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Apparent mineralocorticoid excess-a genetic basis for low renin hypertension
AJH-APRIL 1996-VOL. 9, NO.4, PART 2
ORALS: Hypertensive Heart Disease: Molecular Mechanisms
Thursday, May 16, 2:55 pm
Thursday, May 16, 3:10 pm
APPARENT MINERALOCORTICOID EXCESS-A GENETIC BASIS FOR LOW RENIN HYPERTENSION. RC Wilson, MD Harbison, JQ Wei, and ~•. The New YOlK Hospttal-Comell University Medical Center, New YOlK, NY. Apparent mineralocorticoid excess (AM E) is a syndrome of juvenile hypertension associated wtth hyporenlnemla, hypoaldosteronemia and hypokalemic alkalosis. AME resutts from mutations in the HSDll 62 gene encoding 11 6-hydroxysteroid dehydrogenase type 2, the enzyme responsible for renal conversion of cortisol to cortisone thereby protecting the mineralocorticoid receptor from cortisol activation. HSD1162 Is a 67 kb gene composed of 5 exons which is located on chromosone 16q22. To date, 28 HSDll 62 alleles from 14 klndreds containing 19 patients have been stUdied. Except for a single patient who was a compound heterozygote for two mutallons, all patients were homozygous for one of an addttional eight different mutations. Expression studies have been done on 7 of these 10 mutallons covering 17 of these 19 patients. Only 2, R208C and R213C, have measurable actlvtty. There Is a dlspartty between the In vttro lack of enzymatic actiVity and the severity of the disease clinically. For example, 2 sibs wtth the R208C mutation (13% actlvtty) were severely affected since Infancy. Addttionally, 2 of the 3 patients wtth the R337H, AY338 mutation (0 % actiVity) are mildly affected not having been treated unlll 4to 7 yoa while the 3rd Is severely affected having had a stroke at3 yoa. In 8 of the 13 famlles wtth a homozygous mutation, homozygostty Is explained by consangulntty (3), endogamy (3) and founder effect (2). In the 5 remaining famlles, sufficient pedigrees are not available. We studied an ttallan child who presented at 10 yrs of age with a history of polyuria, polydipsia and hypertension. Sequence analysis of his HSDll 62 gene revealed two new mutations In exon 4, D244N (GAC to MC) and L250R (CTC to CGC). This L250R mutation Involves the same nucleotide In codon 250 as Is Involved In the preViously reported L250P, L251S mutallon (CTC to CCC). The possibility of mutational hot spots within the HSDll 62 gene Is suggested by: 1. The Involvement of codon 250 In the L250R and L250P, L251S mutations. 2. The Involvement of codon 337 In the R337C and the R337H. DY338 mutallons. 3. The appearance of the R208C mutation In two tribal families, a Bedouin from Oman and a Native North American from Canada.
MUTATIONS ALTER TRANSCRIPTION FACTOR BINDING TO THE RENIN GENE FIRST INTRON OF THE SPONTANEOUSLY HYPERTENSIVE RAT. llYll. R Di Nicolantonio. Department of Physiology. The University of Melbourne, Parkville. Victoria 3052, Australia. Overexpression of the renin gene has been found in the spontaneously hypertensive rat (SHR) of the Okamoto strain. We have demonstrated mutations within the renin gene first intron unique to SHR. In the present study we examined whether these mutations disrupt transcriptional factor binding to this region. Synthetic double-strand oligonucleotides were used in gel mobility shift assays. The sequences spanned +490· +514 and +925 - +944 in the rat renin gene intron I. These regions contain consensus sequences for transcriptional factors IF J and E 2A. The probes were radiolabelled at the 5'-end with J2p. Nuclear extracts were prepared from kidney. liver. adrenal gland. heart. brainstem. lung and spleen of Sprague-Dawley (SO) rats. Resultant DNA-protein complexes were analyzed by gel electrophoresis followed by autoradiography. The intensity of DNA-protein bands were semi-quantified by densitometry. Specific nuclear proteins were identified which bound to IF) and ~ motifs and displayed different tissue-specific patterns. The affinities of nuclear protein binding to IF) and ~ elements from SHR renin gene first intron were about 60 and 20 times higher respectively than that from Wistar Kyoto and SO rats. We conclude that the mutations in the renin fIrSt intran of SHR interrupt transcriptional factor binding to this gene and may be the molecular basis for its overexpression.
KeyWords: HSD1162, mutations, apparent mineralocorticoid excess
Key Words:
.
