Reduced bone density in cystic fibrosis: ΔF508 mutation is an independent risk factor

Study subjects and design

The present study was a prospective cross-sectional study of adults with CF, which comprised a randomly selected sample of ∼50% of the population of 174 adults who had not undergone transplantation attending the regional Adult Cystic Fibrosis Service at the Alfred Hospital, Melbourne, Australia. The aim was to obtain a large study sample that was representative of an adult CF population. The sex, age, forced expiratory volume in one second (FEV1) as a percentage of the predicted value, CF genotype, height and pancreatic status of all 174 patients were recorded. The study population of 88 patients showed similar genotypic and phenotypic characteristics to the non-studied population of 86 patients, confirming that the study population was representative of the total clinic population.

The diagnosis of CF had been made previously, using a sweat chloride test and on the basis of an appropriate CF phenotype. CF patients were excluded from the study if they were already receiving treatment for reduced BMD, pregnant or unable to complete the study requirements. Patients (88, all Caucasian, 53 males) aged 19–59 yrs were enrolled in the study between May 2000 and April 2001. Twenty healthy volunteers of similar age to the CF subjects were also recruited, to provide a blood sample. The study was approved by the Alfred Hospital Institutional Ethics Committee and written informed consent was obtained prior to participation.

Clinical characteristics: cystic fibrosis subjects

Clinical characteristic information was collected from the subject's medical record and at interview (table 1⇓). In the present study, pancreatic exocrine status was defined as pancreatic insufficient or pancreatic sufficient according to whether or not pancreatic enzyme replacement therapy was required to control maldigestion, malabsorption or otherwise unexplained nutritional failure, and was reviewed on a periodic basis at routine outpatient clinic consultations.

The median estimated cumulative oral/intravenous corticosteroid dose in prednisolone equivalents was 6,007 mg over a mean of 31.2 patient-yrs. The median age of menarche in females was 14 yrs (range 10–17 yrs). Ten (11%) subjects received ≥400 IU vitamin D daily from supplements. Thirty-five (40%) subjects reported that they had previously sustained one or more bone fractures. The total number of fractures reported was 74. All fractures were associated with significant trauma.

Lung function was assessed by measuring FEV1 using a Lilly pneumotachometer attached to a Jaeger spirometer (Master screen pneumo, version 4.0; Jaeger, Würzburg, Germany). The best lung function in the 3 months prior to the measurement of BMD was recorded. Current height and weight were used to calculate body mass index (BMI) (in kilograms per metre squared) and FEV1 (% pred) using the prediction equations of Knudson et al. 18.

Physical activity was assessed using the Baecke Questionnaire of Habitual Physical Activity 19. Each type of activity is scored 1–5, and the total score for the questionnaire ranges 3–15. Dietary intake was assessed by a trained dietitian using a 4-day household measures food record. Mean daily intakes from the diet and supplements of energy and calcium were estimated using a computerised dietary analysis program (Foodworks, version 2.10; Xyris Software Australia, Brisbane, Australia).

Radiographic assessment and bone mineral density and body composition measurements: cystic fibrosis subjects

Subjects underwent BMD and body composition measurement using dual-energy X-ray absorptiometry scanning (DPX-IQ, version 4.7e; Lunar Radiation Corporation, Madison, WI, USA) of the: 1) whole body, to determine fat mass, fat-free mass and whole-body BMD; and 2) lumbar spine (LS) and femoral neck (FN), to determine their BMDs.

BMDs were expressed as T- and Z-scores. These were calculated using the population reference data provided by the manufacturer (Lunar Radiation Corporation), which were derived from BMD measurements in >4,000 Caucasian adults aged 20–79 yrs from a range of countries. T-scores were calculated by subtracting the sex-specific population mean BMD for young adults (aged 20–40 yrs) from the CF subject's BMD, and this value was divided by the sd of the sex-specific young adult mean. Z-scores were calculated by subtracting the sex- and age-specific population mean BMD from the CF subject's BMD, and this value was divided by the sd of the sex- and age-specific mean. Lateral spinal films of subjects were taken and assessed for osteoporotic fractures using standard radiological criteria 20.

Biochemical measurements

Statistical analysis

Data were analysed for normality, and, where appropriate, underwent logarithmic transformation. Data were summarised as mean±sd or median (interquartile range) as appropriate. Chi-squared or Fisher's exact tests were performed in order to identify differences in categorical variables between subgroups. Unpaired t-tests or Mann–Whitney U-tests were performed to compare means or medians. Univariate analyses using Pearson's correlations, Spearman's correlations and one-way ANOVA were undertaken to assess correlation between variables. The influence of the CF genotype was analysed using three categories: homozygous for ΔF508, heterozygous for ΔF508, and no ΔF508 allele. Oral/intravenous corticosteroid use was analysed as a binomial variable due to the potential for recall and information bias in the estimation of cumulative prednisolone equivalent dose. Multiple linear regression was performed in order to identify which variables were significantly and independently associated with LS and FN BMD Z-scores. This analysis was performed using data from the 85 subjects who had undergone genotype testing. It was undertaken using a backward elimination technique incorporating all variables except those excluded below, in order to incorporate significant correlates in this and other studies, and to reduce the impact of occult confounding in the univariate analyses. Sex-specific variables (age of menarche in females and testosterone level in males) were excluded, as was weight, whose effect was incorporated within other variables (fat-free mass and fat mass), physical activity and dietary intake, for which the data were incomplete and for which there were no correlations with outcome variables on univariate analyses. The largest possible patient group was modelled at each step and the least significant variables eliminated. Regression models were further validated using a stepwise selection approach. The significance level was taken as a p-value of <0.05.

