SALTI: Salt Loading and Thiazide Intervention Study

• Sponsor: University of Maryland, Baltimore (Other)
• Overall Status: Completed
• CT.gov ID: NCT00896389
• Collaborator: National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (NIH)
• Enrollment: 124
• Location: 1
• Arm: 1
• Duration (Months): 41
• Patients Per Site Per Month: 3

Study Details

Study Description

Brief Summary

The investigators of this study propose to examine the relationships between STK39 (Serine Threonine Kinase 39) genotypes and responses to salt loading and to thiazide diuretics, hydrochlorothiazide. The investigators hypothesize that STK39 genotypes will be associated with the outcome of both interventions and can contribute to personalized care for hypertension.

Condition or Disease	Intervention/Treatment	Phase
Hypertension	Procedure: Salt loading; Drug: Hydrochlorothiazide (HCTZ)	Phase 4

Detailed Description

Although hypertension can be easily diagnosed and there are many medications available to treat hypertension, this condition is poorly managed in many patients and is a leading cause of morbidity and mortality worldwide. Because a newly identified hypertension susceptibility gene, STK39 (Serine Threonine Kinase 39), plays a central role in kidney sodium transport, the investigators propose a pharmacogenetics study to examine the relationships between STK39 genotypes and blood pressure responses to salt loading and to thiazide diuretics, hydrochlorothiazide. In addition, STK39 genotypes may also predict those hypertension patients more likely to develop thiazide-induced hyperglycemia. The investigators hypothesize that STK39 genotypes of those single nucleotide polymorphisms (SNPs) that are associated with baseline systolic blood pressure (SBP), diastolic blood pressure (DBP), and hypertension status, will be associated with the outcome of both interventions. Therefore these SNPs can act as markers and contribute to personalized care for hypertension by identifying patients most likely to effectively control their blood pressure by adopting salt-reducing diet versus patients most likely to effectively and safely control their blood pressure by taking thiazide diuretics.

Study Design

• Study Type: Interventional
• Actual Enrollment: 124 participants
• Allocation: N/A
• Intervention Model: Single Group Assignment
• Masking: None (Open Label)
• Primary Purpose: Treatment
• Official Title: The Relationship Between Serine Threonine Kinase 39 (STK39) Genotypes, Salt Sensitivity, Thiazide Diuretics-induced Blood Pressure Response
• Study Start Date: Oct 1, 2009
• Actual Primary Completion Date: Dec 1, 2012
• Actual Study Completion Date: Mar 1, 2013

Arms and Interventions

Arm

Intervention/Treatment

Other: Salt-loading and thiazide diuretic (HCTZ) Salt loading:2 L of 0.9% NaCl. HCTZ:12.5/ 25 mg of HCTZ for 1 week

Procedure: Salt loading; Subjects will arrive at the Amish Research Clinics after overnight fasting. After taking height, weight, BP, and body temperature, subjects will receive 2 liters (L) of 0.9% sodium chloride (NaCl) saline over 4 hours while their blood pressure is monitored every 15 minutes. Blood pressure will be taken every 15 minutes during this procedure. Blood and urine samples will be collected from all subjects pre- and post-infusion.; Drug: Hydrochlorothiazide (HCTZ); We will perform short-term HCTZ intervention on the same 120 subjects. After overnight fasting and having their height, weight, and BP measured, subjects are given seven 12.5 mg HCTZ tablets and instructed to take 1 tablet daily for one week. Ambulatory blood pressure will be measured and blood and urine will be collected on both day 1 and day 8. After a minimum 6-week wash-out period, the subjects will repeat the 7-day HCTZ intervention, taking 25 mg of HCTZ instead. Subjects with plasma potassium levels below 3.6 mmol/L on day 8 of 12.5 mg HCTZ will be given a daily supplement of 16 milliequivalents of potassium to prevent harmful loss of potassium while taking HCTZ.; Other Names: Hydrochlorothiazide HCTZ Apo-Hydro Aquazide H Dichlotride Hydrodiuril HydroSaluric Microzide Esidrex Oretic.

Outcome Measures

Primary Outcome Measures

1. Blood Pressure Change During Salt Loading [Every 15 minutes for 4 hours]

   Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured every 15 minutes for 4 hours. Blood pressure change is calculated by the trapezoid method. Essentially we use the average of blood pressure at each pair of time points (for example, DBP 30min + DBP 15min)/2 + (DBP 45min + DBP 30min)/2 + … up to 4 hours.) normalized by baseline SBP/DBP.

