A. So

Familial hyperaldosteronism type II is linked to the chromosome 7p22 region but also shows predicted heterogeneity.

Background Familial hyperaldosteronism type II (FH-II) is characterized by the familial occurrence of primary aldosteronism; unlike FH-I, it is not glucocorticoid-remediable and not associated with the hybrid CYP11B1/CYP11B2 gene mutation. Linkage to a 5-Mbp region of chromosome 7p22 was previously reported in an Australian family with eight affected members. Mutations in the exons or intron–exon boundaries of PRKAR1B (7p22, closely related to PRKAR1A, which is mutated in Carney complex) have been excluded in this family. Objective To refine the region of linkage, and to seek evidence of linkage in a South American family and in three other Australian families with FH-II, using seven closely spaced markers at 7p22. Methods To establish phenotypes (affected, uncertain or unaffected), blood pressure, plasma aldosterone and plasma renin (activity or concentration) were measured and the aldosterone: renin ratio (ARR) calculated. Individuals with consistently increased ARR underwent fludrocortisone suppression testing. The genotypes of the five pedigrees were analysed using seven closely spaced microsatellite markers at 7p22, and two-point and multipoint logarithm of odds (LOD) scores were calculated to assess linkage with FH-II. Results The combined multipoint LOD score for three families (the original Australian, the South American and a new Australian family) showing linkage at 7p22 was highly significant at 4.61 (&thetas; = 0) for markers D7S462 and D7S517. A newly found recombination event in the first Australian family narrowed the area of linkage by 1.8 Mbp, permitting exclusion of approximately half the candidate genes in the originally reported locus. It was not possible to demonstrate linkage at the 7p22 region in the remaining two Australian families. Conclusion This study provides further evidence for linkage of FH-II to 7p22, refines the locus, and supports the notion that FH-II may be genetically heterogeneous.

Further evidence for linkage of familial hyperaldosteronism type II at chromosome 7p22 in Italian as well as Australian and South American families.

Background Familial hyperaldosteronism type II is a hereditary form of primary aldosteronism not attributable to the hybrid CYP11B1/CYP11B2 mutation that causes glucocorticoid remediable aldosteronism (or familial hyperaldosteronism type I). Although genetic defect(s) underlying familial hyperaldosteronism type II have not yet been elucidated, linkage to chromosome 7p22 was previously reported in two Australian families and a South American family with familial hyperaldosteronism type II. Objective To seek evidence of linkage to chromosome 7p22 in two Italian families with familial hyperaldosteronism type II based on markers that have already yielded evidence of linkage in one South American and two Australian familial hyperaldosteronism type II families and to assess the combined multipoint logarithm of odds score in these five families (two Australian, two Italian, and one South American). Methods Primary aldosteronism was diagnosed or excluded using widely accepted clinical and biochemical criteria. Genotypes of affected and unaffected Italian patients from two families were analysed using seven closely spaced microsatellite markers at 7p22, and multipoint logarithm of odds scores were calculated to assess linkage with familial hyperaldosteronism type II. Results All known affected individuals (four and two, respectively) from each of two Italian families shared identical haplotypes for the seven markers, consistent with linkage of the disease locus with the 7p22 region. The combined multipoint logarithm of odds score for five families showing linkage at 7p22 was highly significant at 5.22 (&thetas; = 0) for markers D7S462 and D7S517. Conclusion Linkage in two Italian families makes this the third geographical area to show linkage of familial hyperaldosteronism type II at 7p22, emphasizing the likely importance of this locus in identifying the causative mutation.

