Sporadic Primary Aldosteronism Research Articles

Differences in the clinical and hormonal presentation of patients with familial and sporadic primary aldosteronism.

To compare the clinical and hormonal characteristics of patients with familial hyperaldosteronism (FH) and sporadic primary aldosteronism (PA). A systematic review of the literature was performed for the identification of FH patients. The SPAIN-ALDO registry cohort of patients with no suspicion of FH was chosen as the comparator group (sporadic group). A total of 360 FH (246 FH type I, 73 type II, 29 type III, and 12 type IV) cases and 830 sporadic PA patients were included. Patients with FH-I were younger than sporadic cases, and women were more commonly affected (P = 0.003). In addition, the plasma aldosterone concentration (PAC) was lower, plasma renin activity (PRA) higher, and hypokalemia (P < 0.001) less frequent than in sporadic cases. Except for a younger age (P < 0.001) and higher diastolic blood pressure (P = 0.006), the clinical and hormonal profiles of FH-II and sporadic cases were similar. FH-III had a distinct phenotype, with higher PAC and higher frequency of hypokalemia (P < 0.001), and presented 45 years before sporadic cases. Nevertheless, the clinical and hormonal phenotypes of FH-IV and sporadic cases were similar, with the former being younger and having lower serum potassium levels. In addition to being younger and having a family history of PA, FH-I and III share other typical characteristics. In this regard, FH-I is characterized by a low prevalence of hypokalemia and FH-III by a severe aldosterone excess causing hypokalemia in more than 85% of patients. The clinical and hormonal phenotype of type II and IV is similar to the sporadic cases.

SAT-549 Identification of Somatic Mutations in CLCN2 as a Cause of Aldosterone-Producing Adenomas

Background: Primary aldosteronism (PA) results from both unilateral and bilateral adrenal disease. Unilateral disease is most often caused by aldosterone-producing adenomas (APAs). We recently identified aldosterone-driver somatic mutations in approximately 90% of APAs using an aldosterone synthase (CYP11B2) immunohistochemistry (IHC)-guided DNA sequencing approach. In the present study, we analyzed DNA from APA samples found to be mutation negative. Methods: Formalin-fixed paraffin-embedded tissue samples from PA patients who underwent adrenalectomy were studied. Genomic DNA was isolated from 118 APAs (identified by CYP11B2 IHC). Next generation sequencing (NGS) was performed to identify known aldosterone-driver mutations in KCNJ5, ATP1A1, ATP2B3, and CACNA1D. APA DNA that was mutation negative and the adjacent normal adrenal tissue DNA were subjected to Whole Exome Sequencing (WES). Results: Targeted NGS and WES detected two variants in the voltage-gated chloride channel ClC-2 (encoded by CLCN2), which were confirmed by Sanger sequencing. One of the CLCN2 mutations (p.Gly24Asp) was identical to that previously found to cause germline early-onset PA. The second CLCN2 mutation, which would affect the same region of the protein, was an unreported PA mutation (p.Met22fs). The presence of these variants in two tumors suggests that CLCN2 mutations as a cause of APAs are rare with an approximate prevalence of 1.7% (2/118 APAs). Conclusion: In this study, we identified somatic mutations in CLCN2, in two of 118 APAs. Germline variants in this gene have previously been shown to cause of familial hyperaldosteronism type II and the current findings indicate that similar mutations cause a small proportion of APAs. These findings also indicate that WES of CYP11B2-guided mutation negative APAs can help determine rarer genetic causes of sporadic PA.

Primary aldosteronism diagnostics: KCNJ5 mutations and hybrid steroid synthesis in aldosterone-producing adenomas.

