COL1A1 Genotypes. The distributions of the COL1A1 Sp1 and –1997 G→T genotypes in postmenopausal women are shown in Tables 1 and 2, respectively; both were shown to be in Hardy-Weinberg equilibrium (p >0.05). Allele frequencies for the Sp1 polymorphism were 65.2% for the S allele and 34.8% for the s, with a heterozygosity rate of 45.6%. The calculated heterozygosity for the –1997 G→T polymorphism was 38.3%, with a frequency of 25.6% for the T and 74.4% for the G allele. Linkage disequilibrium was observed between these two polymorphisms [χ 2 = 12.45; p = 0.01; degrees of freedom (df) = 3] with the combined genotype GG→Ss occurring in 33.6%. When constructing haplotypes, the frequencies of the most common haplotypes were 41.5% for G→S and 23.2% for T→S.

Both polymorphisms in the promoter region of the COL1A1 gene did not show any influence on the LS and FN BMD (Tables 1 and 2). Statistical significance was not reached on tests for genetic models of a dominant or recessive allele (Table 3) or for an allele dose effect by linear regression (p >0.05) (data not shown).

MTHFR C677T Polymorphism. The genotype frequencies for the MTHFR C677T polymorphism observed in postmenopausal women are shown in Table 4. Allele frequencies observed were 63.9% for the C allele and 36.1% for the T, yielding a calculated heterozygosity rate of 46.3%.

The LS, FN, trochanter and Ward’s triangle BMD were lowest in CC homozygotes, although the differences between the genotypes did not reach statistical significance (p >0.05) (Table 4). The C allele was significantly correlated with trochanter BMD and, to a lesser degree, with FN BMD (Table 5). No allele dose effect was observed at any anatomical site for the same allele (p >0.05).

There were no differences in the distribution of genotype frequencies of both COL1A1 polymorphisms between normal postmenopausal women (independent sample t-score >–1.0) and those with a low LS and/or FN BMD when tested by chi-square (p >0.05) (Table 6). Haplotype frequencies were not statistically significant when compared between these two groups (c2 = 1.14; p = 0.76; df = 3) (Table 6). The MTHFR C677T genotype frequencies did not differ significantly between the normal women and those with a low BMD (χ 2 = 0.78; p = 0.68; df = 2) (Table 6).

The MTHFR C677T Frequency in the Maltese Population at Birth. In neonates (n = 245), genotype frequencies for this polymorphism were 46.1% CC, 43.3% CT and 10.6% TT and were in Hardy-Weinberg equilibrium (χ 2 = 0.02; p = 0.88; df = 1).

Table 2. General characteristics, lumbar and femoral bone mineral density in Maltese postmenopausal women according to COL1A1 –1997 G→T polymorphism.

General Characteristics and Genotypes				p Valuea
	TT	TG	GG		TT vs. TG	TT vs. GG	TG vs. GG
Number	10	43	70	0.36a
Per cent	8.1	35.0	56.9	–
Age (years)b,c	57.3 (6.1)	55.6 (6.9)	55.5 (7.5)	0.50	0.27	0.25	0.88
BMI (kg/m2)b	27.3 (5.0)	28.9 (4.5)	28.4 (3.9)	0.60	0.38	0.47	0.55
BMD L2-L4 (g/cm2)b • Adjustedd	0.97 (0.16) 1.01	0.97 (0.22) 0.97	0.94 (0.18) 0.94	0.67 0.37	0.98 0.46	0.58 0.24	0.41 0.34
BMD femoral (g/cm2)b • Adjustedd	0.85 (0.15) 0.88	0.85 (0.14) 0.85	0.83 (0.13) 0.83	0.83 0.43	0.91 0.41	0.67 0.24	0.60 0.49
Ward’s Triangle (g/cm2)b • Adjustedc	0.64 (0.15) 0.69	0.63 (0.16) 0.63	0.62 (0.14) 0.62	0.90 0.45	0.89 0.31	0.72 0.21	0.71 0.75
Trochanter (g/cm2)b • Adjustedd	0.64 (0.15) 0.67	0.68 (0.14) 0.68	0.68 (0.12) 0.67	0.71 096	0.45 0.80	0.46 0.93	0.77 0.83
Procollagen (ng/mL)b,c	88.6 (26.8)	103.9 (86.1)	93.0 (45.4)	0.91	0.85	0.74	0.74
DpD (nmol/mmol creatinine)b	8.3 (2.2)	9.6 (3.4)	9.1 (3.3)	0.66	0.39	0.57	0.57

a p Value obtained for Hardy-Weinberg Equilibrium (chi-square).

b Values are means ± SD in parenthesis; p values obtained by ANOVA or independent sample t-test.

c Kruskal-Wallis or Mann-Whitney used for age and procollagen.

d Values are adjusted for age, BMI, YSM; 95% CI given by univariate analysis (not shown).

