Metabolic Abnormalities in Williams-Beuren Syndrome
Metabolic Abnormalities in Williams-Beuren Syndrome
Significant differences with respect to the expected values were observed for the following biochemical and hormonal parameters: TSH, glucose, triglyceride, cholesterol, TB, direct bilirubin (DB), indirect bilirubin, transferrin, and total protein and albumin levels (Table 1 and see online supplementary table S1 http://jmg.bmj.com/content/52/4/248/suppl/DC1).
Subclinical Hypothyroidism. Subclinical hypothyroidism (defined by mild TSH elevation with normal T3/T4 levels) was present in 31.3% of patients (26/83), with no gender differences. The values followed a bimodal distribution, with a second peak at the 97.5th centile (see online supplementary figure S2 http://jmg.bmj.com/content/52/4/248/suppl/DC1). There were 20 patients with values above 2.5 SDs. When compared by age, these patients were significantly younger (9.35±7.22 years) than the rest of the cohort with TSH values available (15.02±9.94 years) (p=0.021). There was a significant correlation between age and TSH values (r(81)=−0.356, p=0.001) and z-score value (r(81)=−0.356, p=0.001). Increased TSH levels occurred significantly more frequently in children than in adults with WBS (see online supplementary table S3 http://jmg.bmj.com/content/52/4/248/suppl/DC1). Three patients with congenital hypothyroidism and one with autoimmune thyroiditis were excluded from the analysis.
Hyperglycaemia. Basal glucose plasma levels were increased above the 97.5th centile in 7.3% of patients (11/151), 9 children (8%) and 2 adults (5.3%) (Table 1). Only one of these patients had a diagnosis of diabetes mellitus type 1. The histogram of glucose centiles followed a normal distribution, with a small peak at 95–97.5th centiles (see online supplementary figure S3 http://jmg.bmj.com/content/52/4/248/suppl/DC1).
Hypotriglyceridemia and lipid profile. Triglyceride plasma levels were decreased in individuals with WBS; the values were arranged in a bimodal distribution with a first peak at the 2.5–5th centile and a second at the 25–50th centile (figure 1A). Specifically, 18.3% (21/115) of patients with WBS had levels below the 5th centile. There was no relationship between triglyceride levels and body mass index. Only 2.6% of patients (3/115) had hypertriglyceridemia and two of them presented associated hypothyroidism. The mean z-score value for triglyceride levels in our cohort of patients with WBS was below the reference interval mean (−0.146±0.887) (Table 1). The distribution of the z-score values followed a normal distribution with a shift towards the left (figure 1B).
(Enlarge Image)
Figure 1.
(A) Histogram of triglyceride plasma levels in centiles in individuals with Williams–Beuren syndrome. (B) Histogram of the distribution of triglyceride z-score values.
Cholesterol levels followed a bimodal distribution when analysed by centiles or z-score values (see online supplementary figure S4 http://jmg.bmj.com/content/52/4/248/suppl/DC1). Only one patient had total cholesterol values above the 97.5th centile. Cholesterol levels were below the 5th centile in 12.4% (16/129) of patients.
Hyperbilirubinemia. TB levels were increased in 20.2% (18/89) of patients with WBS. Two previously described patients with portal hypertension were excluded from the analysis, as well as a patient diagnosed with beta-thalassaemia minor. TB levels followed a bimodal distribution, with a second peak at the 97.5th centile and a median between the 25th and 50th centiles (figure 2). There were 10 individuals with values above 3 SD (z-score range 3.5–15.78), with equal gender distribution. Indirect hyperbilirubinemia was present in 34% (18/53) of individuals with WBS and the distribution was also bimodal, while DB levels were only increased in 7.5% (4/53) of WBS cases. There were also five individuals with normal TB levels and an increase of either indirect bilirubin (IB) or DB alone. Hepatic function parameters (aspartate aminotransferase (AST), alanine transaminase (ALT), gamma glutamyl transpeptidase (gGT)) were normal in all cases with hyperbilirubinemia but one, who had a slight increase in AST and ALT but normal gGT levels. Out of the 14 individuals with hyperbilirubinemia and fractionated bilirubin levels available, unconjugated hyperbilirubinemia (DB/TB ratio <30%) was present in 28.6% (4/14) while mixed hyperbilirubinemia (DB/TB ratio 30–70%) was found in the remaining 71.4% (10/14). None presented a conjugated hyperbilirubinemia (DB/TB ratio >70%). Individuals with hyperbilirubinemia were significantly older than those with normal bilirubin levels (20.21±10.31 years vs 11.82±8.40 years, respectively. p=0.001). TB and IB levels were more frequently increased in adults compared with children (see online supplementary table S3 http://jmg.bmj.com/content/52/4/248/suppl/DC1).
(Enlarge Image)
Figure 2.
(A) Histogram of total bilirubin plasma levels in centiles in individuals with Williams–Beuren syndrome. (B) Histogram of total bilirubin z-score values.