EXAMINATION OF CHROMOSOME 7p22 CANDIDATE GENES RBaK, PMS2 AND GNA12 IN FAMILIAL HYPERALDOSTERONISM TYPE II

1 There are two types of familial hyperaldosteronism (FH): FH‐I and FH‐II. FH‐I is caused by a hybrid CYP11B1/CYP11B2 gene mutation. The genetic cause of FH‐II, which is more common, is unknown. Adrenal hyperplasia and adenomas are features. We previously reported linkage of FH‐II to a ~5 Mb region on chromosome 7p22. We subsequently reported finding no causative mutations in the retinoblastoma‐associated Kruppel‐associated box gene (RBaK), a candidate at 7p22 involved in tumorigenesis and cell cycle control. 2 In the current study we investigated RBaK regulatory regions and two other candidate genes: postmeiotic segregation increased 2 (PMS2, involved in DNA mismatch repair and tumour predisposition) and guanine nucleotide‐binding protein alpha‐12 (GNA12, a transforming oncogene). 3 The GNA12 and PMS2 genes were examined in two affected (A1, A2) and two unaffected (U1, U2) subjects from a large 7p22‐linked FH‐II family (family 1). No mutations were found. 4 The RBaK and PMS2 distal promoters were sequenced to –2150 bp from the transcription start site for RBaK and–2800 bp for PMS2. Five unreported single nucleotide polymorphisms (SNPs) were found in subjects A1, A2 but not in U1 or U2; A(–2031 bp)T, T(–2030 bp)G, G(–834 bp)C, C(–821 bp)G in RBaK and A(–876 bp)G in PMS2. Additional affected and unaffected subjects from family 1 and from two other 7p22‐linked FH‐II families and 58 unrelated normotensive control subjects were genotyped for these SNPs. 5 The five novel SNPs were found to be present in a significant proportion of normotensive controls. The four RBaK promoter SNPs were found to be in linkage disequilibrium in the normal population. The RBaK promoter (–)2031T/2030G/834C/821T allele was found to be in linkage disequilibrium with the causative mutation in FH‐II family 1, but not in families 2 and 3. The PMS2 promoter (–)876G allele was also found to be linked to affected phenotypes in family 1. 6 The RBaK and PMS2 promoter SNPs alter the binding sites for several transcription factors. Although present in the normal population, it is possible that the RBaK (–)2031T/2030G/834C/821T and PMS2 (–)876G alleles may have functional roles contributing to the FH‐II phenotype in family 1.

Genomic structure of the human gene for protein kinase A regulatory subunit R1‐beta (PRKAR1B) on 7p22: no evidence for mutations in familial hyperaldosteronism type II in a large affected kindred

objective  Familial hyperaldosteronism type II (FH‐II) is characterized by inheritance of primary aldosteronism (PAL) but, unlike FH‐I, is not glucocorticoid remediable and not associated with the hybrid CYP11B1/CYP11B2 gene mutation. Analysis of two pedigrees previously demonstrated linkage of FH‐II with a locus at chromosome 7p22. We sought to determine whether mutations in the exons or intron/exon boundaries in PRKAR1B (encoding protein kinase A regulatory subunit R1‐beta), which resides within the linked locus, are associated with FH‐II.

No evidence for coding region mutations in the retinoblastoma‐associated Kruppel‐associated box protein gene (RBaK) causing familial hyperaldosteronism type II

© 2006 Blackwell Publishing Ltd, Clinical Endocrinology , 65 , 826–831 performed by the same investigator, who was unaware of previous results. Serum TSH was measured on Advia Centaur® analyser (Bayer Diagnostics, Dublin, Ireland). Serum TPOAb and TgAb were measured using commercially available kits (ETI-AB-HTGK and ETI-AB-TPOK, DiaSorin). Reference values were as follows: TSH, 0·35–5·50 mU/l; TPOAb, < 15 KU/l; TgAb, < 100 KU/l. Urinary iodine was measured in 32/56 women using a Urinary Iodine Assay Kit (Bioclone Australia Pty Ltd, Sydney, Australia). In statistical analysis, the paired t -test, the χ 2 -test and Pearson’s analysis were used. P < 0·05 was marked as statistically significant. Body mass index (BMI) in the third trimester of pregnancy was significantly higher than after parturition (mean ± SD, 26·9 ± 4·4 vs 23·7 ± 4·2 kg/m 2 , P < 0·001). Serum TSH and urinary iodine concentrations were within the normal range in the third trimester of pregnancy and after parturition, and were not significantly different ((mean ± SD) 1·522 ± 0·659 vs 1·409 ± 0·741 mU/l, P = 0·41 and (median, range) 198, 68–502 vs 155, 28–485 μ g/g of creatinine, P = 0·2, respectively). As shown in Table 1, mean thyroid volume in the third trimester of pregnancy was 44·5% greater than three to four months after parturition. The mean volume increase (4·4 ± 1·8 ml) in women with previous pregnancies (30/56) did not differ significantly ( P = 0·14) from the value (3·7 ± 1·9 ml) in nulliparous women (26/56). The prevailing CFDS pattern in the third trimester of pregnancy was II, whereas after delivery it was I. Intrathyroid PSV in the third trimester of pregnancy was 56·5% higher than after parturition. A significant correlation between all BMI values and thyroid volumes and between all intrathyroid PSV values and thyroid volumes was found ( r = 0·56, P < 0·0001 and r = 0·41, P = 0·00017, respectively). Our results confirm that thyroid volume and intrathyroidal blood flow decrease after parturition, and therefore must have increased during pregnancy. We have thus provided the first qualitative and quantitative evidence of increased intrathyroidal blood flow during pregnancy. In previous reports, 4 a connection between increased vascularity in the thyroid gland and increased thyroid size was suspected, but never proved. We believe that the present study indirectly proves this hypothesis. Because iodine supply and TSH concentrations did not differ during pregnancy and after parturition, and because PSV correlated well with thyroid volume, it is highly likely that increased intrathyroidal blood flow contributes to thyroid gland enlargement during pregnancy. In conclusion, measurements of intrathyroidal blood flow provide useful additional information on the aetiology of thyroid gland enlargement during pregnancy.