Primary aldosteronism (PA) is characterized by autonomous aldosterone production by renin-independent mechanisms and is most commonly sporadic. While 60-70% of sporadic PA can be attributed to bilateral hyperaldosteronism, the remaining 30-40% is caused by a unilateral aldosterone-producing adenoma (APA). Somatic mutations in or near the selectivity filter the KCNJ5 gene (encoding the potassium channel GIRK4) have been implicated in the pathogenesis of both sporadic and familial PA. Several studies using tumor tissue, peripheral and adrenal vein samples from PA patients have demonstrated that along with aldosterone, the hybrid steroids 18-hydroxycortisol (18OHF) and 18-oxocortisol (18oxoF) are a hallmark of APA harboring KCNJ5 mutations. Herein, we review the recent advances with respect to the molecular mechanisms underlying the pathogenesis of PA and the steroidogenic fingerprints of KCNJ5 mutations. In addition, we present an outlook toward the future of PA subtyping and diagnostic work-up utilizing steroid profiling.

GENETICS IN ENDOCRINOLOGY: The expanding genetic horizon of primary aldosteronism.

Aldosterone is the main mineralocorticoid hormone in humans and plays a key role in maintaining water and electrolyte homeostasis. Primary aldosteronism (PA), characterized by autonomous aldosterone overproduction by the adrenal glands, affects 6% of the general hypertensive population and can be either sporadic or familial. Aldosterone-producing adenoma (APA) and bilateral adrenal hyperplasia (BAH) are the two most frequent subtypes of sporadic PA and 4 forms of familial hyperaldosteronism (FH-I to FH-IV) have been identified. Over the last six years, the introduction of next-generation sequencing has significantly improved our understanding of the molecular mechanisms responsible for autonomous aldosterone overproduction in both sporadic and familial PA. Somatic mutations in four genes (KCNJ5, ATP1A1, ATP2B3 and CACNA1D), differently implicated in intracellular ion homeostasis, have been identified in nearly 60% of the sporadic APAs. Germline mutations in KCNJ5 and CACNA1H cause FH-III and FH-IV, respectively, while germline mutations in CACNA1D cause the rare PASNA syndrome, featuring primary aldosteronism seizures and neurological abnormalities. Further studies are warranted to identify the molecular mechanisms underlying BAH and FH-II, the most common forms of sporadic and familial PA whose molecular basis is yet to be uncovered.

SFE/SFHTA/AFCE consensus on primary aldosteronism, part 1: Epidemiology of PA, who should be screened for sporadic PA?

Depending on the study, the prevalence of primary aldosteronism (PA) in patients with hypertension varies from 6 to 18%. Prevalence is higher in each of the following conditions, any one of which requires screening for PA: severe hypertension (systolic blood pressure [BP]≥180mmHg and/or diastolic BP≥110mmHg); resistant hypertension (systolic BP≥140mmHg and/or diastolic BP≥90mmHg despite adherence to a tritherapy including a thiazide diuretic); hypertension associated with hypokalemia (either spontaneous or associated with a diuretic); Hypertension or hypokalemia associated with adrenal incidentaloma. It should be borne in mind that PA can induce hypertension without hypokalemia or, less frequently, hypokalemia without hypertension. Finally, as cardiovascular and renal morbidity in PA is greater than in essential hypertension of equivalent level, screening for PA is indicated when cardiovascular or renal morbidity is more severe than predicted from BP level.

Immunohistochemical, genetic and clinical characterization of sporadic aldosterone-producing adenomas

Adrenal glands removed for unilateral primary aldosteronism (PA) display marked histological heterogeneity. Recently reported somatic mutations in KCNJ5, ATP1A1, ATP2B3 and CACNA1D can partially account for these differences. In this study we aimed at combining phenotypic and genotypic characteristics, integrating genetic and immunohistochemistry correlates in sporadic PA. Seventy-one adrenal glands have been included in the study and analyzed for mutations in KCNJ5, ATP1A1, ATP2B3 and CACNA1D. Histological examination and immunohistochemical staining for CYP11B1 (11β-hydroxylase) and CYP11B2 (aldosterone synthase) were performed on aldosterone-producing adenomas (APAs) and adjacent adrenal cortex. In our cohort, the final histopathological diagnosis was multinodular hyperplasia in 22.5% of the patients and single nodule in 77.5%. Forty-five percent of the removed adrenals displayed extra-APA CYP11B2-positive cell nests (B2-CN). Among adrenal vein sampling parameters the suppression of contralateral adrenal was more frequent and the lateralization index was higher in the subgroup of patients without extra-APA B2-CN compared to the subgroup with extra-APA B2-CN. KCNJ5-mutated APAs were composed mainly of zona fasciculata-like cells with high expression of CYP11B1, while ATP1A1, ATP2B3 and CACNA1D-mutated APAs presented more frequently a zona-glomerulosa-like phenotype with high expression of CYP11B2. We observed a significant inverse correlation between CYP11B2 expression and the size of the nodules and, if CYP11B2 expression was corrected for tumor volume, a significant correlation with plasma aldosterone and aldosterone to renin ratio. Our findings indicate that combination of genotyping and immunohistochemistry improves the final histopathological diagnosis between single nodule and multinodular hyperplasia of the assessed adrenals.

