Prof Rajesh V Thakker

Research Area: Genetics and Genomics
Technology Exchange: Bioinformatics, Chromosome mapping, ES cell / homologous recombination and Gene therapy
Keywords: Molecular Genetics, Calcium disorders, Neuroendocrine tumours, parathyroid diorders, tubular disorders and MEN syndromes
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The mission of the Academic Endocrine Unit is to investigate the molecular basis of important endocrine and metabolic disorders that principally affect calcium and phosphate homeostasis. These disorders may be due to endocrine neoplasia, renal tubular defects, or abnormalities of bone metabolism. Thus, the identification of the underlying mechanisms is expected to lead to advances in our understanding of a number of clinical disorders that result in endocrine tumour development, kidney stones and different types of bone disease including rickets and osteoporosis. The research activities can be broadly divided into 2 parallel but complementary programmes: 1) Endocrine tumours and related parathyroid; and 2) Renal tubular and hypercalciuric stone disorders.

There are no collaborations listed for this principal investigator.

Kennedy AM, Inada M, Krane SM, Christie PT, Harding B, López-Otín C, Sánchez LM, Pannett AA et al. 2005. MMP13 mutation causes spondyloepimetaphyseal dysplasia, Missouri type (SEMD(MO). J Clin Invest, 115 (10), pp. 2832-2842. Read abstract | Read more

MMPs, which degrade components of the ECM, have roles in embryonic development, tissue repair, cancer, arthritis, and cardiovascular disease. We show that a missense mutation of MMP13 causes the Missouri type of human spondyloepimetaphyseal dysplasia (SEMD(MO)), an autosomal dominant disorder characterized by defective growth and modeling of vertebrae and long bones. Genome-wide linkage analysis mapped SEMD(MO) to a 17-cM region on chromosome 11q14.3-23.2 that contains a cluster of 9 MMP genes. Among these, MMP13 represented the best candidate for SEMD(MO), since it preferentially degrades collagen type II, abnormalities of which cause skeletal dysplasias that include Strudwick type SEMD. DNA sequence analysis revealed a missense mutation, F56S, that substituted an evolutionarily conserved phenylalanine residue for a serine in the proregion domain of MMP13. We predicted, by modeling MMP13 structure, that this F56S mutation would result in a hydrophobic cavity with misfolding, autoactivation, and degradation of mutant protein intracellularly. Expression of wild-type and mutant MMP13s in human embryonic kidney cells confirmed abnormal intracellular autoactivation and autodegradation of F56S MMP13 such that only enzymatically inactive, small fragments were secreted. Thus, the F56S mutation results in deficiency of MMP13, which leads to the human skeletal developmental anomaly of SEMD(MO). Hide abstract

Bowl MR, Nesbit MA, Harding B, Levy E, Jefferson A, Volpi E, Rizzoti K, Lovell-Badge R, Schlessinger D, Whyte MP, Thakker RV. 2005. An interstitial deletion-insertion involving chromosomes 2p25.3 and Xq27.1, near SOX3, causes X-linked recessive hypoparathyroidism. J Clin Invest, 115 (10), pp. 2822-2831. Read abstract | Read more

X-linked recessive hypoparathyroidism, due to parathyroid agenesis, has been mapped to a 906-kb region on Xq27 that contains 3 genes (ATP11C, U7snRNA, and SOX3), and analyses have not revealed mutations. We therefore characterized this region by combined analysis of single nucleotide polymorphisms and sequence-tagged sites. This identified a 23- to 25-kb deletion, which did not contain genes. However, DNA fiber-FISH and pulsed-field gel electrophoresis revealed an approximately 340-kb insertion that replaced the deleted fragment. Use of flow-sorted X chromosome-specific libraries and DNA sequence analyses revealed that the telomeric and centromeric breakpoints on X were, respectively, approximately 67 kb downstream of SOX3 and within a repetitive sequence. Use of a monochromosomal somatic cell hybrid panel and metaphase-FISH mapping demonstrated that the insertion originated from 2p25 and contained a segment of the SNTG2 gene that lacked an open reading frame. However, the deletion-insertion [del(X)(q27.1) inv ins (X;2)(q27.1;p25.3)], which represents a novel abnormality causing hypoparathyroidism, could result in a position effect on SOX3 expression. Indeed, SOX3 expression was demonstrated, by in situ hybridization, in the developing parathyroid tissue of mouse embryos between 10.5 and 15.5 days post coitum. Thus, our results indicate a likely new role for SOX3 in the embryonic development of the parathyroid glands. Hide abstract