2. Blood Pressure Change After 7 Days of Low Dose (12.5 mg) of HCTZ [24-hr Ambulatory blood pressure were measured every hour on day 0 and day 8]

   Blood pressure change is defined as SBP or DBP average over the 24 hour period, Day 8 subtracts Day 0.

3. Blood Pressure Change After 7 Days of High Dose (25 mg) of HCTZ [24-hr Ambulatory blood pressure were measured every hour on day 0 and day 8]

   Blood pressure change is defined as SBP or DBP average over the 24 hour period, Day 8 subtracts Day 0.

4. Fasting Glucose Change After 7 Days of Low Dose (12.5 mg) of HCTZ [Fasting glucose was measured on day 0 and day 8]

   Values on Day 8 subtracts Day 0.

5. Fasting Glucose Change After 7 Days of High Dose (25mg) of HCTZ [Fasting glucose was measured on day 0 and day 8]

   Values on Day 8 subtracts Day 0.

Secondary Outcome Measures

1. Change in Plasma Aldosterone Level Due to Salt-loading [Aldosterone was measured from blood collected pre and post salt loading]

   Aldosterone is a hormone that plays a critical role in homeostatic regulation of blood pressure. Change is defined as the post-salt loading values minus the pre-salt loading values

2. Change in Plasma Renin Activity Due to Salt-loading [Renin was measured from blood collected pre and post salt loading]

   Renin is an enzyme that mediates extracellular fluid and regulates blood pressure. Plasma renin activity (PRA) is a measure of the activity of the plasma enzyme renin. PRA is measured in the laboratory by incubating plasma at physiologic temperature in a buffer that facilitates its enzymatic activity. The natural substrate for the enzyme renin is angiotensinogen. Exogenous angiotensinogen is not added to the reaction mixture. This means that, in effect, the PRA results reported are dependent on both renin concentration and the concentration of its substrate in the patient's plasma. Renin cleaves angiotensinogen to produce a decapeptide, angiotensin I, the concentration of which is assayed using liquid chromatography accompanied by tandem mass spectroscopic detection (LC/MS/MS). PRA levels are reported as the amount of angiotensin I generated per unit of time. Change is defined as the post-salt loading values minus the pre-salt loading values

3. Change in Plasma Sodium/Potassium Level Due to Salt-loading [Plasma sodium and potassium measured from blood collected pre and post salt loading]

   Na/K ratio is a function of kidney function

4. Change in Plasma Sodium/Potassium Level During Low Dose of HCTZ [Plasma sodium and potassium measured from blood collected pre and post salt loading]

   Na/K ratio is a function of kidney function

5. Change in Plasma Sodium/Potassium Level During High Dose of HCTZ [Plasma sodium and potassium measured from blood collected pre and post salt loading]

   Na/K ratio is a function of kidney function

Eligibility Criteria

Criteria

• Ages Eligible for Study: 18 Years to 65 Years
• Sexes Eligible for Study: All
• Accepts Healthy Volunteers: Yes

Inclusion Criteria:

• Old Order Amish

• Age 18 to 65

• Have systolic blood pressure between 120 and 160 and diastolic blood pressure between 80 and 100

Exclusion Criteria:

• History of myocardial infarction, stroke, congestive heart failure, liver disease

• Known cause of secondary hypertension

• Diabetes or Fasting glucose > 100 mg/dL

• Women who are pregnant, on oral contraceptives, or menstruating

• Used hydrochlorothiazide (HCTZ) in the last 8 weeks or known allergy to HCTZ

• Taking non-steroidal anti-inflammatory drugs

• Estimated glomerular filtration rate < 80 mL/m

• Intention to alter dietary habit during the study

• Abuse of alcohol or drug

Contacts and Locations

Locations

	Site	City	State	Country	Postal Code
1	Amish Research Clinics	Lancaster	Pennsylvania	United States	17607

Sponsors and Collaborators

• University of Maryland, Baltimore
• National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)

Investigators

• Principal Investigator: Yen Pei C. Chang, Ph.D., University of Maryland, Baltimore

Study Documents (Full-Text)

None provided.

