretion. A mouse model for FHH2 that would facilitate investigations of the in vivo role of Gα11 and the evaluation of calcimimetic drugs, which are CaSR allosteric activators, is not available. We therefore screened DNA from > 10,000 mice treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU) for GNA11 mutations and identified a Gα11 variant, Asp195Gly (D195G), which downregulated CaSR-mediated intracellular calcium signaling in vitro, consistent with it being a loss-of-function mutation. Treatment with the calcimimetic cinacalcet rectified these signaling responses. In vivo studies showed mutant heterozygous (Gna11+/195G) and homozygous (Gna11195G/195G) mice to be hypercalcemic with normal or increased plasma PTH concentrations and normal urinary calcium excretion. Cinacalcet (30mg/kg orally) significantly reduced plasma albumin-adjusted calcium and PTH concentrations in Gna11+/195G and Gna11195G/195G mice. Thus, our studies have established a mouse model with a germline loss-of-function Gα11 mutation that is representative for FHH2 in humans and demonstrated that cinacalcet can correct the associated abnormalities of plasma calcium and PTH.
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http://dx.doi.org/10.1172/jci.insight.96540DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5846897PMC
October 2017

A mouse model for inherited renal fibrosis associated with endoplasmic reticulum stress.

Dis Model Mech 2017 06 21;10(6):773-786. Epub 2017 Mar 21.

Academic Endocrine Unit, University of Oxford, Oxford Centre for Diabetes, Endocrinology and Metabolism, Churchill Hospital, Headington, Oxford OX3 7LJ, UK

Renal fibrosis is a common feature of renal failure resulting from multiple etiologies, including diabetic nephropathy, hypertension and inherited renal disorders. However, the mechanisms of renal fibrosis are incompletely understood and we therefore explored these by establishing a mouse model for a renal tubular disorder, referred to as autosomal dominant tubulointerstitial kidney disease (ADTKD) due to missense uromodulin () mutations (ADTKD-). ADTKD-, which is associated with retention of mutant uromodulin in the endoplasmic reticulum (ER) of renal thick ascending limb cells, is characterized by hyperuricemia, interstitial fibrosis, inflammation and renal failure, and we used targeted homologous recombination to generate a knock-in mouse model with an ADTKD-causing missense cysteine to arginine uromodulin mutation (C125R). Heterozygous and homozygous mutant mice developed reduced uric acid excretion, renal fibrosis, immune cell infiltration and progressive renal failure, with decreased maturation and excretion of uromodulin, due to its retention in the ER. The ER stress marker 78 kDa glucose-regulated protein (GRP78) was elevated in cells expressing mutant uromodulin in heterozygous and homozygous mutant mice, and this was accompanied, both and , by upregulation of two unfolded protein response pathways in primary thick ascending limb cells from homozygous mutant mice. However, this did not lead to an increase in apoptosis Thus, we have developed a novel mouse model for renal fibrosis, which will be a valuable resource to decipher the mechanisms linking uromodulin mutations with ER stress and renal fibrosis.
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http://dx.doi.org/10.1242/dmm.029488DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5483009PMC
June 2017

G mutation in mice causes hypocalcemia rectifiable by calcilytic therapy.

JCI Insight 2017 02 9;2(3):e91103. Epub 2017 Feb 9.

Academic Endocrine Unit, Radcliffe Department of Medicine, University of Oxford, Oxford, United Kingdom.

Heterozygous germline gain-of-function mutations of G-protein subunit α (Gα), a signaling partner for the calcium-sensing receptor (CaSR), result in autosomal dominant hypocalcemia type 2 (ADH2). ADH2 may cause symptomatic hypocalcemia with low circulating parathyroid hormone (PTH) concentrations. Effective therapies for ADH2 are currently not available, and a mouse model for ADH2 would help in assessment of potential therapies. We hypothesized that a previously reported dark skin mouse mutant () - which has a germline hypermorphic Gα mutation, Ile62Val - may be a model for ADH2 and allow evaluation of calcilytics, which are CaSR negative allosteric modulators, as a targeted therapy for this disorder. Mutant and mice were shown to have hypocalcemia and reduced plasma PTH concentrations, similar to ADH2 patients. In vitro studies showed the mutant Val62 Gα to upregulate CaSR-mediated intracellular calcium and MAPK signaling, consistent with a gain of function. Treatment with NPS-2143, a calcilytic compound, normalized these signaling responses. In vivo, NPS-2143 induced a rapid and marked rise in plasma PTH and calcium concentrations in and mice, which became normocalcemic. Thus, these studies have established mice, which harbor a germline gain-of-function Gα mutation, as a model for ADH2 and have demonstrated calcilytics as a potential targeted therapy.
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http://dx.doi.org/10.1172/jci.insight.91103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5291742PMC
February 2017