EACPT-0015 - Role for Germline Mutations and a Rare Coding Single Nucleotide Polymorphism Within KCNJ5 Potassium Channel in a Large Cohort of Sporadic Cases of Primary Aldosteronism.

Primary aldosteronism (autonomous aldosterone production with suppressed renin) plays an important pathophysiological role in what has been previously labeled as essential hypertension. Besides the recently described germLine mutations in the KCNJ5 potassium channel associated with familial primary aldosteronism, somatic mutations in the same channel have been identified within aldosterone-producing adenomas. In this study, we have resequenced the flanking and coding region of KCNJ5 in peripheral blood DNA from 251 white subjects with primary aldosteronism to look for rare variants that might be important for the pathophysiology of sporadic primary aldosteronism.

Role for Germline Mutations and a Rare Coding Single Nucleotide Polymorphism Within the KCNJ5 Potassium Channel in a Large Cohort of Sporadic Cases of Primary Aldosteronism

Primary aldosteronism (autonomous aldosterone production with suppressed renin) plays an important pathophysiological role in what has been previously labeled as essential hypertension. Besides the recently described germline mutations in the KCNJ5 potassium channel associated with familial primary aldosteronism, somatic mutations in the same channel have been identified within aldosterone-producing adenomas. In this study, we have resequenced the flanking and coding region of KCNJ5 in peripheral blood DNA from 251 white subjects with primary aldosteronism to look for rare variants that might be important for the pathophysiology of sporadic primary aldosteronism. We have identified 3 heterozygous missense mutations (R52H, E246K, and G247R) in the cohort and found that 12 (5% of the cohort) were carriers for the rare nonsynonymous single nucleotide polymorphism rs7102584 causing E282Q substitution of KCNJ5. By expressing the channels in Xenopus oocytes and human adrenal H295R cells, we have shown that the R52H, E246K, and E282Q substitutions are functional, but the G247R mutation is indistinguishable from wild type. Although the functional substitutions are remote from the selectivity filter, they affect the inward-rectification, the ability of the KCNJ5 channels to conduct Na(+) currents and ATII-induced aldosterone release from the H295R cell line. Together these data suggest that germline variation in the KCNJ5 gene has a role to play in the common sporadic form as well as the much rarer syndromic forms of primary aldosteronism.

