ORIGINAL ARTICLE JJBMR

Vitamin D Receptor (VDR) Gene Polymorphism IsAssociated With Left Ventricular (LV) Mass andPredicts Left Ventricular Hypertrophy (LVH)Progression in End-Stage Renal Disease (ESRD) Patients

Alessandra Testa,1 Francesca Mallamaci,1 Francesco A Benedetto,2 Anna Pisano,1 Giovanni Tripepi,1

Lorenzo Malatino ,4 Ravi Thadhani ,3 and Carmine Zoccali1

1CNR-IBIM, Research Unit of Clinical Epidemiology and Physiopathology of Renal Disease and Hypertension, Reggio Calabria, Italy2Cardiology Unit, Morelli Hospital, Reggio Calabria, Italy3Division of Internal Medicine, University of Catania, c/o Ospedale Cannizzaro, Catanua, Italy4Renal Unit and the Center for D-Receptor Activation Research, Massachusetts General Hospital, Harvard Medical School, Boston,MA, USA

ABSTRACTLeft ventricular hypertrophy (LVH) is a strong cardiovascular risk marker in end-stage renal disease (ESRD) patients. Vitamin D deficiency

and/or disturbed vitamin D signaling has been implicated in LVH in experimental models. Because the BsmI vitamin D receptor VDR gene

polymorphism may alter VDR function, we performed a cross-sectional and longitudinal study in a cohort of 182 dialysis patients to

investigate (1) the relationship between BsmI VDR gene polymorphism and left ventricular mass index (LVMI) measured by

echocardiography and (2) the predictive power of this polymorphism for progression in LVH over a 18� 2 months of follow-up. As

a reference group, we used 175 healthy subjects matched to the study population as for age and sex. The distribution of BsmI genotypes

did not significantly deviate from Hardy-Weinberg equilibrium either in patients or in the control group of healthy subjects. The

frequency of the B allele of BsmI polymorphism (40.4%) in dialysis patients was similar to that of healthy control subjects (38.6%), and the

number of B alleles was directly related to LVMI (r¼ 0.20, P¼ .007). This relationship remained robust (b¼ 0.19, P¼ .006) in multivariate

analysis adjusting for traditional and nontraditional risk factors and antihypertensive and calcitriol treatment. In the longitudinal study,

LVMI rose from 60.1� 17.9 to 64.2� 19.3 g/m2.7 (P< .001), and again, the number of B alleles was associated with LVMI changes both in

crude and in fully adjusted analyses. These cross-sectional and longitudinal observations coherently support the hypothesis that altered

vitamin D signaling is implicated in LVH in ESRD patients. � 2010 American Society for Bone and Mineral Research.

KEY WORDS: VITAMIN D; POLYMORPHISM; ASSOCIATION; LEFT VENTRICULAR HYPERTROPHY

Introduction

Left ventricular hypertrophy (LVH) is recognized as one of the

strongest risk factors for all-cause and cardiovascular

mortality in patients with end-stage renal disease (ESRD).(1,2)

The pathogenesis of LVH in ESRD is multifactorial, and arterial

hypertension, hyperparathyroidism, severe anemia, hypoalbu-

minemia, chronic volume overload,(3) sympathetic overactivity,(4)

and accumulation of the endogenous inhibitor of nitric oxide

(NO) synthase asymmetric dimethylarginine (ADMA)(5) have

been implicated as causative mechanisms responsible for LVH in

these patients. However, collectively, these risk factors explain

only in part the variability in left ventricular (LV) mass in the

dialysis population.

Received in original form November 27, 2008; revised form February 4, 2009; acce

Address correspondence to: Professor Carmine Zoccali, CNR-IBIM, CNR e Nefrologia,

89124 Reggio Calabria, Italy. E-mail: carmine.zoccali@tin.it

Journal of Bone and Mineral Research, Vol. 25, No. 2, February 2010, pp 313–319

DOI: 10.1359/jbmr.090717

� 2010 American Society for Bone and Mineral Research

Vitamin D insufficiency or deficiency is pervasive in ESRD

patients and may have substantial health implications in this

population.(6) The myocardium is an important target of vitamin

D. Vitamin D receptor (VDR) knockout mice show myocardial

renin overexpression and marked cardiomyocyte hypertrophy.(7)

In vitro 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] attenuates

cardiomyocyte proliferation(8) and hypertrophy,(9) and treatment

with paricalcitol attenuates the development of LVH and LV

dysfunction in Dahl rats,(10) a reliable animal model of LVH and

vitamin D deficiency. Retrospective analyses in hemodialysis

patients and small clinical trials show that treatment with a

vitamin D analogue may regress LVH in these patients,(11–13)

providing further rationale for a full-fledged randomized clinical

trial in ESRD. Until now, studies in the VDR knockout mice

pted July 6, 2009. Published online July 13, 2009.

