SUPPLEMENTAL DATA
VITAMIN D STATUS IN PRIMARY HYPERPARATHYROIDISM: EFFECT OF
GENETIC BACKGROUND
Claudia Battistaa, Vito Guarnieria, Vincenzo Carnevalea, Filomena Baordaa, Mauro Pileria,
Maria Garrubaa, Antonio S Salcunia, Iacopo Chiodinib, Salvatore Minisolac, Elisabetta
Romagnolid, Cristina Eller-Vainicherb, Stefano A Santinia, Alessandro Parisia Vincenzo
Frusciantea, Andrea Fontanaa, Massimiliano Copettia, Geoffrey N Hendye, Alfredo Scillitania,*
and David EC Colef
a
Endocrinology, Medical Genetics, Internal Medicine, Clinical Pathology, Radiotherapy, Nuclear
Medicine, Biostatistics, IRCCS “Casa Sollievo della Sofferenza”, 71013 San Giovanni Rotondo,
Foggia, Italy
b
Unit of Endocrinology and Metabolic Diseases , Fondazione IRCCS Cà Granda, Department of
Clinical Sciences and Community Health, University of Milan, 20122 Milan, Italy
c,
Department of Internal Medicine and Medical Discipline and
d
Department of Experimental
Medicine, “Sapienza” Rome University, 00185 Rome, Italy
e
Experimental Therapeutics and Metabolism, McGill University Health Centre-Research Institute,
and Departments of Medicine, Physiology and Human Genetics, McGill University, Montreal,
Quebec, H4A 3J1, Canada
f
Departments of Laboratory Medicine and Pathobiology, Medicine and Genetics , University of
Toronto, Ontario, M4N 3M5, Canada
Supplemental methods
Subjects
Family composition
A family was considered a DUO if there was at least one matching relative for the primary hyperparathyroid
(PHPT) proband, and a TRIO if there was at least one matching in-law relative (ILR) and one first-degree
1
relative (FDR). Details of the family composition are to be found in Supplemental Table S1. All possible
pairwise comparisons were tested in the primary analysis of the 88 eligible families with their matching
relatives. We also analysed a TRIO sub-group of 54 families that matched one FDR and one ILR subject for
the single PHPT patient. Controls used for the TRIO analysis (presented here as Supplemental Information)
were selected according to age and sex when there was more than one FDR or ILR for each family.
Laboratory investigation, molecular and statistical analyses
All as described in the main manuscript.
Results
Patient characteristics
Within the TRIO group, PHPT patients and ILR subjects were significantly older than FDR whereas there
was no difference between PHPT patients and ILR subjects. TotalD, DBP, BioD and FreeD values were
significantly lower in patients than in subjects whereas there was no difference between FDR and ILR
subjects (Table S2).
Genotype and DBP
In the TRIO group, the T alleles at rs7041 and the A alleles at rs4588 (additive genetic model) were
negatively associated with DBP in all subjects and in the three different groups. The regression slopes of
genotypes, assumed as the number of risk alleles, in determining DBP (Table S3), were not different across
ILR, FDR and PHPT groups, as indicated by the SNP-by-group interaction variable (Table S3) which
measures the degree of heterogeneity.
FreeD and BioD
After adjustment for season, gender and serum creatinine, FreeD and BioD values were different
among the 3 groups (Table S4).
2
Table S1. Composition of families entered into the study*
$
Number of recruited
controls in each family
2
3
4
5
6
7
10
TOTAL
#Number of DUO families
@
Number of TRIO families
19
3
6
4
1
1
0
-33
7
10
1
2
1
34
54
* All families (DUO and TRIO) consisted of a PHPT proband and at least one matched family member, either
related (FDR) or not (ILR)
$
Specific numbers of controls included in the analyses are given here
#
The DUO families with only two family members consisted of proband and one matched control. Those
with 3 or more family members contributed 2 or more controls to the pair-wise comparison, necessitating
adjustment for multiple comparisons
@
TRIO families all consisted of at least three individuals—a PHPT patient proband, a first degree relative
(FDR) and an unrelated in-law relative (ILR).
Those with additional matched controls are enumerated here.