Van Esch H, Groenen P, Nesbit MA, Schuffenhauer S, Lichtner P, Vanderlinden G, Harding B, Beetz R et al. 2000. GATA3 haplo-insufficiency causes human HDR syndrome. Nature, 406 (6794), pp. 419-422. Read abstract | Read more

Terminal deletions of chromosome 10p result in a DiGeorge-like phenotype that includes hypoparathyroidism, heart defects, immune deficiency, deafness and renal malformations. Studies in patients with 10p deletions have defined two non-overlapping regions that contribute to this complex phenotype. These are the DiGeorge critical region II (refs 1, 2), which is located on 10p13-14, and the region for the hypoparathyroidism, sensorineural deafness, renal anomaly (HDR) syndrome (Mendelian Inheritance in Man number 146255), which is located more telomeric (10p14-10pter). We have performed deletion-mapping studies in two HDR patients, and here we define a critical 200-kilobase region which contains the GATA3 gene. This gene belongs to a family of zinc-finger transcription factors that are involved in vertebrate embryonic development. Investigation for GATA3 mutations in three other HDR probands identified one nonsense mutation and two intragenic deletions that predicted a loss of function, as confirmed by absence of DNA binding by the mutant GATA3 protein. These results show that GATA3 is essential in the embryonic development of the parathyroids, auditory system and kidneys, and indicate that other GATA family members may be involved in the aetiology of human malformations. Hide abstract

Pearce SH, Bai M, Quinn SJ, Kifor O, Brown EM, Thakker RV. 1996. Functional characterization of calcium-sensing receptor mutations expressed in human embryonic kidney cells. J Clin Invest, 98 (8), pp. 1860-1866. Read abstract | Read more

The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that plays a key role in extracellular calcium ion homeostasis. We have engineered 11 CaR mutants that have been described in the disorders familial benign hypercalcemia (FBH), neonatal severe hyperparathyroidism (NSHPT), and autosomal dominant hypocalcaemia (ADH), and studied their function by characterizing intracellular calcium [Ca2+]i transients in response to varying concentrations of extracellular calcium [Ca2+]o or gadolinium [Gd3+]o. The wild type receptor had an EC50 for calcium (EC50[Ca2+]o) (the value of [Ca2+]o producing half of the maximal increase in [Ca2+]i) of 4.0 mM (+/- 0.1 SEM). However, five missense mutations associated with FBH or NSHPT, (P55L, N178D, P221S, R227L, and V817I) had significantly higher EC50[Ca2+]os of between 5.5 and 9.3 mM (all P < 0.01). Another FBH mutation, Y218S, had an EC50[Ca2+]o of > 50 mM but had only a mildly attenuated response to gadolinium, while the FBH mutations, R680C and P747fs, were unresponsive to either calcium or gadolinium. In contrast, three mutations associated with ADH, (F128L, T151M, and E191K), showed significantly reduced EC50[Ca2+]os of between 2.2 and 2.8 mM (all P < 0.01). These findings provide insights into the functional domains of the CaR and demonstrate that mutations which enhance or reduce the responsiveness of the CaR to [Ca2+]o cause the disorders ADH, FBH, and NSHPT, respectively. Hide abstract

Heath D. 1996. Familial hypocalcemia -- not hypoparathyroidism. N Engl J Med, 335 (15), pp. 1144-1145. | Read more