Ospedali Riuniti, c/o EUROLINE di Ascrizzi Vincenzo, Via Vallone Petrara 55-57,

313

represent the most convincing evidence of the fundamental role

of vitamin D in the regulation of LV mass.

While the clinical trial remains the ultimate test for establishing

whether or not vitamin D is implicated in LVH in ESRD, examining

the association between VDR gene polymorphisms and LV mass

represents an additional relevant step for unraveling the role of

vitamin D in cardiac disease in these patients. An important

advantage of genetic association studies is that the transmission

of genes occurs at random (Mendelian randomization), thus

mimicking the design of classical intervention trials.(14) Three

common polymorphisms (BsmI, ApaI, and TaqI) at the 3’ end of

the VDR have been intensively investigated, and one of these

(BsmI) already has been associated with survival in the dialysis

population,(15) suggesting that this polymorphism may be

implicated in the high risk of this population. However, the

association between the BsmI polymorphism and LVH has never

been tested. Therefore, we performed an association study

investigating the relationship between this polymorphism and

LV mass in a cohort of ESRD patients. To minimize the risk that

the observed association are merely due to chance, we

performed a study including both a cross-sectional analysis of

the association between BsmI VDR gene polymorphism and LV

mass index (LVMI) and a longitudinal observation aimed at

establishing whether or not this polymorphism predicts LVH

progression in ESRD. For the first time, we demonstrate that the

BsmI VDR gene polymorphism is independently related to LVH

and to LVH progression in ESRD patients.

Methods

The study protocol was in conformity with the local ethical

guidelines of our institution and informed consent was obtained

from each participant.

Patients

One-hundred and eighty-two hemodialysis patients (104 males

and 78 females, all Caucasians) who had been on regular dialysis

treatment (RDT) for at least 6 months and who were free of overt

infections (i.e., fever, infected vascular access, or peritonitis or exit-

site infection) were recruited for the study. Patients were being

treated thrice weekly with standard bicarbonate dialysis (Na

138mmol/L, HCO3 5mmol/L, K 1.5mmol/L, Ca 1.25mmol/L, Mg

0.75mmol/L) with either Cuprophan or semisynthetic membranes.

Control subjects

As a control group, we enrolled a series of 175 age- and sex-

matched healthy blood donors from the same geographic area

of patients.

Genotyping of the BsmI VDR gene polymorphism

The participants were genotyped for the G ! A change

identified as BsmI polymorphism (bB alleles) in intron 8 of the

VDR gene on chromosome 12. This polymorphism, described

under identification number rs1544410, was studied by high-

throughput TaqMan allelic discrimination assay. Genomic DNA

was extracted from peripheral blood leukocytes by the salting-

314 Journal of Bone and Mineral Research

out technique.(16) The assay mix (including unlabeled PCR

primers FAM and VIC dye-labeled TaqMan MGB probes) of the

Assays-on-Demand was designed and provided by Applied

Biosystems (Applied Biosystems, Inc., Foster City, CA, USA). The

reaction system contained 1 to 5 ng of genomic DNA, 12.5mL of

TaqMan Universal PCR Master Mix, 2�No AmpErase UNG, and

1.25mL 40�Assay Mix and was adjusted with H2O for a total

volume of 25mL. The genotyping was performed on an ABI Prism

7300, and a random 5% of samples were repeated indepen-

dently to confirm genotyping results. The genotype results for

these samples were completely consistent.