3
Table S2. Clinical characteristics and GC genotypes of the TRIO samples and comparisons among groups
All subjects
PHPT
FDR
ILR
(n=162)
(n=54)
(n=54)
(n=54)
53.3±14.2
58.9±11.1a
45.4±14.8b
55.6±12.8a
87(54)
46(85)a
26(48)b
15(28)b
Creatinine (mg/dL)
0.88±0.19
0.87±0.24a
0.88±0.17a
0.90±0.15a
Albumin (g/dL)
4.24±0.41
4.00±0.38a
4.38±0.40b
4.34±0.35b
TotalD (ng/mL)
18.4±12.2
12.9±6.9a
19.3±10.8b
23.1±15.3b
DBP (mg/L)
365±101
334±72a
378±114b
384±105b
FreeD (pg/mL)
4.23±3.02
3.26±2.00a
4.52±3.39b
4.90±3.26b
BioD (ng/mL)
1.64±1.20
1.19±0.76a
1.77±1.29b
1.96±1.34b
iCa (mmol/L)
1.33±0.18
1.52±0.19a
1.24±0.05b
1.23±0.04b
CaAlb-adj (mg/dL)
9.88±1.29
11.3±1.13a
9.30±0.64b
9.05±0.64b
PTH (pg/mL)
132±251
300±384a
54.4±20.6b
50.7±20.7b
GC rs4588 genotype*
85, 52, 9
30, 18, 5
26, 18, 2
29, 16, 2
CC, CA, AA n, (%)
(58,36,6)
(57,34,11)
(57, 39, 4)
(62, 34, 4)
GC rs7041 genotype*
45, 70, 31
16, 26, 11
11, 24, 11
18, 20, 9
(31, 48, 21)
(30, 49, 21)
(24, 52, 24)
(38, 43, 19)
Age (years)
Women n, (%)
GG, GT, TT n, (%)
PHPT, primary hyperparathyroid group; FDR, first-degree relative group; ILR, in-law relative group; TotalD,
serum total 25-hydroxyvitamin D (25OHD); DBP, vitamin D binding protein; BioD, bioavailable 25OHD;
FreeD, free 25OHD; iCa, blood ionized calcium (corrected to pH 7.4); CaAlb-adj, serum total calcium adjusted
for albumin; PTH, serum parathyroid hormone; GC, group-specific component gene encoding DBP.
a-b
, Pairwise mean values followed by different superscript letters are significantly different from one
another. p-values were derived from HGLM, accounting for family clustering. p-values were adjusted for
multiple comparisons using Bonferroni correction.
*
, No significant differences between groups (PHPT, FDR, ILR) under an additive genetic model.
4
Table S3. Sample means ± standard deviations of serum DBP levels (mg/L) for each GC genotype and
regression slopes from additive genetic models, along with their standard errors (SE), in the TRIO samples
Groups
Regression slope
(SE)*
p-value#
Rs4588a
0 - CC
1 - CA
2 - AA
All
392 ± 90#
339 ± 108
289 ± 70
-50.2 (12.9)
<0.001
ILR
408 ± 112
389 ± 83
243 ± 52
-41.2 (23.4)
0.090
FDR
398 ± 80
342 ± 150
381 ± 58
-32.0 (23.4)
0.078
PHPT
371 ± 71
293 ± 39
271 ± 50
-59.8 (19.1)
0.001
Rs7041b
0 - GG
1 - GT
2 - TT
All
397 ± 93
366 ± 107
325 ± 80
-36.6 (11.2)
0.001
ILR
408 ± 109
399 ± 109
358 ± 90
-23.0 (18.4)
0.222
FDR
404 ± 91
392 ± 127
311 ± 82
-42.5 (19.7)
0.023
PHPT
381 ± 78
316 ± 58
312 ± 68
-41.4 (18.0)
0.021
DBP, vitamin D binding protein; GC, group-specific component
gene encoding DBP; ILR, in-law relative group; FDR, first-degree
relative group; PHPT, primary hyperparathyroid group.
*
, regression slopes were derived from hierarchical generalized linear models (which accounts for family
cluster) using DBP levels in their original scale (mg/L)
#
, p-value for regression slopes were derived from hierarchical generalized linear models (which accounts
for family cluster) using log-transformed DBP levels.
5
The SNP-by-group interaction variable was included to evaluate whether regression slopes were different
across the ILR, FDR and PHPT groups.
a
, interaction p=0.616. b, interaction p=0.636.
Table S4. Adjusted FreeD and BioD levels with 95% confidence intervals in the TRIO samples
Variable
PHPT
FDR
ILR
FreeD (pg/mL)
2.7 (2.16-3.37)a
3.27 (2.68-3.99)b
3.55 (2.88-4.37)b
BioD (ng/mL)
0.98 (0.78-1.22)a
1.28 (1.05-1.57)b
1.4 (1.14-1.73)b
FreeD, free 25-hydroxyvitamin D, BioD, bioavailable 25-hydroxyvitamin D; PHPT, primary hyperparathyroid
group; FDR, first-degree relative group; ILR, in-law relative group.
a-b
, Pairwise mean values followed by different superscript letters are significantly different from one
another. p-values were derived from multivariable HGLM, accounting for family cluster, adjusting for
season, gender, serum creatinine, and adjusting for multiple comparisons using Bonferroni correction.
6