More Information

Publications

• Chiga M, Rai T, Yang SS, Ohta A, Takizawa T, Sasaki S, Uchida S. Dietary salt regulates the phosphorylation of OSR1/SPAK kinases and the sodium chloride cotransporter through aldosterone. Kidney Int. 2008 Dec;74(11):1403-9. doi: 10.1038/ki.2008.451. Epub 2008 Sep 17.
• Delpire E, Gagnon KB. SPAK and OSR1: STE20 kinases involved in the regulation of ion homoeostasis and volume control in mammalian cells. Biochem J. 2008 Jan 15;409(2):321-31. Review.
• Wang Y, O'Connell JR, McArdle PF, Wade JB, Dorff SE, Shah SJ, Shi X, Pan L, Rampersaud E, Shen H, Kim JD, Subramanya AR, Steinle NI, Parsa A, Ober CC, Welling PA, Chakravarti A, Weder AB, Cooper RS, Mitchell BD, Shuldiner AR, Chang YP. From the Cover: Whole-genome association study identifies STK39 as a hypertension susceptibility gene. Proc Natl Acad Sci U S A. 2009 Jan 6;106(1):226-31. doi: 10.1073/pnas.0808358106. Epub 2008 Dec 29.

• Responsible Party: Yen-Pei Christy Chang, Associate Professor, University of Maryland, Baltimore
• ClinicalTrials.gov Identifier: NCT00896389
• Other Study ID Numbers: HP-00040712; R21DK084566
• First Posted: May 11, 2009
• Last Update Posted: Mar 25, 2022
• Last Verified: Mar 1, 2022

• Keywords provided by Yen-Pei Christy Chang, Associate Professor, University of Maryland, Baltimore: hypertension; hydrochlorothiazide; HCTZ; Salt sensitivity; Hydrochlorothiazide induced hyperglycemia
• Additional relevant MeSH terms: Hypertension; Hydrochlorothiazide

Study Results

Participant Flow

Arm/Group Title	Salt-loading and Thiazide Diuretics (HCTZ)
Arm/Group Description	Salt loading: 2 liters (L) of 0.9% sodium chloride (NaCl). HCTZ:12.5 or 25 mg of HCTZ for 1 week
Period Title: Salt Loading
STARTED	124
COMPLETED	124
NOT COMPLETED	0
Period Title: Salt Loading
STARTED	28
COMPLETED	28
NOT COMPLETED	0
Period Title: Salt Loading
STARTED	25
COMPLETED	25
NOT COMPLETED	0

Baseline Characteristics

Arm/Group Title	Salt-loading and Thiazide Diuretic (HCTZ)
Arm/Group Description	Salt loading: Subjects will arrive at the Amish Research Clinics after overnight fasting. After taking height, weight, BP, and body temperature, subjects will receive 2 L of 0.9% NaCl (sodium chloride) saline over 4 hours while their blood pressure is monitored every 15 minutes. Blood pressure will be taken every 15 minutes during this procedure. Blood and urine samples will be collected from all subjects pre- and post-infusion. Hydrochlorothiazide (HCTZ): After overnight fasting and having their height, weight, and BP measured, subjects are given 12.5 mg HCTZ tablets and instructed to take 1 tablet daily for one week. Ambulatory blood pressure, blood and urine will be collected on both day 1 and day 8. After a wash-out period, the subjects will repeat the HCTZ intervention, taking 25 mg HCTZ instead.
Overall Participants	124
Age (years) [Mean (Standard Deviation) ]
Saline infusion only	50 (7)
saline infusion and HCTZ	53 (9)
Sex: Female, Male (Count of Participants)
Female	71 57.3%
Male	53 42.7%
Systolic blood pressure (mmHg) [Mean (Standard Deviation) ]
Salt loading	114 (11)
HCTZ	126 (10)
Diastolic blood pressure (mmHg) [Mean (Standard Deviation) ]
Salt Loading	71 (7)
HCTZ	80 (7)
BMI (kg/m2) [Mean (Standard Deviation) ]
Mean (Standard Deviation) [kg/m2]	26.1 (2.8)
creatinine (mg/dL) [Mean (Standard Deviation) ]
Mean (Standard Deviation) [mg/dL]	0.74 (0.12)

Outcome Measures

1. Primary Outcome

Title	Blood Pressure Change During Salt Loading
Description	Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured every 15 minutes for 4 hours. Blood pressure change is calculated by the trapezoid method. Essentially we use the average of blood pressure at each pair of time points (for example, DBP 30min + DBP 15min)/2 + (DBP 45min + DBP 30min)/2 + … up to 4 hours.) normalized by baseline SBP/DBP.
Time Frame	Every 15 minutes for 4 hours

Outcome Measure Data

Analysis Population Description

124 subjects were divided into 3 genotype groups based on their rs35929607 genotypes. Genotype GG is homozygous for the minor allele G, genotype AG is heterozygous, and genotype AA is homozygous for the major allele A.