Laboratory measurements

A fasting blood sampling was performed during the midweek

nondialysis day. Serum cholesterol, albumin, phosphate, para-

thormone (PTH), and hemoglobin measurements were made

using standard methods in a routine clinical laboratory. Serum C-

reactive protein (CRP), plasma total homocysteine, and asym-

metric dimethylarginine (ADMA) were measured as previously

reported.(17) The plasma concentration of norepinephrine was

measured by a commercially available radioimmunoassay (RIA)

kit (Amicyl-test TM, Immunological Laboratories, Hamburg,

Germany). The intraassay coefficient of variation was 7% to 15%.

Echocardiography and blood pressure

These studies were performed on a nondialysis day within 2

hours of blood sampling. All echocardiographic measurements

were carried out according to the recommendations of the

American Society of Echocardiography by an observer unaware

of biochemical results. Left ventricular mass (LVM) was calculated

according to the Devereux formula and indexed to height2.7

(LVMI).(18) LVH was defined by an LVMI of over 47 g/m2.7 in

women or over 50 g/m2.7 in men. The height-based indexing of

LVM was specifically chosen to minimize any potential distortion

attributable to extracellular volume expansion (surface area

indexing being weight sensitive).(2)

Blood pressure (BP) was measured before dialysis and during

all dialysis sessions of the month (i.e., 12 measurements)

preceding the baseline echocardiographic study. Average values

were considered for statistical analysis. Average monthly BP is

associated with LVmass equally well as 24 hour ambulatory BP in

dialysis patients.(19)

Patients who repeated the echocardiographic study

Thirty-four of the 182 patients who entered into this study did

not repeat the second echocardiography because of death.

Therefore, 148 patients were left for the longitudinal part of this

study aimed at examining the relationship between the BsmI

polymorphism and changes in LVMI. These patients did not

materially differ from the original study population (data not

shown).

Statistical analysis

Power analysis

We assumed additivity of the effects of the B allele on LV mass

index. Such an assumption is preferred when allele dominance

TESTA ET AL.

cannot be assumed.(20) Nonrandomized parallel studies indicate

that the lack of 1,25(OH)2D3 treatment may be responsible for a

15% increase in LV mass in ESRD patients.(13) We hypothesised

that homozygotes for the protective allele of the BmsI

polymorphism gene (bb) have a LV mass that is 15% lower

than homozygotes for the risk allele (BB) and that LV mass in

heterozygotes (Bb) takes an intermediate value (7.5% higher

than in bb homozygotes). We estimated that LVMI in Bb patients

would be 61 g/m2.7 (� SD 15), that is the average value of LVMI

in hemodialysis patients in our center. On the basis of these data,

we calculated that the average value of LVMI would be 56 g/m2.7

in bb (7.5% lower than in Bb) and 66 g/m2.7 in BB (7.5% higher

than in Bb) genotypes. Power analysis based on these data

indicated that a sample size of 180 dialysis patients had an 80%

probability to detect as statistically significant (a error¼ 0.05) a

5 g/m2.7 average increase in LVMI for each unitary increase in the

number of B alleles.

Statistical tests

The relationship between the number of B alleles of BsmI VDR

polymorphism and LV mass at baseline and with LV mass

changes during the follow-up was investigated by univariate and

multiple linear regression analyses (Models 1 to 4 in Tables 2

and 3). As potential confounders we considered Framingham risk

factors (e.g., age, sex, smoking, diabetes, systolic pressure and

antihypertensive treatment, cholesterol, and previous cardio-

Table 1. Main Demographic, Clinical, Biochemical, and Echocardiogr

Vita

bb (n¼ 61

Age (years) 60� 13

Duration of RDT (months) 47 (21–12

Male sex, n (%) 34 (56%)

Smoking, n (%) 22 (36%)

Diabetes, n (%) 10 (16%)

Patients with previous cardiovascular events, n (%) 33 (54%)

On antihypertensive treatment, n (%) 23 (38%)

On EPO treatment, n (%) 29 (47%)

On calcitriol treatment, n (%) 28 (46%)

Systolic pressure (mmHg) 137� 25

Diastolic pressure (mmHg) 74� 14

Heart rate (b/min) 80� 10

Cholesterol (mmol/L) 5.26� 1.6

Haemoglobin (g/L) 107� 22

Albumin (g/L) 41� 6

Calcium phosphate (mmol2/L2) 4.5� 1.3

Phosphate (mmol/L) 1.96� 0.5

PTH (pg/mL) 161 (64–34

Norepinephrine (nmol/L) 3.3 (2.0–6.