Genetic Forms of Primary Aldosteronism

Numerous recent reports suggest that primary aldosteronism (PAL) is much more common than previously thought, accounting for 5–10% of hypertensive patients with most being normokalaemic. A recent Framingham study analysis has revealed the aldosterone/renin ratio, a marker of autonomous aldosterone production, to be an independent predictor of blood pressure progression and hypertension development. The description of two familial forms and Framingham results showing significant heritability of the aldosterone/renin ratio suggests a genetic basis for PAL. One rare, glucocorticoid-remediable, familial form (familial hyperaldosteronism type I [FH-I]), is caused by an adrenocorticotropic hormone-regulated, hybrid CYP11B1/CYP11B2 gene mutation and is associated with a wide spectrum of phenotypic expression from normotension to severe hypertension, which may cause early death from stroke, but is readily controlled by giving low-dose glucocorticoids. Identification of the underlying mutation has permitted development of genetic tests, greatly facilitating diagnosis. Familial hyperaldosteronism type II (FH-II), which is not glucocorticoid-remediable and not associated with the hybrid gene mutation, is at least five times more common than FH-I. Linkage studies have implicated a locus at chromosome 7p22 in three of five families with FH-II so far studied, and candidate genes within the linked locus are currently being closely examined. Since FH-II is clinically indistinguishable from apparently non-familial PAL, mutations causing FH-II are likely to be operative in the wider PAL population. As has occurred with FH-I, the search for its genetic basis brings with it the hope of new, more streamlined genetic methods of detection and a better understanding of its pathophysiology.

Examination of candidate genes at chromosome 7p22 in familial hyperaldosteronism type II