Arm/Group Title	rs35929607 SNP
Arm/Group Description	Single nucleotide polymorphism (SNP) rs35929607, located in an intron of serine threonine kinase 39 (STK39) gene, is associated with blood pressure in the Amish population. This SNP, rs35929607, has 2 alleles, A and G. Allele G is found less frequently and has an allele frequency of approximately 0.2-0.4 in populations studied thus far. We genotyped all participants of this study and determine if they are homozygous for the G allele, i.e., have the GG genotype, or are heterozygous (AG genotype), or are homozygous for the A allele and have the AA genotype.
Measure Participants	124
DBP, Genotype GG	-25.5 (4.3)
DBP, Genotype AG	34.6 (11.6)
DBP, Genotype AA	23.2 (7.7)
SBP, Genotype GG	-41.4 (8.4)
SBP, Genotype AG	62.9 (15.7)
SBP, Genotype AA	44.8 (10.6)

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	rs35929607 SNP
	Comments	Null hypothesis: There is no association between STK39 SNP rs35929607 genotype group and phenotypes collected in this study.
	Type of Statistical Test	Other
	Comments	Association between genotypes and phenotypes were tested using a linear mixed model, coding genotype as an independent variable with an additive genetic effect, and including age, age^2, and sex as covariates in the model. All association analyses were run using the Mixed Models for Pedigree Analysis (MMAP) software program to account for the relatedness among study subjects. MMAP User Guide is available from http://edn.som.umaryland.edu/mmap/index.php.
Statistical Test of Hypothesis	p-Value	>0.05
	Comments	Because no P-values less than 0.05 was observed, we did not adjust them for multiple comparison to avoid false positives.
	Method	Chi-squared
	Comments

2. Secondary Outcome

Title	Change in Plasma Aldosterone Level Due to Salt-loading
Description	Aldosterone is a hormone that plays a critical role in homeostatic regulation of blood pressure. Change is defined as the post-salt loading values minus the pre-salt loading values
Time Frame	Aldosterone was measured from blood collected pre and post salt loading

Outcome Measure Data

Analysis Population Description

124 subjects were divided into 3 genotype groups based on their rs35929607 genotypes. Genotype GG is homozygous for the minor allele G, genotype AG is heterozygous, genotype AA is homozygous for the major allele A.

Arm/Group Title	rs35929607 SNP
Arm/Group Description	SNP rs35929607, located in an intron of STK39 gene, is associated with blood pressure in the Amish population. This SNP, rs35929607, has 2 alleles, A and G. Allele G is found less frequently and has an allele frequency of approximately 0.2-0.4 in populations studied thus far. We genotyped all participants of this study and determine if they are homozygous for the G allele, i.e., have the GG genotype, or are heterozygous (AG genotype), or are homozygous for the A allele and have the AA genotype.
Measure Participants	124
Genotype GG	-7.5 (4.5)
Genotype AG	-1.2 (0.7)
Genotype AA	-1.9 (0.6)

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	rs35929607 SNP
	Comments
	Type of Statistical Test	Other
	Comments	Association between genotype and phenotype were tested using a linear mixed model, coding genotype as an independent variable with an additive genetic effect, and including age, age^2, and sex as covariates in the model. All association analyses were run using the Mixed Models for Pedigree Analysis (MMAP) software program to account for the relatedness among study subjects. MMAP User Guide is available from http://edn.som.umaryland.edu/mmap/index.php.
Statistical Test of Hypothesis	p-Value	<0.05
	Comments	Because no P-values less than 0.05 was observed, we did not adjust them for multiple comparison to avoid false positives.
	Method	Chi-squared
	Comments

3. Secondary Outcome

Title	Change in Plasma Renin Activity Due to Salt-loading
Description	Renin is an enzyme that mediates extracellular fluid and regulates blood pressure. Plasma renin activity (PRA) is a measure of the activity of the plasma enzyme renin. PRA is measured in the laboratory by incubating plasma at physiologic temperature in a buffer that facilitates its enzymatic activity. The natural substrate for the enzyme renin is angiotensinogen. Exogenous angiotensinogen is not added to the reaction mixture. This means that, in effect, the PRA results reported are dependent on both renin concentration and the concentration of its substrate in the patient's plasma. Renin cleaves angiotensinogen to produce a decapeptide, angiotensin I, the concentration of which is assayed using liquid chromatography accompanied by tandem mass spectroscopic detection (LC/MS/MS). PRA levels are reported as the amount of angiotensin I generated per unit of time. Change is defined as the post-salt loading values minus the pre-salt loading values
Time Frame	Renin was measured from blood collected pre and post salt loading

Outcome Measure Data

Analysis Population Description

124 subjects were divided into 3 genotype groups based on their rs35929607 genotypes. Genotype GG is homozygous for the minor allele G, genotype AG is heterozygous, genotype AA is homozygous for the major allele A.