Mice with an N-Ethyl-N-Nitrosourea (ENU) Induced Tyr209Asn Mutation in Natriuretic Peptide Receptor 3 (NPR3) Provide a Model for Kyphosis Associated with Activation of the MAPK Signaling Pathway.

PLoS One 2016 13;11(12):e0167916. Epub 2016 Dec 13.

Oxford Centre for Diabetes, Endocrinology and Metabolism, Radcliffe Department of Medicine, University of Oxford, Oxford, United Kingdom.

Non-syndromic kyphosis is a common disorder that is associated with significant morbidity and has a strong genetic involvement; however, the causative genes remain to be identified, as such studies are hampered by genetic heterogeneity, small families and various modes of inheritance. To overcome these limitations, we investigated 12 week old progeny of mice treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU) using phenotypic assessments including dysmorphology, radiography, and dual-energy X-ray absorptiometry. This identified a mouse with autosomal recessive kyphosis (KYLB). KYLB mice, when compared to unaffected littermates, had: thoraco-lumbar kyphosis, larger vertebrae, and increased body length and increased bone area. In addition, female KYLB mice had increases in bone mineral content and plasma alkaline phosphatase activity. Recombination mapping localized the Kylb locus to a 5.5Mb region on chromosome 15A1, which contained 51 genes, including the natriuretic peptide receptor 3 (Npr3) gene. DNA sequence analysis of Npr3 identified a missense mutation, Tyr209Asn, which introduced an N-linked glycosylation consensus sequence. Expression of wild-type NPR3 and the KYLB-associated Tyr209Asn NPR3 mutant in COS-7 cells demonstrated the mutant to be associated with abnormal N-linked glycosylation and retention in the endoplasmic reticulum that resulted in its absence from the plasma membrane. NPR3 is a decoy receptor for C-type natriuretic peptide (CNP), which also binds to NPR2 and stimulates mitogen-activated protein kinase (MAPK) signaling, thereby increasing the number and size of hypertrophic chondrocytes. Histomorphometric analysis of KYLB vertebrae and tibiae showed delayed endochondral ossification and expansion of the hypertrophic zones of the growth plates, and immunohistochemistry revealed increased p38 MAPK phosphorylation throughout the growth plates of KYLB vertebrae. Thus, we established a model of kyphosis due to a novel NPR3 mutation, in which loss of plasma membrane NPR3 expression results in increased MAPK pathway activation, causing elongation of the vertebrae and resulting in kyphosis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0167916PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5154531PMC
June 2017

Autosomal dominant osteopetrosis associated with renal tubular acidosis is due to a CLCN7 mutation.

Am J Med Genet A 2016 11 19;170(11):2988-2992. Epub 2016 Aug 19.

Academic Endocrine Unit, Oxford Centre for Diabetes, Endocrinology and Metabolism, Churchill Hospital, University of Oxford, Oxford, United Kingdom.

The aim of this study was to identify the causative mutation in a family with an unusual presentation of autosomal dominant osteopetrosis (OPT), proximal renal tubular acidosis (RTA), renal stones, epilepsy, and blindness, a combination of features not previously reported. We undertook exome sequencing of one affected and one unaffected family member, followed by targeted analysis of known candidate genes to identify the causative mutation. This identified a missense mutation (c.643G>A; p.Gly215Arg) in the gene encoding the chloride/proton antiporter 7 (gene CLCN7, protein CLC-7), which was confirmed by amplification refractory mutation system (ARMS)-PCR, and to be present in the three available patients. CLC-7 mutations are known to cause autosomal dominant OPT type 2, also called Albers-Schonberg disease, which is characterized by osteosclerosis, predominantly of the spine, pelvis and skull base, resulting in bone fragility and fractures. Albers-Schonberg disease is not reported to be associated with RTA, but autosomal recessive OPT type 3 (OPTB3) with RTA is associated with carbonic anhydrase type 2 (CA2) mutations. No mutations were detected in CA2 or any other genes known to cause proximal RTA. Neither CLCN7 nor CA2 mutations have previously been reported to be associated with renal stones or epilepsy. Thus, we identified a CLCN7 mutation in a family with autosomal dominant osteopetrosis, RTA, renal stones, epilepsy, and blindness. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/ajmg.a.37755DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5132132PMC
November 2016