Somatic ATP1A1 , ATP2B3 , and KCNJ5 Mutations in Aldosterone-Producing Adenomas

Aldosterone-producing adenomas (APAs) cause a sporadic form of primary aldosteronism and somatic mutations in the KCNJ5 gene, which encodes the G-protein-activated inward rectifier K(+) channel 4, GIRK4, account for ≈40% of APAs. Additional somatic APA mutations were identified recently in 2 other genes, ATP1A1 and ATP2B3, encoding Na(+)/K(+)-ATPase 1 and Ca(2+)-ATPase 3, respectively, at a combined prevalence of 6.8%. We have screened 112 APAs for mutations in known hotspots for genetic alterations associated with primary aldosteronism. Somatic mutations in ATP1A1, ATP2B3, and KCNJ5 were present in 6.3%, 0.9%, and 39.3% of APAs, respectively, and included 2 novel mutations (Na(+)/K(+)-ATPase p.Gly99Arg and GIRK4 p.Trp126Arg). CYP11B2 gene expression was higher in APAs harboring ATP1A1 and ATP2B3 mutations compared with those without these or KCNJ5 mutations. Overexpression of Na(+)/K(+)-ATPase p.Gly99Arg and GIRK4 p.Trp126Arg in HAC15 adrenal cells resulted in upregulation of CYP11B2 gene expression and its transcriptional regulator NR4A2. Structural modeling of the Na(+)/K(+)-ATPase showed that the Gly99Arg substitution most likely interferes with the gateway to the ion binding pocket. In vitro functional assays demonstrated that Gly99Arg displays severely impaired ATPase activity, a reduced apparent affinity for Na(+) activation of phosphorylation and K(+) inhibition of phosphorylation that indicate decreased Na(+) and K(+) binding, respectively. Moreover, whole cell patch-clamp studies established that overexpression of Na(+)/K(+)-ATPase Gly99Arg causes membrane voltage depolarization. In conclusion, somatic mutations are common in APAs that result in an increase in CYP11B2 gene expression and may account for the dysregulated aldosterone production in a subset of patients with sporadic primary aldosteronism.

Genetics in endocrinology: Genetics of mineralocorticoid excess: an update for clinicians

Aldosterone plays a major role in the regulation of sodium and potassium homeostasis and blood pressure. More recently, aldosterone has emerged as a key hormone mediating end organ damage. In extreme cases, dysregulated aldosterone production leads to primary aldosteronism (PA), the most common form of secondary hypertension. However, even within the physiological range, high levels of aldosterone are associated with an increased risk of developing hypertension over time. PA represents the most common and curable form of hypertension, with a prevalence that increases with the severity of hypertension. Although genetic causes underlying glucocorticoid-remediable aldosteronism, one of the three Mendelian forms of PA, were established some time ago, somatic and inherited mutations in the potassium channel GIRK4 have only recently been implicated in the formation of aldosterone-producing adenoma (APA) and in familial hyperaldosteronism type 3. Moreover, recent findings have shown somatic mutations in two additional genes, involved in maintaining intracellular ionic homeostasis and cell membrane potential, in a subset of APAs. This review summarizes our current knowledge on the genetic determinants that contribute to variations in plasma aldosterone and renin levels in the general population and the genetics of familial and sporadic PA. Various animal models that have significantly improved our understanding of the pathophysiology of excess aldosterone production are also discussed. Finally, we outline the cardiovascular, renal, and metabolic consequences of mineralocorticoid excess beyond blood pressure regulation.

Genetic Variations in the KCNJ5 Gene in Primary Aldosteronism Patients from Xinjiang, China

BackgroundPrimary aldosteronism (PA) is the most common endocrine form of secondary hypertension, and one of the most common subtypes of sporadic PA is aldosterone-producing adenoma (APA). Recently, two somatic mutations of the KCNJ5 gene were implicated in APA, and two germline mutations were associated with familial hyperaldosteronism III.ObjectivesThis case-control study was designed to investigate the relationship between genetic variations in the KCNJ5 gene and sporadic PA patients in Xinjiang, China.MethodsFive common single nucleotide polymorphisms (SNPs) of the KCNJ5 gene (rs6590357, rs4937391, rs3740835, rs2604204, and rs11221497) were detected in patients with sporadic PA (n = 235) and essential hypertension (EH; n = 913) by the TaqMan polymerase chain reaction method.ResultsThe EH group and the PA group showed significant differences in the distributions of genotypes and alleles of rs4937391 and rs2604204 in total and male subjects (P<0.05), as well as rs3740835 in male subjects (P<0.05). However, only the association between the rs2604204 genotype and male sporadic PA remained significant after Bonferroni’s correction (P<0.01). Furthermore, logistic regression analysis demonstrated that the CC genotype of rs2604204 was a risk factor for male patients with sporadic PA, after adjusting for age and body mass index (odds ratio = 2.228, 95% CI: 1.300–3.819, P = 0.004).ConclusionThe genetic variant rs2604204 of KCNJ5 is associated with sporadic PA in Chinese males, suggesting that KCNJ5 may be involved in the pathogenesis of sporadic PA in these particular patients.