Arm/Group Title	rs35929607 SNP
Arm/Group Description	SNP rs35929607, located in an intron of STK39 gene, is associated with blood pressure in the Amish population. This SNP, rs35929607, has 2 alleles, A and G. Allele G is found less frequently and has an allele frequency of approximately 0.2-0.4 in populations studied thus far. We genotyped all participants of this study and determine if they are homozygous for the G allele, i.e., have the GG genotype, or are heterozygous (AG genotype), or are homozygous for the A allele and have the AA genotype.
Measure Participants	124
Genotype GG	-0.8 (0.3)
Genotype AG	-0.3 (0.1)
Genotype AA	-0.4 (0.1)

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	rs35929607 SNP
	Comments	Null hypothesis: There is no association between STK39 SNP rs35909607 genotype group and phenotypes collected in this study.
	Type of Statistical Test	Other
	Comments	Association between genotype and phenotype were tested using a linear mixed model, coding genotype as an independent variable with an additive genetic effect, and including age, age^2, and sex as covariates in the model. All association analyses were run using the Mixed Models for Pedigree Analysis (MMAP) software program to account for the relatedness among study subjects. MMAP User Guide is available from http://edn.som.umaryland.edu/mmap/index.php.
Statistical Test of Hypothesis	p-Value	>0.05
	Comments	Because no P-values less than 0.05 was observed, we did not adjust them for multiple comparison to avoid false positives.
	Method	Chi-squared
	Comments

4. Secondary Outcome

Title	Change in Plasma Sodium/Potassium Level Due to Salt-loading
Description	Na/K ratio is a function of kidney function
Time Frame	Plasma sodium and potassium measured from blood collected pre and post salt loading

Outcome Measure Data

Analysis Population Description

124 subjects were divided into 3 genotype groups based on their rs35929607 genotypes. Genotype GG is homozygous for the minor allele G, genotype AG is heterozygous, genotype AA is homozygous for the major allele A.

Arm/Group Title	rs35929607 SNP
Arm/Group Description	SNP rs35929607, located in an intron of STK39 gene, is associated with blood pressure in the Amish population. This SNP, rs35929607, has 2 alleles, A and G. Allele G is found less frequently and has an allele frequency of approximately 0.2-0.4 in populations studied thus far. We genotyped all participants of this study and determine if they are homozygous for the G allele, i.e., have the GG genotype, or are heterozygous (AG genotype), or are homozygous for the A allele and have the AA genotype.
Measure Participants	124
Genotype GG	0.3 (0.8)
Genotype AG	-0.3 (0.4)
Genotype AA	0.1 (0.2)

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	rs35929607 SNP
	Comments	Null hypothesis: There is no association between STK39 SNP rs35909607 genotype group and phenotypes collected in this study.
	Type of Statistical Test	Other
	Comments	Association between genotype and phenotype were tested using a linear mixed model, coding genotype as an independent variable with an additive genetic effect, and including age, age^2, and sex as covariates in the model. All association analyses were run using the Mixed Models for Pedigree Analysis (MMAP) software program to account for the relatedness among study subjects. MMAP User Guide is available from http://edn.som.umaryland.edu/mmap/index.php.
Statistical Test of Hypothesis	p-Value	>0.05
	Comments	Because no P-values less than 0.05 was observed, we did not adjust them for multiple comparison to avoid false positives.
	Method	Chi-squared
	Comments

5. Secondary Outcome

Title	Change in Plasma Sodium/Potassium Level During Low Dose of HCTZ
Description	Na/K ratio is a function of kidney function
Time Frame	Plasma sodium and potassium measured from blood collected pre and post salt loading

Outcome Measure Data

Analysis Population Description

28 subjects were divided into 3 genotype groups based on their rs35929607 genotypes. Genotype 11 is homozygous for the minor allele G, genotype 12 is heterozygous, genotype 22 is homozygous for the major allele A.