Identification of a G-Protein Subunit-α11 Gain-of-Function Mutation, Val340Met, in a Family With Autosomal Dominant Hypocalcemia Type 2 (ADH2).

J Bone Miner Res 2016 06;31(6):1207-14

Academic Endocrine Unit, University of Oxford, Oxford Centre for Diabetes, Endocrinology and Metabolism, Churchill Hospital, Oxford, UK.

Autosomal dominant hypocalcemia (ADH) is characterized by hypocalcemia, inappropriately low serum parathyroid hormone concentrations and hypercalciuria. ADH is genetically heterogeneous with ADH type 1 (ADH1), the predominant form, being caused by germline gain-of-function mutations of the G-protein coupled calcium-sensing receptor (CaSR), and ADH2 caused by germline gain-of-function mutations of G-protein subunit α-11 (Gα11 ). To date Gα11 mutations causing ADH2 have been reported in only five probands. We investigated a multigenerational nonconsanguineous family, from Iran, with ADH and keratoconus which are not known to be associated, for causative mutations by whole-exome sequencing in two individuals with hypoparathyroidism, of whom one also had keratoconus, followed by cosegregation analysis of variants. This identified a novel heterozygous germline Val340Met Gα11 mutation in both individuals, and this was also present in the other two relatives with hypocalcemia that were tested. Three-dimensional modeling revealed the Val340Met mutation to likely alter the conformation of the C-terminal α5 helix, which may affect G-protein coupled receptor binding and G-protein activation. In vitro functional expression of wild-type (Val340) and mutant (Met340) Gα11 proteins in HEK293 cells stably expressing the CaSR, demonstrated that the intracellular calcium responses following stimulation with extracellular calcium, of the mutant Met340 Gα11 led to a leftward shift of the concentration-response curve with a significantly (p < 0.0001) reduced mean half-maximal concentration (EC50 ) value of 2.44 mM (95% CI, 2.31 to 2.77 mM) when compared to the wild-type EC50 of 3.14 mM (95% CI, 3.03 to 3.26 mM), consistent with a gain-of-function mutation. A novel His403Gln variant in transforming growth factor, beta-induced (TGFBI), that may be causing keratoconus was also identified, indicating likely digenic inheritance of keratoconus and ADH2 in this family. In conclusion, our identification of a novel germline gain-of-function Gα11 mutation, Val340Met, causing ADH2 demonstrates the importance of the Gα11 C-terminal region for G-protein function and CaSR signal transduction. © 2016 American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbmr.2797DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4915495PMC
June 2016

Factors influencing success of clinical genome sequencing across a broad spectrum of disorders.

Nat Genet 2015 Jul 18;47(7):717-726. Epub 2015 May 18.

Institute of Physiology, Zurich Center for Integrative Human Physiology, University of Zurich, Zurich, Switzerland.

To assess factors influencing the success of whole-genome sequencing for mainstream clinical diagnosis, we sequenced 217 individuals from 156 independent cases or families across a broad spectrum of disorders in whom previous screening had identified no pathogenic variants. We quantified the number of candidate variants identified using different strategies for variant calling, filtering, annotation and prioritization. We found that jointly calling variants across samples, filtering against both local and external databases, deploying multiple annotation tools and using familial transmission above biological plausibility contributed to accuracy. Overall, we identified disease-causing variants in 21% of cases, with the proportion increasing to 34% (23/68) for mendelian disorders and 57% (8/14) in family trios. We also discovered 32 potentially clinically actionable variants in 18 genes unrelated to the referral disorder, although only 4 were ultimately considered reportable. Our results demonstrate the value of genome sequencing for routine clinical diagnosis but also highlight many outstanding challenges.
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http://dx.doi.org/10.1038/ng.3304DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4601524PMC
July 2015

Association between genotype and phenotype in uromodulin-associated kidney disease.