Clinical measurements: cystic fibrosis subjects

Table 2⇓ shows CF subject demographics, dietary intake and physical activity results.

Lateral LS films were taken for 75 subjects. Of these, four (5%) met the radiological criteria for osteoporotic fractures 20.

Biochemical results

The biochemical results for CF subjects are shown in table 3⇓. There was a trend towards higher 25-hydroxyvitamin D levels in subjects studied in the summer months compared to those studied in the winter months (October–March 71 nM (median) versus April–September 52 nM; p<0.05 (Mann–Whitney test)). A low 25-hydroxyvitamin D level did not correlate with pancreatic status (p = 0.25).

IL-1β was not detected in any CF subject or healthy volunteer (<5 pg·ml⁻¹). IL-6 was not detected in 15 (75%) of the volunteers (<5 pg·ml⁻¹). TNF-α was not detected in 10 (50%) of the volunteers (<4 pg·ml⁻¹). The lower 95% ranges for IL-6 and TNF-α in volunteers were taken as the reference ranges, the upper limits of which were 10.4 and 11.7 pg·ml⁻¹ respectively. TNF-α was not detected in 29 (36%) of the CF subjects, whereas IL-6 was not detected in 39 (48%) of the CF subjects. IL-6 levels were above the reference range in 19 (23%) of the CF subjects, whereas TNF-α levels were elevated in five (6%) of the CF subjects. IL-6 and TNF-α levels did not correlate in the 28 CF subjects in whom both were detected.

Bone mineral density measurements

Results of the BMD and body composition measurements are shown in table 4⇓. Thirty-seven (42%) subjects had a BMD Z-score of <−1 at one or more of the three sites tested, with 15 (17%) having a BMD Z-score of <−2. The prevalence of osteoporotic T-scores (<−2.5) was 15%, with a further 42% of subjects having a T-score ranging −1.0– −2.5 at one or more sites. Compared to females, males had significantly lower BMD Z-scores at all sites, indicating that males exhibited greater deficits in bone mineral relative to the age-matched normal population.

Correlates of bone mineral density

Mean BMD Z-scores at the LS and FN were significantly lower in subjects who were homozygous (−0.67±1.33 and −0.37±1.24, respectively) or heterozygous (−0.73±1.16 and −0.44±1.03, respectively) for the ΔF508 mutation, compared to those with no ΔF508 allele (0.43±1.26 and 0.80±1.26, respectively; p<0.05 (one-way ANOVA)) (fig. 1⇓).

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Fig. 1—

Bone mineral density (BMD) Z-scores at the lumbar spine (□) and femoral neck (▓) in adult cystic fibrosis (CF) patients for each CF genotype category (homozygous: n = 39; heterozygous: n = 36; absent: n = 10). Boxes represent the median and interquartile range, and whiskers the 5th and 95th percentiles. *: p<0.05 (one-way ANOVA).

Correlation of all variables significantly associated with LS and/or FN BMD Z-score are shown in tables 5⇓ and 6⇓, as is the correlation of age with BMD. There was no significant difference in mean BMD Z-scores in subjects who were pancreatic insufficient compared with those who were pancreatic sufficient, at either the LS (−0.58±1.33 and −0.61±1.14, respectively; p = 0.94) or the FN (−0.23±1.24 and −0.29±0.89, respectively; p = 0.88). Age, serum levels of calcium, phosphate, parathyroid hormone, 25-hydroxyvitamin D, osteocalcin, TNF-α, IL-6, testosterone in males and oestradiol in females, physical activity score, and dietary intakes of energy and calcium were not associated with either LS or FN BMD Z-score. The physical activity score did not correlate significantly with disease severity, as measured by FEV1 (% pred).

Multiple linear regression analysis of associations with lumbar spine and femoral neck bone mineral density Z-scores

Multiple linear regression analysis showed that the most significant independent predictors of lower LS BMD Z-scores were presence of the ΔF508 CF genotype (homozygote or heterozygote), malnutrition as indicated by the requirement for supplementary enteral nutrition, male sex, lower serum phosphate level, lower serum 25-hydroxyvitamin D level and higher fat mass (table 7⇓). For FN BMD Z-score, the best predictors were male sex, lower fat-free mass, ΔF508 genotype and greater disease severity as measured by FEV1 (% pred) (table 7⇓). The significant variables identified accounted for 44% of the variability in LS BMD Z-score and 34% of the that in FN BMD Z-score.

Further analysis showed that CF genotype was not associated with sex (Chi-squared test), and that there were no significant differences in age, BMI, or serum 25-hydroxyvitamin D, osteocalcin, parathyroid hormone or urinary N-telopeptide level across the three genotype categories (one-way ANOVA). There was no association between CF genotype and FEV1 (% pred), even when the effect of age was accounted for. However, pancreatic status and genotype were associated, with 100% of the subjects who were homozygous for ΔF508 being pancreatic insufficient, compared to 78% for those who were heterozygous for ΔF508 and 90% for those who had no ΔF508 allele (p<0.01; Chi-squared test).