	Dominant Modela			Recessive Modela
COL1A1 Sp1 Polymorphism	SS	Ss and ss	p Valueb	SS and Ss	ss	p Valueb
Number	49	71	–	106	14	–
BMD L2-L4 (g/cm2)c • Adjustedd	0.97 (0.22) 0.97	0.95 (0.17) 0.94	0.61 0.37	0.96 (0.20) 0.96	0.90 (0.16) 0.89	0.28 0.11
BMD femoral (g/cm2)c • Adjustedd	0.86 (0.14) 0.86	0.83 (0.13) 0.83	0.30 0.13	0.84 (0.14) 0.84	0.85 (0.12) 0.84	0.83 0.98
Ward’s Triangle (g/cm2)c • Adjustedd	0.64 90.17) 0.64	0.62 (0.13) 0.62	0.69 0.31	0.63 (0.15) 0.63	0.64 (0.12) 0.62	0.84 0.79
Trochanter (g/cm2)c • Adjustedd	0.67 (0.14) 0.68	0.69 (0.12) 0.68	0.47 0.83	0.68 (0.13) 0.68	0.70 (0.09) 0.68	0.64 0.94
–1997 T→G Polymorphism	TT	TG and GG	p Valueb	TT and TG	GG	p Valueb
Number	10	113	–	53	70	–
BMD L2-L4 (g/cm2)c • Adjustedd	0.97 (0.16) 1.01	0.95 (0.20) 0.95	0.74 0.31	0.97 (0.21) 0.98	0.94 (0.18) 0.94	0.37 0.21
BMD femoral (g/cm2)c • Adjustedd	0.85 (0.15) 0.88	0.84 (0.14) 0.84	0.75 0.28	0.85 (0.14) 0.85	0.83 (0.13) 0.83	0.55 0.30
Ward’s Triangle (g/cm2)c • Adjustedd	0.64 (0.15) 0.69	0.63 (0.15) 0.62	0.78 0.22	0.63 (0.14) 064	0.62 (0.14 0.62	0.66 0.49
Trochanter (g/cm2)c • Adjustedd	0.64 (0.15) 0.67	0.68 (0.13) 0.68	0.44 0.88	0.68 (0.14) 0.68	0.68 (0.12) 0.66	0.96 0.86

	Dominant Modela			Recessive Modela
C6776 Polymorphism	CC	CC and TT	p Valueb	CC and CT	TT	p Valueb
Number	47	77	–	110	14	–
BMD L2-L4 (g/cm2)c • Adjustedd	0.94 (0.22) 0.94	0.96 (0.17) 0.96	0.42 0.42	0.95 (0.20) 0.96	0.97 (0.12) 0.93	0.71 0.64
BMD femoral (g/cm2)c • Adjustedd	0.81 (0.14) 0.82	0.86 (0.13) 0.86	0.07 0.05	0.84 (0.14) 0.84	0.89 (0.13) 0.86	0.18 0.49
Ward’s Triangle (g/cm2)c • Adjustedd	0.60 (0.16) 0.61	0.64 (0.14) 0.64	0.14 0.22	0.62 (0.15) 0.63	0.68 (0.09) 0.63	0.29 0.87
Trochanter (g/cm2)c • Adjustedd	0.65 (0.14) 0.65	0.70 (0.11) 0.70	0.07 0.03	0.67 (0.13) 0.67	0.74 (0.08) 0.71	0.12 0.33

a Mean BMD compared between genotype groups assuming hypothesis: A) dominant C allele; B) recessive C allele.

b p Values obtained by unpaired independent sample t-test (two-tailed).

c Values are means ± SD in parenthesis.

d Values are adjusted for age, BMI, YSM; 95% CI given by univariate analysis (not shown).

Table 6. Genotype and haplotype frequencies (%) in normal postmenopausal women and women having a low bone mineral density.

	Normal	Low BMD	p Valuea
COL1A1 Sp1 • SS • Ss • ss	46.9 42.9 10.2	36.6 50.7 12.7	0.53
COL1A1 –1997 G→T • TT • TG • GG	10.0 36.0 54.0	5.6 33.8 60.6	0.60
MTHFR • CC • CT • TT	33.3 54.9 11.8	41.1 47.9 11.0	0.68
COL1A1 Haplotype • T/S • G/S • T/s • G/s	25.6 42.1 1.5 30.8	21.0 40.6 2.2 36.2	0.76

a p Values obtained by the chi-squared test.