Clin J Am Soc Nephrol 2013 Aug 30;8(8):1349-57. Epub 2013 May 30.

Renal Research Institute, 207 East 94th Street, New York, NY 10128, USA.

Background And Objectives: Uromodulin-associated kidney disease (UAKD) is an autosomal dominant disease caused by uromodulin (UMOD) gene mutations. This study explored genotype-phenotype correlations by examining the relationship between the type of UMOD mutation and the age at onset of ESRD.

Design, Setting, Participants & Measurements: Extensive bibliographic research was used to ascertain patient-level data of all patients with UAKD published up to October 2011. Data included sex; ages at onset of hyperuricemia, gout, and ESRD; and UMOD genotype. Kaplan-Meier analysis and Cox proportional hazards models fitted with shared gamma frailty terms to adjust for within-family correlations were used to model time to event.

Results: Thirty-one peer-reviewed publications reporting on 202 patients from 74 families with 59 different UMOD mutations were included. Median ages at onset of hyperuricemia, gout, and ESRD were 24, 40, and 56 years, respectively. Men developed gout and ESRD significantly earlier than did women (age at ESRD was 50 years for men and 60 for women; P=0.04, shared frailty model). Median ages at ESRD development were lowest with Cys77Tyr (37.5 years) and highest with Gln316Pro (65.5 years) UMOD mutations. Onset of ESRD was significantly earlier with UMOD mutations located within the epidermal growth factor domains 2 and 3 (range, 45-52 years; P<0.01 and 0.04, respectively) compared with the cysteine-rich domains (range, 60-65 years; by shared frailty model).

Conclusions: The UMOD genotype is related to the clinical phenotype of UAKD. This finding may assist in counseling of patients.
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http://dx.doi.org/10.2215/CJN.11151012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3731914PMC
August 2013

Receptor-mediated endocytosis and endosomal acidification is impaired in proximal tubule epithelial cells of Dent disease patients.

Proc Natl Acad Sci U S A 2013 Apr 9;110(17):7014-9. Epub 2013 Apr 9.

Academic Endocrine Unit, Oxford Centre for Diabetes, Endocrinology, and Metabolism, Nuffield Department of Clinical Medicine, University of Oxford, Churchill Hospital, Oxford OX3 7LJ, United Kingdom.

Receptor-mediated endocytosis, involving megalin and cubilin, mediates renal proximal-tubular reabsorption and is decreased in Dent disease because of mutations of the chloride/proton antiporter, chloride channel-5 (CLC-5), resulting in low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis, and renal failure. To facilitate studies of receptor-mediated endocytosis and the role of CLC-5, we established conditionally immortalized proximal-tubular epithelial cell lines (ciPTECs) from three patients with CLC-5 mutations (30:insH, R637X, and del132-241) and a normal male. Confocal microscopy using the tight junction marker zona occludens-1 (ZO-1) and end-binding protein-1 (EB-1), which is specific for the plus end of microtubules demonstrated that the ciPTECs polarized. Receptor-mediated endocytic uptake of fluorescent albumin and transferrin in 30:insH and R637X ciPTECs was significantly decreased, compared with normal ciPTECs, and could be further reduced by competition with 10-fold excess of unlabeled albumin and transferrin, whereas in the del132-241 ciPTEC, receptor-mediated endocytic uptake was abolished. Investigation of endosomal acidification by live-cell imaging of pHluorin-VAMP2 (vesicle-associated membrane protein-2), a pH-sensitive-GFP construct, revealed that the endosomal pH in normal and 30:insH ciPTECs was similar, whereas in del132-241 and R637X ciPTECs, it was significantly more alkaline, indicating defective acidification in these ciPTECs. The addition of bafilomycin-A1, a V-ATPase inhibitor, raised the pH significantly in all ciPTECs, demonstrating that the differences in acidification were not due to alterations in the V-ATPase, but instead to abnormalities of CLC-5. Thus, our studies, which have established human Dent disease ciPTECs that will facilitate studies of mechanisms in renal reabsorption, demonstrate that Dent disease-causing CLC-5 mutations have differing effects on endosomal acidification and receptor-mediated endocytosis that may not be coupled.
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http://dx.doi.org/10.1073/pnas.1302063110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3637698PMC
April 2013

Autosomal dominant hypercalciuria in a mouse model due to a mutation of the epithelial calcium channel, TRPV5.

