1. Human Hypertension caused by Mutations in WNK Kinases Science Paper Wilson et al 2001

2. Why is the study of Mendelian Forms of Hypertension important? • Hypertension affects 25% of most adult population and is a common risk factor for common causes of morbidity and mortality • Molecular Pathogenesis of common forms of hypertension is poorly understood • Studying rare Mendelian form of hypetension provide clues to pathways • Identify new drug targets and approaches to treatment

3. Features of PHAII/Gordons Syndrome • Autosomal Dominant • Hypertension; ↑ renal salt reabsorption • Hyperkalemia; ↓ renal K+ excretion • Metabolic acidosis; ↓ renal H+ secretion • These features suggest a defect in RENAL ELECTROLYTE HANDLING. • Treatment – Thiazide diuretics.

4. Genetics of Affected Families Linkage ↓ Genotype - (Look for mutations) (Size of fragment) PCR Sequencing Southern blotting ↓ Deletions Missense mutations

5. Genetics of Affected Families – K22 • Genome wide scan shows linkage to Chromosome 12 • PHA II Kindreds (affected have typical features of PHAII) • Null allele D12s94 • 3 Additional marker close to D12s94 • PCR: • - using primers usually 42 kb apart in WT, • - only 600bp in affected individuals • confirm deletion and 41kb fragment in exon 1 of WNK1

6. Genetics of affected families – K4 • Null alleles at • STS45K • STS60K • PCR • Novel 24Kb product • Absent in WT • DNA sequencing • Deletion of 21Kb • NB – this deletion is located within the deletion shown in the K22 family. • Expression studies in K4 and WT show there I s 5 fold increase in WNK1 expression is affected individuals

7. Families with WNK 1 paralog mutations • Kindreds K13, K23, K11, K21 • Paralogs of WNK1 ↓ ↓ ↓ WNK2 WNK3 WNK4 Chr9 Chr X Chr 17 • Additional PHAII loci have previously been mapped to Chromosome 1 and Chromosome 17 (Mansfield et al 1997) • 4 missense mutations in WNK4 found in PHAII kindreds.

8. Causes of PHAII No mutation in WNK 1 or WNK 4 Affected Families Chromosome 17 WNK 4 mutation Chromosome 1 ? Chromosome 12 WNK 1 mutation Missense mutations Deletion a.a 564 Asp -> Ala Exon 7 41kb deletion Intron 1 a.a 562 Glu -> Lys Exon 7 a.a 1185 Arg -> Cys Exon 7 21kb deletion Intron 1 a.a 565 Gln -> Glu Exon 7 K4 K13 K23 K11 K21 K22

9. Expression of WNK 1 and 4 WNK 1 10kb transcript-kidney 12kb transcript- predominantly in heart and skeletal muscle WNK 4 Approx 4.4kb expressed exclusively in kidney

10. Localisation WNK 1 WNK 4 WNK 4 WNK 1 Distal Convoluted Tubule Staining Anti WNK4 -RED Anti NCCT- GREEN Distal Convoluted Tubule Staining Anti WNK1-RED Anti NCCT- GREEN Renal Cortex Staining Anti WNK1-RED Anti AQP2- GREEN Renal Cortex Staining Anti WNK4-RED Anti AQP2- GREEN Results not shown Anti WNK4-RED ZO-1GREEN – A tight junction complex Results show that WNK is found in the intercellular junctions Anti AQP2- GREEN- Marker of connecting tubule and collecting duct Anti NCCT- GREEN- Marker of DCT

11. Summary • Mutations in WNK1 & WNK4 cause PHAII leads to a genetic gain of function mechanism. • Clustering of mutations in highly conserved domain suggest disruption at this site normally required for normal regulation. • Both kinases are found in the distal nephron which is well known to play a key role in electrolyte homeostasis, This process is disrupted in PHAII individuals. • Identification of upstream and downstream targets will help to understand WNK signalling pathways.

12. WNK1 in vitro Prevents WNK4 interacting with NCCT WNK 4 Missense mutations NCCT │ ↑overactivity │ ↑net sodium Reabsorption │ ↑plasma volume │ ↑cardiac output │ ↑ BP Hypertension NCCT In vitro WNK4 interacts with NCCT and Loses its ability to suppress it Hyperkaleamia WNK4 directly Interacts with K+ channel ROMK