Arm/Group Title	rs35929607 SNP
Arm/Group Description	SNP rs35929607, located in an intron of STK39 gene, is associated with blood pressure in the Amish population. This SNP, rs35929607, has 2 alleles, A and G. Allele G is found less frequently and has an allele frequency of approximately 0.2-0.4 in populations studied thus far. We genotyped all participants of this study and determine if they are homozygous for the G allele, i.e., have the GG genotype, or are heterozygous (AG genotype), or are homozygous for the A allele and have the AA genotype.
Measure Participants	28
Genotype GG	0 (0)
Genotype AG	1.0 (0.6)
Genotype AA	0.6 (0.4)

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	rs35929607 SNP
	Comments	Null hypothesis: There is no association between STK39 SNP rs35909607 genotype group and phenotypes collected in this study.
	Type of Statistical Test	Other
	Comments	Association between genotype and phenotype were tested using a linear mixed model, coding genotype as an independent variable with an additive genetic effect, and including age, age^2, and sex as covariates in the model. All association analyses were run using the Mixed Models for Pedigree Analysis (MMAP) software program to account for the relatedness among study subjects. MMAP User Guide is available from http://edn.som.umaryland.edu/mmap/index.php.
Statistical Test of Hypothesis	p-Value	>0.05
	Comments	Because no P-values less than 0.05 was observed, we did not adjust them for multiple comparison to avoid false positives.
	Method	Chi-squared
	Comments

6. Primary Outcome

Title	Blood Pressure Change After 7 Days of Low Dose (12.5 mg) of HCTZ
Description	Blood pressure change is defined as SBP or DBP average over the 24 hour period, Day 8 subtracts Day 0.
Time Frame	24-hr Ambulatory blood pressure were measured every hour on day 0 and day 8

Outcome Measure Data

Analysis Population Description

28 subjects were divided into 3 genotype groups based on their rs35929607 genotypes. Genotype GG is homozygous for the minor allele G, genotype AG is heterozygous, genotype AA is homozygous for the major allele A.

Arm/Group Title	rs35929607 SNP
Arm/Group Description	SNP rs35929607, located in an intron of STK39 gene, is associated with blood pressure in the Amish population. This SNP, rs35929607, has 2 alleles, A and G. Allele G is found less frequently and has an allele frequency of approximately 0.2-0.4 in populations studied thus far. We genotyped all participants of this study and determine if they are homozygous for the G allele, i.e., have the GG genotype, or are heterozygous (AG genotype), or are homozygous for the A allele and have the AA genotype.
Measure Participants	28
DBP, Genotype GG	0 (0)
DBP, Genotype AG	2.0 (2.4)
DBP, Genotype AA	-1.1 (1.1)
SBP, Genotype GG	1.0 (0)
SBP, Genotype AG	-0.7 (4.3)
SBP, Genotype AA	-4.2 (1.3)

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	rs35929607 SNP
	Comments	Null hypothesis: There is no association between STK39 SNP rs35929607 genotype group and phenotypes collected in this study.
	Type of Statistical Test	Other
	Comments	Association between genotype and phenotype were tested using a linear mixed model, coding genotype as an independent variable with an additive genetic effect, and including age, age^2, and sex as covariates in the model. All association analyses were run using the Mixed Models for Pedigree Analysis (MMAP) software program to account for the relatedness among study subjects. MMAP User Guide is available from http://edn.som.umaryland.edu/mmap/index.php.
Statistical Test of Hypothesis	p-Value	>0.05
	Comments	Because no P-values less than 0.05 was observed, we did not adjust them for multiple comparison to avoid false positives.
	Method	Chi-squared
	Comments

7. Primary Outcome

Title	Blood Pressure Change After 7 Days of High Dose (25 mg) of HCTZ
Description	Blood pressure change is defined as SBP or DBP average over the 24 hour period, Day 8 subtracts Day 0.
Time Frame	24-hr Ambulatory blood pressure were measured every hour on day 0 and day 8

Outcome Measure Data

Analysis Population Description

25 subjects were divided into 3 genotype groups based on their rs35929607 genotypes. Genotype GG is homozygous for the minor allele G, genotype AG is heterozygous, genotype AA is homozygous for the major allele A.