PLoS One 2013 30;8(1):e55412. Epub 2013 Jan 30.

Academic Endocrine Unit, Nuffield Department of Medicine, University of Oxford, Oxford Centre for Diabetes, Endocrinology and Metabolism-OCDEM, Churchill Hospital, Headington, Oxford, United Kingdom.

Hypercalciuria is a major cause of nephrolithiasis, and is a common and complex disorder involving genetic and environmental factors. Identification of genetic factors for monogenic forms of hypercalciuria is hampered by the limited availability of large families, and to facilitate such studies, we screened for hypercalciuria in mice from an N-ethyl-N-nitrosourea mutagenesis programme. We identified a mouse with autosomal dominant hypercalciuria (HCALC1). Linkage studies mapped the Hcalc1 locus to a 11.94 Mb region on chromosome 6 containing the transient receptor potential cation channel, subfamily V, members 5 (Trpv5) and 6 (Trpv6) genes. DNA sequence analysis of coding regions, intron-exon boundaries and promoters of Trpv5 and Trpv6 identified a novel T to C transition in codon 682 of TRPV5, mutating a conserved serine to a proline (S682P). Compared to wild-type littermates, heterozygous (Trpv5(682P/+)) and homozygous (Trpv5(682P/682P)) mutant mice had hypercalciuria, polyuria, hyperphosphaturia and a more acidic urine, and ∼10% of males developed tubulointerstitial nephritis. Trpv5(682P/682P) mice also had normal plasma parathyroid hormone but increased 1,25-dihydroxyvitamin D(3) concentrations without increased bone resorption, consistent with a renal defect for the hypercalciuria. Expression of the S682P mutation in human embryonic kidney cells revealed that TRPV5-S682P-expressing cells had a lower baseline intracellular calcium concentration than wild-type TRPV5-expressing cells, suggesting an altered calcium permeability. Immunohistological studies revealed a selective decrease in TRPV5-expression from the renal distal convoluted tubules of Trpv5(682P/+) and Trpv5(682P/682P) mice consistent with a trafficking defect. In addition, Trpv5(682P/682P) mice had a reduction in renal expression of the intracellular calcium-binding protein, calbindin-D(28K), consistent with a specific defect in TRPV5-mediated renal calcium reabsorption. Thus, our findings indicate that the TRPV5 S682P mutant is functionally significant and study of HCALC1, a novel model for autosomal dominant hypercalciuria, may help further our understanding of renal calcium reabsorption and hypercalciuria.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0055412PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3559602PMC
July 2013

A mouse model of early-onset renal failure due to a xanthine dehydrogenase nonsense mutation.

PLoS One 2012 14;7(9):e45217. Epub 2012 Sep 14.

Nuffield Department of Clinical Medicine, Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Oxford, United Kingdom.

Chronic kidney disease (CKD) is characterized by renal fibrosis that can lead to end-stage renal failure, and studies have supported a strong genetic influence on the risk of developing CKD. However, investigations of the underlying molecular mechanisms are hampered by the lack of suitable hereditary models in animals. We therefore sought to establish hereditary mouse models for CKD and renal fibrosis by investigating mice treated with the chemical mutagen N-ethyl-N-nitrosourea, and identified a mouse with autosomal recessive renal failure, designated RENF. Three-week old RENF mice were smaller than their littermates, whereas at birth they had been of similar size. RENF mice, at 4-weeks of age, had elevated concentrations of plasma urea and creatinine, indicating renal failure, which was associated with small and irregularly shaped kidneys. Genetic studies using DNA from 10 affected mice and 91 single nucleotide polymorphisms mapped the Renf locus to a 5.8 Mbp region on chromosome 17E1.3. DNA sequencing of the xanthine dehydrogenase (Xdh) gene revealed a nonsense mutation at codon 26 that co-segregated with affected RENF mice. The Xdh mutation resulted in loss of hepatic XDH and renal Cyclooxygenase-2 (COX-2) expression. XDH mutations in man cause xanthinuria with undetectable plasma uric acid levels and three RENF mice had plasma uric acid levels below the limit of detection. Histological analysis of RENF kidney sections revealed abnormal arrangement of glomeruli, intratubular casts, cellular infiltration in the interstitial space, and interstitial fibrosis. TUNEL analysis of RENF kidney sections showed extensive apoptosis predominantly affecting the tubules. Thus, we have established a mouse model for autosomal recessive early-onset renal failure due to a nonsense mutation in Xdh that is a model for xanthinuria in man. This mouse model could help to increase our understanding of the molecular mechanisms associated with renal fibrosis and the specific roles of XDH and uric acid.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0045217PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3443222PMC
February 2013