CRP (mg/L) 9.5 (3.4–20

ADMA (mmol/L) 23.2 (17.4–3

Homocysteine (mmol/L) 2.42 (1.55–3

Kt/V 1.23� 0.2

LVMI (g/m2.7) 56.4� 17.

Data are expressed as mean� SD, median and interquartile range, or as pe

VITAMIN D POLYMORPHISM AND LVH IN ESRD

vascular events), risk factors peculiar to ESRD (e.g., duration of

dialysis treatment, haemoglobin, albumin, phosphate, PTH, and

fractional urea clearance [Kt/V]), and emerging risk factors (e.g.,

CRP, homocysteine, ADMA, and norepinephrine). To control for

the potential confounding effect of current calcitriol therapy, we

also included this factor in the multiple regression analysis. Data

are expressed as standardized regression coefficient b and P

value. The deviation of Hardy-Weinberg equilibrium was

investigated by calculating the chi-square test between

observed and expected frequencies. Since we did not test

other VDR single-nucleotide polymorphisms (SNPs), no adjust-

ment for multiple testing was done.

Data are expressed as mean� SD (normally distributed data),

median and interquartile range (nonnormally distributed data),

or percent frequency, as appropriate, and by trend analysis.

Positively skewed distributed data (e.g., CRP, homocysteine, and

ADMA) were log transformed (lg10) before data analysis.

All calculations were done using two standard statistical

packages (SPSS for Windows, Version 9.0.1,� SPSS Inc., Chicago,

IL, USA, and NCSS PASS (2004), Number Cruncher Statistical

Systems, Kaisville, UT, USA).

Results

In the entire study population (see Table 1), the median duration

of RDT was 45 months (interquartile range 20 to 110 months).

The average urea Kt/V in these patients was 1.22� 0.28.

aphic Data in Hemodialysis Patients

min D receptor (BsmI polymorphism)

) Bb (n¼ 95) BB (n¼ 26) P for trend

57� 16 60� 14 .63

1) 43 (20–92) 46 (14–121) .72

54 (57%) 16 (62%) .65

38 (40%) 8 (31%) .83

13 (14%) 3 (11%) .52

44 (46%) 14 (54%) .74

38 (40%) 8 (31%) .69

59 (62%) 12 (46%) .62

55 (58%) 12 (46%) .61

143� 24 147� 22 .05

78� 13 78� 12 .12

80� 10 76� 9 .27

5 5.38� 1.37 5.75� 1.47 .19

106� 17 107� 20 .96

42� 4 41� 6 .47

4.6� 1.2 4.4� 1.8 .95

0 2.02� 0.44 1.99� 0.49 .73

5) 171 (70–405) 145 (53–436) .51

4) 3.1 (1.6–5.3) 3.7 (1.9–5.6) .60

.1) 8.1 (3.4–15.0) 8.0 (3.4–21.1) .66

5.2) 28.4 (21.0–44.5) 25.2 (19.3–36.7) .34

.88) 2.88 (1.68–4.08) 3.33 (1.38–4.15) .23

9 1.20� 0.25 1.27� 0.32 .74

7 62.7� 19.1 66.9� 15.4 .007

rcent frequency, as appropriate.

Journal of Bone and Mineral Research 315

Fig. 1. Distribution of left ventricular mass index (LVMI) in the study

cohort.

Twenty-six patients were diabetics, and 68 were habitual

smokers (22� 16 cigarettes/day). Sixty-nine patients were on

antihypertensive drugs [48 on monotherapy with angiotensin-

converting enzyme (ACE) inhibitors, AT-1 antagonists, calcium

channel blockers, and alpha and beta blockers and the remaining

21 on double or triple therapy with various combinations of

these drugs]. One hundred patients were on treatment with

erythropoietin and 95 with calcitriol. At baseline, LVMI was on

average 61� 18 g/m2.7. The distribution of LVMI in the study

population is presented graphically in Fig. 1.