Arm/Group Title	rs35929607 SNP
Arm/Group Description	SNP rs35929607, located in an intron of STK39 gene, is associated with blood pressure in the Amish population. This SNP, rs35929607, has 2 alleles, A and G. Allele G is found less frequently and has an allele frequency of approximately 0.2-0.4 in populations studied thus far. We genotyped all participants of this study and determine if they are homozygous for the G allele, i.e., have the GG genotype, or are heterozygous (AG genotype), or are homozygous for the A allele and have the AA genotype.
Measure Participants	25
DBP, Genotype GG	-2 (0)
DBP, Genotype AG	0.7 (2.4)
DBP, Genotype AA	-1.7 (1.1)
SBP, Genotype GG	1 (0)
SBP, Genotype AG	-1.3 (4.2)
SBP, Genotype AA	-4.5 (1.3)

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	rs35929607 SNP
	Comments	Null hypothesis: There is no association between STK39 SNP rs35929607 genotype group and phenotypes collected in this study.
	Type of Statistical Test	Other
	Comments	Association between genotype and phenotype were tested using a linear mixed model, coding genotype as an independent variable with an additive genetic effect, and including age, age^2, and sex as covariates in the model. All association analyses were run using the Mixed Models for Pedigree Analysis (MMAP) software program to account for the relatedness among study subjects. MMAP User Guide is available from http://edn.som.umaryland.edu/mmap/index.php.
Statistical Test of Hypothesis	p-Value	>0.05
	Comments	Because no P-values less than 0.05 was observed, we did not adjust them for multiple comparison to avoid false positives.
	Method	Chi-squared
	Comments

8. Primary Outcome

Title	Fasting Glucose Change After 7 Days of Low Dose (12.5 mg) of HCTZ
Description	Values on Day 8 subtracts Day 0.
Time Frame	Fasting glucose was measured on day 0 and day 8

Outcome Measure Data

Analysis Population Description

28 subjects were divided into 3 genotype groups based on their rs35929607 genotypes. Genotype GG is homozygous for the minor allele G, genotype AG is heterozygous, genotype AA is homozygous for the major allele A.

Arm/Group Title	rs35929607 SNP
Arm/Group Description	SNP rs35929607, located in an intron of STK39 gene, is associated with blood pressure in the Amish population. This SNP, rs35929607, has 2 alleles, A and G. Allele G is found less frequently and has an allele frequency of approximately 0.2-0.4 in populations studied thus far. We genotyped all participants of this study and determine if they are homozygous for the G allele, i.e., have the GG genotype, or are heterozygous (AG genotype), or are homozygous for the A allele and have the AA genotype.
Measure Participants	28
Fasting glucose, GG Genotype	9 (0)
Fasting glucose, AG Genotype	2 (2.5)
Fasting glucose, AA Genotype	-0.1 (1.2)

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	rs35929607 SNP
	Comments	Null hypothesis: There is no association between STK39 SNP rs35929607 genotype group and phenotypes collected in this study.
	Type of Statistical Test	Other
	Comments	Association between genotype and phenotype were tested using a linear mixed model, coding genotype as an independent variable with an additive genetic effect, and including age, age^2, and sex as covariates in the model. All association analyses were run using the Mixed Models for Pedigree Analysis (MMAP) software program to account for the relatedness among study subjects. MMAP User Guide is available from http://edn.som.umaryland.edu/mmap/index.php.
Statistical Test of Hypothesis	p-Value	>0.05
	Comments	Because no P-values less than 0.05 was observed, we did not adjust them for multiple comparison to avoid false positives.
	Method	Chi-squared
	Comments

9. Primary Outcome

Title	Fasting Glucose Change After 7 Days of High Dose (25mg) of HCTZ
Description	Values on Day 8 subtracts Day 0.
Time Frame	Fasting glucose was measured on day 0 and day 8

Outcome Measure Data

Analysis Population Description

25 subjects were divided into 3 genotype groups based on their rs35929607 genotypes. Genotype 11 is homozygous for the minor allele G, genotype 12 is heterozygous, genotype 22 is homozygous for the major allele A.

Arm/Group Title	rs35929607 SNP
Arm/Group Description	SNP rs35929607, located in an intron of STK39 gene, is associated with blood pressure in the Amish population. This SNP, rs35929607, has 2 alleles, A and G. Allele G is found less frequently and has an allele frequency of approximately 0.2-0.4 in populations studied thus far. We genotyped all participants of this study and determine if they are homozygous for the G allele, i.e., have the GG genotype, or are heterozygous (AG genotype), or are homozygous for the A allele and have the AA genotype.
Measure Participants	25
Fasting glucose, GG Genotype	1.0 (0)
Fasting glucose, AG Genotype	1.0 (2.3)
Fasting glucose, AA Genotype	2.3 (2.1)