There are two types of familial hyperaldosteronism: FH-I and FH-II. FH-I is caused by a hybrid CYP11B1/CYP11B2 gene mutation. The genetic cause of FH-II, which is more common, is unknown. Adrenal hyperplasia and adenomas are features. We reported linkage of FH-II to a 4 Mb region on chr 7p22. Candidate genes at 7p22 involved in cell cycle control include retinoblastoma-associated Kruppel-associated box gene (RBaK), postmeiotic segregation increased 2 (PMS2) and guanine nucleotide-binding protein alpha-12 (GNA12). RBaK interacts with the retinoblastoma gene product to repress expression of genes activated by E2F transcription factors. PMS2 is involved in DNA mismatch repair and tumor predisposition. GNA12 is a transforming oncogene. We previously reported finding no causative mutations in RBaK and PMS2 coding regions. In the current study, (1) GNA12 exons and proximal promoter were examined in two affected (A1, A2) and two unaffected (U1, U2) subjects from FH-II family 1, and a normotensive control. No mutations were found. (2) The RBaK promoter was sequenced to -1300bp from the transcription start site. Two unreported single nucleotide polymorphisms (SNPs; C-1034G and T-1021C) were found in subjects A1, A2 but not in U1, U2 or the control. Additional subjects from 7p22-linked FH-II families 1, 2 and 3 and 68 controls were therefore genotyped. Results (see table) suggest that the –1034C/-1021T allele may be in linkage disequilibrium with the causative mutation in family 1. Its frequency among controls does not exclude it, since, based on recent data from the Framingham offspring study linking aldosterone/renin ratio to rising BP and chr 7p, it could indicate those predisposed to become hypertensive. Since this sequence alters the binding sites for several transcription factors in the RBaK promoter and may contribute to FH-II phenotype, these SNPs will be genotyped in additional FH-II subjects.Due to differences in pressure amplification, central BP can differ greatly between individuals with similar brachial BP. Recent large trials have highlighted an independent role of central BP for predicting CV events. However, measuring central BP requires extra effort and dedicated equipment. This study sought to identify individuals most likely to clinically benefit from assessment of central BP. Supine brachial BP was recorded by sphygmomanometry and central BP by validated radial tonometry in a heterogeneous population of 765 people (214 healthy, 207 with known or suspected CAD, 219 with type 2 diabetes, 125 at increased risk of CVD). Normal central SBP was defined as 115 mmHg (men) or 109 mmHg (women) based on Framingham data. Amplification of SBP (SBPamp) was the difference between brachial and central SBP. Across all levels of brachial BP, there was wide variation in SBPamp (2 – 33 mmHg, mean SD, 12 5 mmHg). Normal or high-normal brachial SBP was evident in 68% (n 521) of the population. However, 47% (n 246) of these 521 people had above normal central SBP. There was no additional value (in terms of categorizing individuals as having “normal” or “high” BP) in assessing central BP in people with brachial SBP 160 mmHg because central SBP was high (139 mmHg) in all. The table shows individuals grouped according to brachial SBP and the impact of central SBP measurement on BP categorization in. In terms of further assessing whether patients have, or do not have, elevated SBP, people with normal to mild hypertension (160 mmHg) are those most likely to benefit from central BP monitoring. This does not exclude central BP monitoring as being useful in selected individuals from these or other groups.AVS plays a critical role in the diagnostic workup of primary aldosteronism (PAL) as it is the most reliable means of differentiating unilateral forms (e.g. aldosterone-producing adenoma) correctable by unilateral adrenalectomy, from bilateral forms usually treated with aldosterone antagonist medications. Examination of the adrenal/peripheral venous (AV/PV) cortisol ratio permits assessment of the adequacy of AVS. Ratios of 3 indicate adequate sampling. The right adrenal vein (RAV) is often harder to locate than the left (LAV) as it usually is smaller and empties into the inferior vena cava (IVC) rather than the renal vein at a level ranging from upper T11 to mid L1. Thus, even in highly experienced hands, the RAV cannulation success rate (87% at Princess Alexandra Hospital) is lower than that for LAV (94%). Use of contrast CT prior to AVS has contributed to high success rates achieved in our institutions by permitting visualization of the RAV at its point of entry into the IVC. We recently instituted an on-the-spot method of measuring plasma cortisol that permits determination of AV levels within 12 min of collection. Rapid cortisol estimation was performed by competitive fluorescence polarization assay using a TDx analyser and the TDx reagent system for cortisol. The standard assay for cortisol was modified by reducing the original 16 min incubation time to 6 min by following a test protocol on the analyser originally used for measuring ethosuximide. The requirement for only 50 L sample volumes allowed rapid centrifugation (4 min). Measurement of RAV and simultaneously collected PV cortisol levels was undertaken while the radiologist collected samples from the LAV, resulting in minimal or no prolongation of the AVS procedure. Cortisol levels of 1500 nmol/L could be estimated accurately, permitting reliable assessment of cannulation success provided PV levels were 500 nmol/L (which was almost always the case). This method proved accurate when compared with an established competitive chemiluminescent immunoassay (ADVIA Centaur). This approach offers a means of definitively establishing, at the time of AVS, whether AV cannulation has been successful, and thereby promises to reduce the number of samples required and the need for repeat procedures.