Epidemiology of uromodulin-associated kidney disease - results from a nation-wide survey.

Nephron Extra 2012 Jan 1;2(1):147-58. Epub 2012 Jun 1.

Department of Nephrology and Dialysis, Academic Teaching Hospital Feldkirch, Austria.

Background/aims: Uromodulin-associated kidney disease (UAKD) is caused by uromodulin mutations and leads to end-stage renal disease. Our objective was to examine the epidemiology of UAKD.

Methods: Data from all UAKD families in Austria were collected. Patients included in the Austrian Dialysis and Transplantation Registry (OEDTR) with unclear diagnoses or genetic diseases were asked whether they had (1) a family history of kidney disease or (2) had suffered from gout. Patients with gout and autosomal dominant renal disease underwent mutational analysis. Kaplan-Meier and Cox analysis was employed to estimate time to renal failure.

Results: Of the 6,210 patients in the OEDTR, 541 were approached with a questionnaire; 353 patients answered the questionnaire. Nineteen of them gave two affirmative answers. In 7 patients, an autosomal dominant renal disease was found; in 1 patient a UMOD mutation was identified. One family was diagnosed through increased awareness as a consequence of the study. At present, 14 UAKD patients from 5 families are living in Austria (1.67 cases per million), and 6 of them require renal replacement therapy (0.73 per 1,000 patients). Progression to renal failure was significantly associated with UMOD genotype.

Conclusion: UAKD patients can be identified by a simple questionnaire. UMOD genotype may affect disease progression.
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http://dx.doi.org/10.1159/000339102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3383240PMC
January 2012

Mouse models for inherited endocrine and metabolic disorders.

J Endocrinol 2011 Dec 15;211(3):211-30. Epub 2011 Jul 15.

Academic Endocrine Unit, Oxford Centre for Diabetes, Endocrinology and Metabolism, Churchill Hospital, University of Oxford, Headington, Oxford OX3 7LJ, UK.

In vivo models represent important resources for investigating the physiological mechanisms underlying endocrine and metabolic disorders, and for pre-clinical translational studies that may include the assessments of new treatments. In the study of endocrine diseases, which affect multiple organs, in vivo models provide specific advantages over in vitro models, which are limited to investigation of isolated systems. In recent years, the mouse has become the popular choice for developing such in vivo mammalian models, as it has a genome that shares ∼85% identity to that of man, and has many physiological systems that are similar to those in man. Moreover, methods have been developed to alter the expression of genes in the mouse, thereby generating models for human diseases, which may be due to loss- or gain-of-function mutations. The methods used to generate mutations in the mouse genome include: chemical mutagenesis; conventional, conditional and inducible knockout models; knockin models and transgenic models, and these strategies are often complementary. This review describes some of the different strategies that are utilised for generating mouse models. In addition, some mouse models that have been successfully generated by these methods for some human hereditary endocrine and metabolic disorders are reviewed. In particular, the mouse models generated for parathyroid disorders, which include: the multiple endocrine neoplasias; hyperparathyroidism-jaw tumour syndrome; disorders of the calcium-sensing receptor and forms of inherited hypoparathyroidism are discussed. The advances that have been made in our understanding of the mechanisms of these human diseases by investigations of these mouse models are described.
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http://dx.doi.org/10.1530/JOE-11-0193DOI Listing
December 2011

Genome-wide study of familial juvenile hyperuricaemic (gouty) nephropathy (FJHN) indicates a new locus, FJHN3, linked to chromosome 2p22.1-p21.