Lloyd SE, Pearce SH, Fisher SE, Steinmeyer K, Schwappach B, Scheinman SJ, Harding B, Bolino A et al. 1996. A common molecular basis for three inherited kidney stone diseases. Nature, 379 (6564), pp. 445-449. Read abstract | Read more

Kidney stones (nephrolithiasis), which affect 12% of males and 5% of females in the western world, are familial in 45% of patients and are most commonly associated with hypercalciuria. Three disorders of hypercalciuric nephrolithiasis (Dent's disease, X-linked recessive nephrolithiasis (XRN), and X-linked recessive hypophosphataemic rickets (XLRH)) have been mapped to Xp11.22 (refs 5-7). A microdeletion in one Dent's disease kindred allowed the identification of a candidate gene, CLCN5 (refs 8,9) which encodes a putative renal chloride channel. Here we report the investigation of 11 kindreds with these renal tubular disorders for CLCN5 abnormalities; this identified three nonsense, four missense and two donor splice site mutations, together with one intragenic deletion and one microdeletion encompassing the entire gene. Heterologous expression of wild-type CLCN5 in Xenopus oocytes yielded outwardly rectifying chloride currents, which were either abolished or markedly reduced by the mutations. The common aetiology for Dent's disease, XRN and XLRH indicates that CLCN5 may be involved in other renal tubular disorders associated with kidney stones. Hide abstract

Parkinson DB, Thakker RV. 1992. A donor splice site mutation in the parathyroid hormone gene is associated with autosomal recessive hypoparathyroidism. Nat Genet, 1 (2), pp. 149-152. Read abstract | Read more

Investigation of one kindred with autosomal recessive isolated hypoparathyroidism, which had resulted from a consanguineous marriage, has identified a g to c substitution in the first nucleotide of intron 2 of the parathyroid hormone (PTH) gene. This donor splice mutation could be detected by restriction enzyme cleavage with Ddel, and this revealed that the patients were homozygous for the mutant alleles, the unaffected relatives were heterozygous, and unrelated normals were homozygous for the wild type alleles. Defects in messenger RNA splicing were investigated by the detection of illegitimate transcription of the PTH gene in lymphoblastoid cells. The mutation resulted in exon skipping with a loss of exon 2, which encodes the initiation codon and the signal peptide, thereby causing parathyroid hormone deficiency. Hide abstract

Thakker RV, Bouloux P, Wooding C, Chotai K, Broad PM, Spurr NK, Besser GM, O'Riordan JL. 1989. Association of parathyroid tumors in multiple endocrine neoplasia type 1 with loss of alleles on chromosome 11. N Engl J Med, 321 (4), pp. 218-224. Read abstract | Read more

Familial multiple endocrine neoplasia type 1 (MEN-1) is an autosomal dominant disorder characterized by the combined occurrence of tumors of the parathyroid glands, the pancreas, and the pituitary gland. Pancreatic tumors have previously been shown to be associated with the loss of alleles on chromosome 11; we therefore looked for similar genetic alterations in specimens of parathyroid tumors, which are the most common feature of MEN-1. We obtained parathyroid tumors and peripheral-blood leukocytes from six patients with MEN-1; 18 cloned human DNA sequences from chromosome 11 were then used to identify restriction-fragment-length polymorphisms. A loss of heterozygosity was detected in parathyroid tumors from three of the six patients with MEN-1; this finding demonstrated that allelic deletions on chromosome 11 are involved in the monoclonal development of parathyroid tumors in patients with MEN-1. In addition, studies of three affected families (with 17 affected members and 51 unaffected members) established linkage with the oncogene INT2 (peak lod score, 3.30, at 0 percent recombination); the MEN-1 gene was thus mapped to the pericentromeric region of the long arm of chromosome 11 (11q13). Our location of the MEN-1 gene at 11q13 is close to the location previously reported. We conclude that a single inherited locus on chromosome 11, band q13, causes MEN-1 and that the monoclonal development of parathyroid and pancreatic tumors in patients with MEN-1 involves similar allelic deletions on chromosome 11. Hide abstract

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