Genetic basis of familial hyperaldosteronism type II: Further evidence of linkage at chromosome 7p22

There are two types of familial hyperaldosteronism: FH-I and FH-II. FH-I is caused by a hybrid CYP11B1/CYP11B2 gene mutation. The genetic cause of FH-II, which is more common, is unknown. Adrenal hyperplasia and adenomas are features. We reported linkage of FH-II to a 4 Mb region on chr 7p22. Candidate genes at 7p22 involved in cell cycle control include retinoblastoma-associated Kruppel-associated box gene (RBaK), postmeiotic segregation increased 2 (PMS2) and guanine nucleotide-binding protein alpha-12 (GNA12). RBaK interacts with the retinoblastoma gene product to repress expression of genes activated by E2F transcription factors. PMS2 is involved in DNA mismatch repair and tumor predisposition. GNA12 is a transforming oncogene. We previously reported finding no causative mutations in RBaK and PMS2 coding regions. In the current study, (1) GNA12 exons and proximal promoter were examined in two affected (A1, A2) and two unaffected (U1, U2) subjects from FH-II family 1, and a normotensive control. No mutations were found. (2) The RBaK promoter was sequenced to -1300bp from the transcription start site. Two unreported single nucleotide polymorphisms (SNPs; C-1034G and T-1021C) were found in subjects A1, A2 but not in U1, U2 or the control. Additional subjects from 7p22-linked FH-II families 1, 2 and 3 and 68 controls were therefore genotyped. Results (see table) suggest that the –1034C/-1021T allele may be in linkage disequilibrium with the causative mutation in family 1. Its frequency among controls does not exclude it, since, based on recent data from the Framingham offspring study linking aldosterone/renin ratio to rising BP and chr 7p, it could indicate those predisposed to become hypertensive. Since this sequence alters the binding sites for several transcription factors in the RBaK promoter and may contribute to FH-II phenotype, these SNPs will be genotyped in additional FH-II subjects.Due to differences in pressure amplification, central BP can differ greatly between individuals with similar brachial BP. Recent large trials have highlighted an independent role of central BP for predicting CV events. However, measuring central BP requires extra effort and dedicated equipment. This study sought to identify individuals most likely to clinically benefit from assessment of central BP. Supine brachial BP was recorded by sphygmomanometry and central BP by validated radial tonometry in a heterogeneous population of 765 people (214 healthy, 207 with known or suspected CAD, 219 with type 2 diabetes, 125 at increased risk of CVD). Normal central SBP was defined as 115 mmHg (men) or 109 mmHg (women) based on Framingham data. Amplification of SBP (SBPamp) was the difference between brachial and central SBP. Across all levels of brachial BP, there was wide variation in SBPamp (2 – 33 mmHg, mean SD, 12 5 mmHg). Normal or high-normal brachial SBP was evident in 68% (n 521) of the population. However, 47% (n 246) of these 521 people had above normal central SBP. There was no additional value (in terms of categorizing individuals as having “normal” or “high” BP) in assessing central BP in people with brachial SBP 160 mmHg because central SBP was high (139 mmHg) in all. The table shows individuals grouped according to brachial SBP and the impact of central SBP measurement on BP categorization in. In terms of further assessing whether patients have, or do not have, elevated SBP, people with normal to mild hypertension (160 mmHg) are those most likely to benefit from central BP monitoring. This does not exclude central BP monitoring as being useful in selected individuals from these or other groups.AVS plays a critical role in the diagnostic workup of primary aldosteronism (PAL) as it is the most reliable means of differentiating unilateral forms (e.g. aldosterone-producing adenoma) correctable by unilateral adrenalectomy, from bilateral forms usually treated with aldosterone antagonist medications. Examination of the adrenal/peripheral venous (AV/PV) cortisol ratio permits assessment of the adequacy of AVS. Ratios of 3 indicate adequate sampling. The right adrenal vein (RAV) is often harder to locate than the left (LAV) as it usually is smaller and empties into the inferior vena cava (IVC) rather than the renal vein at a level ranging from upper T11 to mid L1. Thus, even in highly experienced hands, the RAV cannulation success rate (87% at Princess Alexandra Hospital) is lower than that for LAV (94%). Use of contrast CT prior to AVS has contributed to high success rates achieved in our institutions by permitting visualization of the RAV at its point of entry into the IVC. We recently instituted an on-the-spot method of measuring plasma cortisol that permits determination of AV levels within 12 min of collection. Rapid cortisol estimation was performed by competitive fluorescence polarization assay using a TDx analyser and the TDx reagent system for cortisol. The standard assay for cortisol was modified by reducing the original 16 min incubation time to 6 min by following a test protocol on the analyser originally used for measuring ethosuximide. The requirement for only 50 L sample volumes allowed rapid centrifugation (4 min). Measurement of RAV and simultaneously collected PV cortisol levels was undertaken while the radiologist collected samples from the LAV, resulting in minimal or no prolongation of the AVS procedure. Cortisol levels of 1500 nmol/L could be estimated accurately, permitting reliable assessment of cannulation success provided PV levels were 500 nmol/L (which was almost always the case). This method proved accurate when compared with an established competitive chemiluminescent immunoassay (ADVIA Centaur). This approach offers a means of definitively establishing, at the time of AVS, whether AV cannulation has been successful, and thereby promises to reduce the number of samples required and the need for repeat procedures.