I
h
.
remn gene. spontaneous y ypertenslve rat. transcriptional factor. first intron.
9A
ORALS: Hypertensive Heart Disease: Molecular Mechanisms
Thursday, May 16, 2:55 pm
Thursday, May 16, 3:10 pm
APPARENT MINERALOCORTICOID EXCESS-A GENETIC BASIS FOR LOW RENIN HYPERTENSION. RC Wilson, MD Harbison, JQ Wei, and ~•. The New YOlK Hospttal-Comell University Medical Center, New YOlK, NY. Apparent mineralocorticoid excess (AM E) is a syndrome of juvenile hypertension associated wtth hyporenlnemla, hypoaldosteronemia and hypokalemic alkalosis. AME resutts from mutations in the HSDll 62 gene encoding 11 6-hydroxysteroid dehydrogenase type 2, the enzyme responsible for renal conversion of cortisol to cortisone thereby protecting the mineralocorticoid receptor from cortisol activation. HSD1162 Is a 67 kb gene composed of 5 exons which is located on chromosone 16q22. To date, 28 HSDll 62 alleles from 14 klndreds containing 19 patients have been stUdied. Except for a single patient who was a compound heterozygote for two mutallons, all patients were homozygous for one of an addttional eight different mutations. Expression studies have been done on 7 of these 10 mutallons covering 17 of these 19 patients. Only 2, R208C and R213C, have measurable actlvtty. There Is a dlspartty between the In vttro lack of enzymatic actiVity and the severity of the disease clinically. For example, 2 sibs wtth the R208C mutation (13% actlvtty) were severely affected since Infancy. Addttionally, 2 of the 3 patients wtth the R337H, AY338 mutation (0 % actiVity) are mildly affected not having been treated unlll 4to 7 yoa while the 3rd Is severely affected having had a stroke at3 yoa. In 8 of the 13 famlles wtth a homozygous mutation, homozygostty Is explained by consangulntty (3), endogamy (3) and founder effect (2). In the 5 remaining famlles, sufficient pedigrees are not available. We studied an ttallan child who presented at 10 yrs of age with a history of polyuria, polydipsia and hypertension. Sequence analysis of his HSDll 62 gene revealed two new mutations In exon 4, D244N (GAC to MC) and L250R (CTC to CGC). This L250R mutation Involves the same nucleotide In codon 250 as Is Involved In the preViously reported L250P, L251S mutallon (CTC to CCC). The possibility of mutational hot spots within the HSDll 62 gene Is suggested by: 1. The Involvement of codon 250 In the L250R and L250P, L251S mutations. 2. The Involvement of codon 337 In the R337C and the R337H. DY338 mutallons. 3. The appearance of the R208C mutation In two tribal families, a Bedouin from Oman and a Native North American from Canada.
MUTATIONS ALTER TRANSCRIPTION FACTOR BINDING TO THE RENIN GENE FIRST INTRON OF THE SPONTANEOUSLY HYPERTENSIVE RAT. llYll. R Di Nicolantonio. Department of Physiology. The University of Melbourne, Parkville. Victoria 3052, Australia. Overexpression of the renin gene has been found in the spontaneously hypertensive rat (SHR) of the Okamoto strain. We have demonstrated mutations within the renin gene first intron unique to SHR. In the present study we examined whether these mutations disrupt transcriptional factor binding to this region. Synthetic double-strand oligonucleotides were used in gel mobility shift assays. The sequences spanned +490· +514 and +925 - +944 in the rat renin gene intron I. These regions contain consensus sequences for transcriptional factors IF J and E 2A. The probes were radiolabelled at the 5'-end with J2p. Nuclear extracts were prepared from kidney. liver. adrenal gland. heart. brainstem. lung and spleen of Sprague-Dawley (SO) rats. Resultant DNA-protein complexes were analyzed by gel electrophoresis followed by autoradiography. The intensity of DNA-protein bands were semi-quantified by densitometry. Specific nuclear proteins were identified which bound to IF) and ~ motifs and displayed different tissue-specific patterns. The affinities of nuclear protein binding to IF) and ~ elements from SHR renin gene first intron were about 60 and 20 times higher respectively than that from Wistar Kyoto and SO rats. We conclude that the mutations in the renin fIrSt intran of SHR interrupt transcriptional factor binding to this gene and may be the molecular basis for its overexpression.
KeyWords: HSD1162, mutations, apparent mineralocorticoid excess
Key Words:
.
I
h
.
remn gene. spontaneous y ypertenslve rat. transcriptional factor. first intron.
9A