Hum Genet 2011 Jan 26;129(1):51-8. Epub 2010 Oct 26.

Academic Endocrine Unit, Oxford Centre for Diabetes Endocrinology and Metabolism, Churchill Hospital, University of Oxford, Headington, Oxford, OX3 7LJ, UK.

Familial juvenile hyperuricaemic (gouty) nephropathy (FJHN), is an autosomal dominant disease associated with a reduced fractional excretion of urate, and progressive renal failure. FJHN is genetically heterogeneous and due to mutations of three genes: uromodulin (UMOD), renin (REN) and hepatocyte nuclear factor-1beta (HNF-1β) on chromosomes 16p12, 1q32.1, and 17q12, respectively. However, UMOD, REN or HNF-1β mutations are found in only approximately 45% of FJHN probands, indicating the involvement of other genetic loci in approximately 55% of probands. To identify other FJHN loci, we performed a single nucleotide polymorphism (SNP)-based genome-wide linkage analysis, in six FJHN families in whom UMOD, HNF-1β and REN mutations had been excluded. Parametric linkage analysis using a 'rare dominant' model established linkage in five of the six FJHN families, with a LOD score >+3, at 0% recombination, between FJHN and SNPs at chromosome 2p22.1-p21. Analysis of individual recombinants in two unrelated affected individuals defined a approximately 5.5 Mbp interval, flanked telomerically by SNP RS372139 and centromerically by RS896986 that contained the locus, designated FJHN3. The interval contains 28 genes, and DNA sequence analysis of the most likely candidate, solute carrier family 8 member 1 (SLC8A1), did not identify any abnormalities in the FJHN3 probands. FJHN3 is likely located within a approximately 5.5 Mbp interval on chromosome 2p22.1-p21, and identifying the genetic abnormality will help to further elucidate mechanisms predisposing to gout and renal failure.
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http://dx.doi.org/10.1007/s00439-010-0897-1DOI Listing
January 2011

Identification and characterization of novel parathyroid-specific transcription factor Glial Cells Missing Homolog B (GCMB) mutations in eight families with autosomal recessive hypoparathyroidism.

Hum Mol Genet 2010 May 27;19(10):2028-38. Epub 2010 Feb 27.

Academic Endocrine Unit, Nuffield Department of Clinical Medicine, Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Churchill Hospital, Headington, Oxford OX3 7LJ, UK.

GCMB is a member of the small transcription factor family GCM (glial cells missing), which are important regulators of development, present in vertebrates and some invertebrates. In man, GCMB encodes a 506 amino acid parathyroid gland-specific protein, mutations of which have been reported to cause both autosomal dominant and autosomal recessive hypoparathyroidism. We ascertained 18 affected individuals from 12 families with autosomal recessive hypoparathyroidism and have investigated them for GCMB abnormalities. Four different homozygous germline mutations were identified in eight families that originate from the Indian Subcontinent. These consisted of a novel nonsense mutation R39X; a missense mutation, R47L in two families; a novel missense mutation, R110W; and a novel frameshifting deletion, I298fsX307 in four families. Haplotype analysis, using polymorphic microsatellites from chromosome 6p23-24, revealed that R47L and I298fsX307 mutations arose either as ancient founders, or recurrent de novo mutations. Functional studies including: subcellular localization studies, EMSAs and luciferase-reporter assays, were undertaken and these demonstrated that: the R39X mutant failed to localize to the nucleus; the R47L and R110W mutants both lost DNA-binding ability; and the I298fsX307 mutant had reduced transactivational ability. In order to gain further insights, we undertook 3D-modeling of the GCMB DNA-binding domain, which revealed that the R110 residue is likely important for the structural integrity of helix 2, which forms part of the GCMB/DNA binding interface. Thus, our results, which expand the spectrum of hypoparathyroidism-associated GCMB mutations, help elucidate the molecular mechanisms underlying DNA-binding and transactivation that are required for this parathyroid-specific transcription factor.
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http://dx.doi.org/10.1093/hmg/ddq084DOI Listing
May 2010