Further evidence of linkage at 7p22 with familial hyperaldosteronism type II and exclusion of genetic defects in the RBAK coding regions

Once thought rare, primary aldosteronism (PAL) is now reported to be responsible for 5–10% of hypertension. Unlike familial hyperaldosteronism type I (FH-I), FH-II is not glucocorticoidremediable and not associated with the hybrid CYP11B1/CYP11B2 gene mutation. At least five times more common than FH-I, FH-II is clinically indistinguishable from apparently sporadic PAL, suggesting an even higher incidence. Studies performed in collaboration with C Stratakis (NIH, Bethesda) on our largest Australian family (eight affected members) demonstrated linkage at chromosome 7p22. Linkage at this region was also found in a South American family (DNA provided by MI New, Mount Sinai School of Medicine, New York) and in a second Australian family. The combined multipoint LOD score for these 3 families is 4.61 (q = 0) with markers D7S462 and D7S517, providing strong support for this locus harbouring mutations responsible for FH-II. A newly identified recombination event in our largest Australian family has narrowed the region of linkage by 1.8 Mb, permitting exclusion of approximately half the genes residing in the originally reported 5 Mb linked locus. Candidate genes that are involved in cell cycle control are of interest as adrenal hyperplasia and adrenal adenomas are common in FH-II patients. A novel candidate gene in this linked region produces the retinoblastoma-associated Kruppel-associated box protein (RBaK) which interacts with the retinoblastoma gene product to repress the expression of genes activated by members of the E2F family of transcription factors.

Further evidence of linkage at 7p22 with familial hyperaldosteronism type II

Primary aldosteronism (PAL) is caused by the autonomous over-production of aldosterone. Once thought rare, it is now reported to be responsible for 5–10% of hypertension. Familial hyperaldosteronism type II (FH-II), unlike familial hyperaldosteronism type I, is not glucocorticoid-remediable and not associated with the hybrid CYP11B1/CYP11B2 gene mutation. At least five times more common than FH-I, FH-II is clinically, biochemically and morphologically indistinguishable from apparently sporadic PAL, suggesting that its incidence maybe even higher. Studies performed in collaboration with C Stratakis (NIH, Bethesda) on our largest Australian FH-II family (eight affected members) demonstrated linkage at chromosome 7p22. Similar linkage at this region was also found in a South American FH-II family (DNA provided by MI New, Presbyterian Hospital, New York). Mutations in the exons and intron/exon boundaries of the PRKARIB gene (which resides at 7p22 and is closely related to PRKARIA gene mutated in Carney complex) have been excluded in our largest Australian FH-II family. Using more finely spaced markers, we have confirmed linkage at 7p22 in these 2 families, and identified a second Australian family with evidence of linkage at this locus. The combined multipoint LOD score for these 3 families is 4.87 (θ=0) with markers D7S462 and D7S2424, which exceeds the critical threshold for genome-wide significance suggested by Lander and Kruglyak (1995), providing strong support for this locus harbouring mutations responsible for FH-II. A newly identified recombination event in our largest Australian family has narrowed the region of linkage by 1.8 Mb, permitting exclusion of approximately half the genes residing in the original reported 5Mb linked locus. In addition, we have strongly excluded linkage to these key markers in two Australian families (maximum multipoint LOD scores −3.51 and −2.77), supporting the notion that FH-II may be genetically heterogeneous. In order to identify candidate genes at 7p22, more closely spaced markers will be used to refine the locus, as well as single nucleotide polymorphism analysis.