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	rs35929607 SNP
	Comments	Null hypothesis: There is no association between STK39 SNP rs35929607 genotype group and phenotypes collected in this study.
	Type of Statistical Test	Other
	Comments	Association between genotype and phenotype were tested using a linear mixed model, coding genotype as an independent variable with an additive genetic effect, and including age, age^2, and sex as covariates in the model. All association analyses were run using the Mixed Models for Pedigree Analysis (MMAP) software program to account for the relatedness among study subjects. MMAP User Guide is available from http://edn.som.umaryland.edu/mmap/index.php.
Statistical Test of Hypothesis	p-Value	>0.05
	Comments	Because no P-values less than 0.05 was observed, we did not adjust them for multiple comparison to avoid false positives.
	Method	Chi-squared
	Comments

10. Secondary Outcome

Title	Change in Plasma Sodium/Potassium Level During High Dose of HCTZ
Description	Na/K ratio is a function of kidney function
Time Frame	Plasma sodium and potassium measured from blood collected pre and post salt loading

Outcome Measure Data

Analysis Population Description

25 subjects were divided into 3 genotype groups based on their rs35929607 genotypes. Genotype GG is homozygous for the minor allele G, genotype AG is heterozygous, genotype AA is homozygous for the major allele A.

Arm/Group Title	rs35929607 SNP
Arm/Group Description	SNP rs35929607, located in an intron of STK39 gene, is associated with blood pressure in the Amish population. This SNP, rs35929607, has 2 alleles, A and G. Allele G is found less frequently and has an allele frequency of approximately 0.2-0.4 in populations studied thus far. We genotyped all participants of this study and determine if they are homozygous for the G allele, i.e., have the GG genotype, or are heterozygous (AG genotype), or are homozygous for the A allele and have the AA genotype.
Measure Participants	25
Genotype GG	1 (0)
Genotype AG	0 (2.3)
Genotype AA	2.3 (2.1)

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	rs35929607 SNP
	Comments	Null hypothesis: There is no association between STK39 SNP rs35929607 genotype group and phenotypes collected in this study.
	Type of Statistical Test	Other
	Comments	Association between genotype and phenotype were tested using a linear mixed model, coding genotype as an independent variable with an additive genetic effect, and including age, age^2, and sex as covariates in the model. All association analyses were run using the Mixed Models for Pedigree Analysis (MMAP) software program to account for the relatedness among study subjects. MMAP User Guide is available from http://edn.som.umaryland.edu/mmap/index.php.
Statistical Test of Hypothesis	p-Value	>0.05
	Comments
	Method	Chi-squared
	Comments	Because no P-values less than 0.05 was observed, we did not adjust them for multiple comparison to avoid false positives.

Adverse Events

Time Frame	1 year, 9 months
Arm/Group Title	Salt Loading		HCTZ
Arm/Group Description	Subjects will arrive at the Amish Research Clinics after overnight fasting. After taking height, weight, BP, and body temperature, subjects will receive 2 L of 0.9% NaCl (sodium chloride) saline over 4 hours while their blood pressure is monitored every 15 minutes. Blood pressure will be taken every 15 minutes during this procedure. Blood and urine samples will be collected from all subjects pre- and post-infusion.		After overnight fasting and having their height, weight, and BP measured, subjects are given seven 12.5 mg HCTZ tablets and instructed to take 1 tablet daily for one week. Ambulatory blood pressure will be measured and blood and urine will be collected on both day 1 and day 8. After a minimum 6-week wash-out period, the subjects will repeat the 7-day HCTZ intervention, taking 25 mg of HCTZ instead. Subjects with plasma potassium levels below 3.6 mmol/L on day 8 of 12.5 mg HCTZ will be given a daily supplement of 16 milliequivalents of potassium to prevent harmful loss of potassium while taking HCTZ.
All Cause Mortality
	Salt Loading		HCTZ
	Affected / at Risk (%)	# Events	Affected / at Risk (%)	# Events
Total	0/124 (0%)		0/28 (0%)
Serious Adverse Events
	Salt Loading		HCTZ
	Affected / at Risk (%)	# Events	Affected / at Risk (%)	# Events
Total	0/124 (0%)		0/28 (0%)
Other (Not Including Serious) Adverse Events
	Salt Loading		HCTZ
	Affected / at Risk (%)	# Events	Affected / at Risk (%)	# Events
Total	0/124 (0%)		0/28 (0%)

Limitations/Caveats

[Not Specified]

More Information

Certain Agreements

Principal Investigators are NOT employed by the organization sponsoring the study.

There is NOT an agreement between Principal Investigators and the Sponsor (or its agents) that restricts the PI's rights to discuss or publish trial results after the trial is completed.