The distribution of BsmI genotypes did not significantly

deviate from Hardy-Weinberg equilibrium either in patients (bb

genotype 33.5%, Bb genotype 52.2%, and BB genotype 14.3%;

x2¼ 1.29, P¼NS) or in controls (bb genotype 37.8%, Bb

genotype 47.4%, and BB genotype 14.9%; x2¼ 0.01, P¼NS).

The frequency of the B allele (38.6%) in healthy subjects was

almost identical to that of dialysis patients (40.4%). Data analysis

according to BsmI genotypes (see Table 1) revealed that there

was a dose-dependent relationship between the number of BsmI

B alleles and systolic arterial pressure (bb genotype

137� 25mmHg, Bb genotype 143� 24mmHg, BB genotype

147� 22mmHg; P for trend¼ 0.048) (see Table 1). No significant

difference among the three groups was observed for the

remaining variables listed in Table 1.

Fig. 2. Relationship between BsmI polymorphism and LVMI and with LVMI ch

316 Journal of Bone and Mineral Research

BsmI genotypes and baseline LVMI: linear regressionanalyses

On univariate analysis, the number of B alleles was directly and

significantly related to LVMI (r¼ 0.20, P¼ .007) (Fig. 2, left panel).

The independent association between the BsmI polymorphism

and LVMI was tested in multiple linear regression models of

increasing complexity (Table 2). Data adjustment for age, sex,

and duration of RDT did not modify the strength of the B allele–

LVMI link (b¼ 0.21, P¼ .004) (Model 1), and this also was true

when we added into the model other Framingham risk factors

(e.g., smoking, systolic pressure, diabetes, cholesterol, and

previous cardiovascular events), as well as antihypertensive

and calcitriol treatment (b¼ 0.20, P¼ .004) (Model 2). Further

data adjustment for factors peculiar to ESRD (e.g., hemoglobin,

albumin, phosphate, PTH, and Kt/V) (Model 3) and nontraditional

risk factors (e.g., CRP, homocysteine, norepinephrine, and ADMA)

(Model 4) did not change the strength of the association. In the

fully adjusted multiple regression model (Model 4), besides the

BsmI polymorphism, serum albumin (b¼�0.24, P¼ .003),

systolic arterial pressure (b¼ 0.24, P¼ .002), and plasma ADMA

(b¼ 0.17, P¼ .02) also were significantly and independently

related to LVMI.

BsmI genotypes and changes in LVMI

Since baseline LVMI was inversely associated with LVMI changes

(r¼�0.18, P¼ .03), indicating regression to the mean, LVMI

changes therefore were adjusted for the corresponding baseline

values before analyses. As shown in Table 3, the number of B

alleles was associated with LVMI changes both in crude (see

Fig. 2, right panel) and in adjusted analyses, the correlation

coefficient of this relationship remaining virtually unchanged

after full data adjustment across models of increasing complexity

(b¼ 0.19, P¼ .03).

Discussion

This study shows that in chronic dialysis patients the frequency

of the B allele of the BsmI VDR gene polymorphism is

independently related to LVH and associated with higher

progression rate of LVH. Overall, these genetic associations are

compatible with the hypothesis that altered vitamin D

anges. Data are mean� SD.

TESTA ET AL.

Table 2. Multiple Linear Regression Models for Baseline LVMI

Dependent variable: LVMI

Unadjusted r (P) Model 1 b (P) Model 2 b (P) Model 3 b (P) Model 4 b (P)

BsmI polymorphism 0.20 (.007) 0.21 (.004) 0.20 (.004) 0.19 (.004) 0.18 (.006)

Age 0.27 (<.001) 0.21 (.06) 0.13 (.10) 0.12 (.12)

Sex 0.07 (.37) 0.07 (.40) 0.13 (.10) 0.09 (.28)

Duration of RDT 0.04 (.58) 0.08 (.28) 0.07 (.35) 0.03 (.67)

Smoking 0.01 (.85) 0.04 (.60) 0.07 (.35)

Diabetes 0.05 (.48) 0.10 (.16) 0.09 (.21)

Cholesterol �0.12 (.09) 0.02 (.80) �0.04 (.58)

Systolic pressure 0.23 (.003) 0.22 (.003) 0.24 (.002)

Previous cardiovascular events 0.18 (.02) 0.16 (.04) 0.14 (.07)