Role of KCNJ5 in familial and sporadic primary aldosteronism

Primary aldosteronism is characterised by the dysregulation of aldosterone production and comprises both sporadic forms, caused by an aldosterone-producing adenoma or bilateral adrenal hyperplasia, and familial forms (familial hyperaldosteronism types I, II and III). The two principal physiological regulators of aldosterone synthesis are angiotensin II and serum K(+), which reverse the high resting K(+) conductance and hyperpolarized membrane potential of adrenal glomerulosa cells. The resulting membrane depolarization causes the opening of voltage-gated Ca(2+) channels and an increase in intracellular Ca(2+) that stimulates aldosterone biosynthesis. Point mutations in the KCNJ5 gene, which encodes the G-protein-activated inward rectifier K(+) channel 4 (GIRK4), have been implicated in the pathogenesis of both sporadic and familial forms of primary aldosteronism. These mutations interfere with the selectivity filter of GIRK4 causing Na(+) entry, cell depolarization and Ca(2+) channel opening, resulting in constitutive aldosterone production. Seven families with familial hyperaldosteronism caused by KCNJ5 germline mutations have so far been described, and multicentre studies have reported KCNJ5 mutations in approximately 40% of sporadic aldosterone-producing adenomas. Herein, we review the role of GIRK4 in adrenal pathophysiology and provide an overview of the clinical and biochemical phenotypes resulting from KCNJ5 mutations in patients with sporadic and familial primary aldosteronism.

The Prevalence of Familial Hyperaldosteronism in Apparently Sporadic Primary Aldosteronism in Germany: a Single Center Experience

Primary aldosteronism (PA) is the most frequent cause of secondary arterial hypertension. To date 3 forms of familial hyperaldosteronism (FH) have been described accounting for a small percentage of all PA cases. In Germany, the prevalence of FH is currently unknown. Our aim was to determine the prevalence of familiarity in a large cohort of patients with PA. A total of 166 patients with apparently sporadic PA in Munich were investigated. FH types I, II, and III were identified using established clinical, biochemical, and molecular criteria. Among the 166 patients with PA, 2 patients (1.2%) reported a family history suggestive of FH. None of the 166 patients showed clinical, endocrine, or genetic evidence of FH type I. The 2 families had characteristic features of FH type II. Family A had 3 subjects affected out of 11 evaluated family members. Family B had 3 out of 4. Bilateral adrenal hyperplasia and unilateral adrenal adenoma were found within the same family. FH type I and FH type III are rare in Germany. With a prevalence of 1.2%, FH type II seems to be more common in apparently sporadic PA than had been assumed so far.

KCNJ5 Mutations in European Families With Nonglucocorticoid Remediable Familial Hyperaldosteronism

Primary aldosteronism is the most frequent cause of endocrine hypertension. Three forms of familial hyperaldosteronism (FH) have been described, named FH-I to -III. Recently, a mutation of KCNJ5 has been shown to be associated with FH-III, whereas the cause of FH-II is still unknown. In this study we searched for mutations in KCNJ5 in 46 patients from 21 families with FH, in which FH-I was excluded. We identified a new germline G151E mutation in 2 primary aldosteronism-affected subjects from an Italian family and 3 somatic mutations in aldosterone-producing adenomas, T158A described previously as a germline mutation associated with FH-III, and G151R and L168R both described as somatic mutations in aldosterone-producing adenoma. The phenotype of the family with the G151E mutation was remarkably milder compared with the previously described American family, in terms of both clinical and biochemical parameters. Furthermore, patients with somatic KCNJ5 mutations displayed a phenotype indistinguishable from that of sporadic primary aldosteronism. The functional characterization of the effects of the G151E mutation in vitro showed a profound alteration of the channel function, with loss of K(+) selectivity, Na(+) influx, and membrane depolarization. These alterations have been postulated to be responsible for voltage gate Ca(2+) channel activation, increase in cytosolic calcium, and stimulation of aldosterone production and adrenal cell proliferation. In conclusion, we describe herein a new mutation in the KCNJ5 potassium channel associated with FH-III, responsible for marked alterations of channel function but associated with a mild clinical and hormonal phenotype.