Antihypertensive treatment 0.12 (.12) 0.08 (.31) 0.05 (.55)

On treatment with calcitriol �0.03 (.62) �0.01 (.83) �0.05 (.47)

Hemoglobin �0.12 (.09) �0.11 (.12)

Albumin �0.28 (.001) �0.24 (.003)

Phosphate 0.10 (.15) 0.08 (.22)

PTH 0.12 (.08) 0.07 (.31)

Kt/V �0.001 (.98) �0.02 (.75)

CRP �0.008 (.91)

Homocysteine �0.008 (.91)

ADMA 0.17 (.02)

Norepinephrine 0.06 (.43)

Data are expressed as beta regression coefficients (b) and P values.

metabolism exerts an important influence on the control of LV

mass in this population.

LVH is considered to be a clinical indicator integrating the

long-term exposure not only to pressure overload but also to

Table 3. Multiple Linear Regression Models for Changes in LVMI

Dependent variable: chang

Unadjusted b (P) Model 1 b

BsmI polymorphism 0.19 (.02) 0.19 (.0

Age 0.01 (.8

Sex 0.003 (.9

Duration of RDT �0.03 (.6

Smoking

Diabetes

Cholesterol

Systolic pressure

Previous cardiovascular events

Antihypertensive treatment

On treatment with calcitriol

Hemoglobin

Albumin

Phosphate

PTH

Kt/V

CRP

Homocysteine

ADMA

Norepinephrine

Data are expressed as beta regression coefficients (b) and P values.

VITAMIN D POLYMORPHISM AND LVH IN ESRD

several hemodynamic and nonhemodynamic factors. Regression

of LVH is associated with a 59% lower risk of subsequent adverse

events compared with the persistence or new development of

LVH.(21) LVH is exceedingly frequent in dialysis patients, with a

es in LVMI (g/m2.7/month) adjusted for LVMI at baseline

(P) Model 2 b (P) Model 3 b (P) Model 4 b (P)

2) 0.18 (.04) 0.19 (.03) 0.19 (.03)

9) 0.04 (.68) 0.05 (.62) 0.07 (.51)

6) �0.05 (.62) �0.10 (.32) �0.12 (.30)

9) �0.05 (.58) �0.04 (.69) �0.06 (.57)

0.06 (.53) 0.03 (.76) 0.04 (.70)

�0.11 (.19) �0.12 (.17) �0.10 (.31)

0.08 (.33) �0.12 (.21) �0.15 (.15)

0.03 (.74) 0.04 (.70) 0.03 (.74)

�0.02 (.82) 0.004 (.97) �0.02 (.80)

0.007 (.94) 0.04 (.68) 0.03 (.74)

0.15 (.07) 0.16 (.07) 0.16 (.07)

0.12 (.20) 0.11 (.25)

0.10 (.34) 0.08 (.47)

�0.08 (.35) �0.07 (.48)

0.17 (.05) 0.12 (.19)

�0.06 (.57) �0.06 (.56)

�0.003 (.97)

0.13 (.19)

0.02 (.85)

0.07 (.49)

Journal of Bone and Mineral Research 317

prevalence ranging from 60% to 78%. In line with studies in other

conditions, cohort studies in dialysis patients solidly link LVH

progression and mortality(22) and improved survival when LVH

regresses.(23) Even though several causative factors are impli-

cated in high LV mass in the dialysis population, regression of

LVH in this population is difficult to achieve, and even a most

intensive treatment strategy including long nocturnal dialysis

failed to normalize LV mass in several patients.(24)

Even though experimental evidence linking vitamin D

deficiency and alterations in LV mass and function appears

coherent and convincing,(7–9) the issue received only scant

attention in dialysis patients. In an uncontrolled series of 12

dialysis patients,(11) treatment with 1a-hydroxycholecalciferol for

6 weeks elicited an increase in LV contractility, as measured by

fractional fiber shortening and mean velocity of fiber shortening.

In a nonrandomized parallel study, intravenous calcitriol caused

a marked reduction in LVH, whereas LVH did not regress in

untreated patients. Apart from these small nonrandomized

studies, until now, there has been no study exploring the link

between the vitamin D system and LV mass in this population.