Prevalence and Characteristics of Familial Hyperaldosteronism

Primary aldosteronism (PA) is the most frequent cause of secondary hypertension, and patients display an increased prevalence of cardiovascular events compared with essential hypertensives. To date, 3 familial forms of PA have been described and termed familial hyperaldosteronism types I, II, and III (FH-I to -III). The aim of this study was to investigate the prevalence and clinical characteristics of the 3 forms of FH in a large population of PA patients. Three-hundred consecutive PA patients diagnosed in our unit were tested by long-PCR of the CYP11B1/CYP11B2 hybrid gene that causes FH-I, and all of the available relatives of PA patients were screened to confirm or exclude PA and, thus, FH-II. Urinary 18-hydroxycortisol and 18-oxocortisol were measured in all of the familial PA patients. Two patients were diagnosed with FH-I (prevalence: 0.66%), as well as 21 of their relatives, and clinical phenotypes of the 2 affected families varied markedly. After exclusion of families who refused testing and those who were not informative, 199 families were investigated, of which 12 were diagnosed with FH-II (6%) and an additional 15 individuals had confirmed PA; clinical and biochemical phenotypes of FH-II families were not significantly different from sporadic PA patients. None of the families displayed a phenotype compatible with FH-III diagnosis. Our study demonstrates that familial forms of hyperaldosteronism are more frequent than previously expected and reinforces the recommendation of the Endocrine Society Guidelines to screen all first-degree hypertensive relatives of PA patients.

Further study of chromosome 7p22 to identify the molecular basis of familial hyperaldosteronism type II

Familial hyperaldosteronism type II (FH-II) is an inherited form of hyperaldosteronism associated with hypertension in most patients. The mutations that cause FH-II are unknown, but linkage analysis has mapped them to chromosome 7p22. As FH-II is clinically indistinguishable from sporadic primary aldosteronism, a common and treatable condition, unravelling the cause of FH-II has important implications for these sporadic cases. To investigate whether FH-II is caused by large deletions or insertions, we examined the virtual karyotype of four pairs of affected and unaffected individuals using high-density bead chips. We also sequenced the coding regions of five 7p22 candidate genes that were prioritized because of their putative role in cell growth. We found no evidence of single-nucleotide polymorphism (SNP) copy number variation between pairs, and from the widest gap on the chip, chromosome 7p22 deletions or insertions exceeding ∼50 kb in these pedigrees can be excluded. We found 15 SNPs (two of which were novel), but none of them were non-synonymous and segregated with the disease in the FH-II pedigrees. We have been able to exclude large genomic deletions or insertions at 7p22 and refine the candidate gene list for this locus, but the mutations causing FH-II remain elusive.

K001 Is primary aldosteronism a channelopathy ?

In mouse models the genetic deletion of TWIK-related acid-sensitive K (TASK)-1 and TASK-3 channels removes an important background K current that results in a marked depolarization of adrenal zona glomerulosa cell membrane potential, leading to the autonomous overproduction of aldosterone. The importance of TASK channel dysfunction in human primary aldosteronism (PAL) however, is uncertain, motivating their molecular analysis. We screened coding exons and flanking intronic sequences of KCNK3 and KCNK9 (genes coding for TASK 1 and TASK 3 respectively) in 825 PAL patients for germline DNA sequence variants. A total of 14 different coding sequence variants were found in 19 patients. The variants include 8 different synonymous mutations and 6 different missense variants. In silico predictions (PolyPhen, SIFT and Alamut) suggested the non-synonymous mutations to be potentially damaging. Analysis of mutated channel function by heterologous expression, however, revealed the missense mutations detected were non-functional. As somatic mutations may be involved in some cases of sporadic PAL, sequencing 90 patients for tumoral DNA sequence variants is underway. We are also investigating expression of TASK 1 and TASK 3 by in-situ hybridization and immunohistochemistry on adrenal tissue sections of 150 PAL patients who have undergone surgery.

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.