The response to vitamin D compounds critically depends on

the VDR functioning. In humans, three common polymorphisms

(BsmI, ApaI, and TaqI) at the 3’ end of the VDR have been

identified, and these polymorphisms have been linked to a

variety of diseases, from low bone mass density (BMD)(25) to type

1 diabetes and cancer.(26) In ESRD, the B allele of the BsmI

polymorphism was associated with a low BMD in patients

younger than 65 year of age(26) and with inflammation, anemia,

and resistance to erythropietin.(27) Of note, patients homozygous

for the B allele display a substantially shorter survival than

heterozygotes or homozygotes for the b allele.(15) Given the

exceedingly high rate of cardiovascular complications in ESRD, it

is plausible that such an association underlies disturbed control

of a vitamin D–dependent mechanism affecting the cardiovas-

cular system. To date, there have been no studies investigating

the BsmI polymorphism as related to cardiovascular disease

neither in the general population nor in the dialysis population.

The consequences of vitamin D deficiency and vitamin D

resistance in ESRD depend on a large number of factors.

Therefore, confounding is a real possibility in observational

studies based on the measurement of vitamin D plasma levels or

on vitamin D treatment status (treated versus untreated).

Mendelian randomization(14) is an interesting option to explore

the nature of the LV mass– vitamin D association. Unlike classic

epidemiology studies, SNP association studies are uncon-

founded by behavioral and environmental factors because

these factors usually do not alter genotype,(14) thus representing

a useful approach in studies focusing on the ESRD population.(15)

As predicated by Mendialian randomization theory, with the

exception of blood pressure (see below), risk factors were evenly

distributed among patients categorized on the basis of the B

allele. We hypothesised that the B allele of the BmsI

polymorphism may serve as a marker of altered vitamin D

signaling in ESRD patients and assumed that this alteration in BB

homozygotes produces an increase in LV mass quantitatively

similar to the difference in LV mass between vitamin D–treated

and untreated patients.(13) At baseline, in our cohort we found an

association between the B allele and LVMI. This association was

318 Journal of Bone and Mineral Research

almost unmodified after extensive statistical adjustment for

other risk factors, including calcitriol treatment, serum phos-

phate, and PTH. The independent association of the B allele with

systolic blood pressure is of interest in that hypertension is a

phenotypic characteristic of the VDR knockout model and

because a link between this polymorphism and BP has been

noted very recently in another study in ESRD.(28) This

phenomenon suggests that altered signaling may also con-

tribute to raise BP in ESRD patients. Both 25-hydroxycholecalci-

ferol and 1,25(OH)2D3 levels are strongly associated with arterial

distensibility, pulse wave velocity, and endothelial function, as

measured by the forearm blood flow response to hand warming

in ESRD patients,(29) indicating a relevant impact of vitamin D

status on the control of large and small arterial vessels. Beyond

baseline associations, we also found that the B allele predicts

LVH progression. This relationship is remarkable in that it also

persisted virtually unchanged in a multivariate model consider-

ing the regression-to-the-mean phenomenon, which is an

important confounder in longitudinal studies of LV mass in

ESRD patients.(22) Over half the patients in this study were on

long-term treatment with calcitriol, but apparently this treatment

had no effect on LV mass index nor modified the association

between the B allele and this index. Because confounding by

indication cannot be eliminated safely by statistical adjustment,

we believe that no conclusion can be drawn on the basis of our

data on the effect of treatment with activated vitamin D

compounds on LVH in ESRD. An ongoing double-blind, placebo-

controlled, randomized clinical trial testing the effect of an active

form of vitamin D (paracalcitol) on LVMI in ESRD patients

(http://clinicaltrials.gov/ct2/show/NCT00616902?term¼PRIMO&

rank ¼2) will provide a definitive answer to the question of

whether vitamin D administration can reverse LVH in ESRD

patients.

In conclusion, in dialysis patients, the B allele of the BsmI VDR

gene polymorphism is strongly and independently related to

LVH and is associated with a higher progression rate of LVH.

Since genes are randomly transmitted (Mendelian randomiza-

tion), our cross-sectional and longitudinal observations consis-

tently support the hypothesis that altered vitamin D signaling is

implicated in LVH in ESRD patients.

Disclosures

The authors state that they have no conflicts of interest.

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