Abnormal Protein and Iron Levels. A high proportion of individuals with WBS displayed elevated total protein (27.3%) and elevated albumin levels (11%). Only 7 of the 22 cases with hyperproteinemia and albumin levels available had an elevation of this parameter. Total protein levels followed a bimodal distribution with a second peak at 97.5th centile.
We also found alterations in iron metabolism, with 21.4% (15/70) of individuals showing iron concentrations above the 97.5th centile. Haemoglobin and haematocrit levels were normal in all cases. Iron concentration had a bimodal distribution with a first peak at the 25–50th centile and a second at >p97.5th centile. Iron z-score values followed a normal distribution with a shift towards the right.
TSH z-score values correlated significantly with TB z-score values in an inverse manner (r(66)=−0.328, p=0.006). Triglyceride z-score value correlated significantly with TB z-score (r(78)=−0.220, p=0.050) and with total protein z-score value (r(79)=−0.321, p=0.003) in an inverse manner. Cholesterol z-score values also correlated with DB z-score values (r(51)=−0.436, p=0.001). As expected, total protein z-score values correlated significantly with albumin z-score values (r(71)=0.666, p<0.001). We did not find any correlation of the biochemical alterations detected and molecular data at 7q11.23, either the size or the parental origin of the deletion.
In order to test whether genetic variants at the non-deleted allele might be responsible for the variation in the lipid profile of individuals with WBS, we genotyped an SNP of the MLXIPL gene previously associated with hypotriglyceridemia. We compared allelic frequencies of individuals with WBS with triglyceride values below the 5th centile (n=21) with those with triglyceride values above the 50th centile that were considered controls (n=24). No significant differences were found between cases and controls (see online supplementary table S4 http://jmg.bmj.com/content/52/4/248/suppl/DC1).
We also tested whether the hyperbilirubinemia observed in patients with WBS was related to known genetic susceptibility factors: a dinucleotide repeat polymorphism at the UGT1A1 promoter and an SNP at the SLCO1B1 gene. The UGT1A1 polymorphism was studied in 79 patients with WBS with bilirubin values available and 94 Spanish population controls (no bilirubin levels available). Allelic frequencies were not significantly different in individuals with WBS and controls (see online supplementary table S5 http://jmg.bmj.com/content/52/4/248/suppl/DC1). UGT1A1 genotype was significantly associated with elevated plasma concentrations (>2.5SD) of TB (p<0.001) and IB (p<0.001) in WBS (DB (p=0.055)). The penetrance of hyperbilirubinemia in individuals with WBS homozygous for the UGT1A1-promoter allele [(TA)7] was of 72.7%. Hyperbilirubinemia was present in 13.3% of [(TA)6/(TA)7] heterozygous individuals with WBS, either unconjugated or mixed hyperbilirubinemia, and 13.2% of [(TA)6] homozygous individuals with WBS, all with unconjugated hyperbilirubinemia (figure 3). Although there was a significant correlation between UGT1A1 genotypes and bilirubin levels, specifically increased bilirubin in patients homozygous for [(TA)7], there was a great variability of bilirubin levels among individuals with the same UGT1A1 genotype. However, no significant differences in the allelic frequencies of the SLCO1B1 SNP genotypes were observed in WBS with respect to bilirubin levels.
(Enlarge Image)
Figure 3.
Total bilirubin (TB), direct bilirubin (DB) and IB z-score values with respect to UGT1A1 genotype (mean±SD). Significant differences were found between genotypes with respect to TB (F(2,76)=6.412, p=0.003), DB (F(2,49)=5.497, p=0.007) and IB (F(2,49)=4.397, p=0.018).
TSH was measured in all mouse models at 25 weeks of age (Table 2). Only PD mice had a significant increase in TSH levels compared with WT (p=0.004). Basal blood glucose level at 25 weeks and the intraperitoneal glucose tolerance test in 11-week-old mice showed no significant differences between WT animals and deletion mouse models (figure 4A). A morphological analysis of the pancreatic Langerhans islets revealed that CD mice had a higher percentage of smaller islets compared with the WT (66% in CD vs 48.8% in WT) and less bigger islets (0.51% in CD vs 6.51% in WT) (figure 4B).
(Enlarge Image)
Figure 4.
(A) Intraperitoneal glucose tolerance test. Results represent mean±SD (n=8–16). (B) Langerhans islets area analysis. Results represent mean±SD (n=3–6).
Triglyceride plasma concentration was significantly decreased only in the DD and PD mice at 4.5 weeks of age compared with WT (p=0.003 and 0.005, respectively), a decrement that was milder and non-significant at 26 weeks of age (Table 2). Total cholesterol, high-density lipoprotein and low-density lipoprotein levels were not significantly different in any group of animals at any age (Table 2 and see online supplementary table S6 http://jmg.bmj.com/content/52/4/248/suppl/DC1).
An anthropometric analysis was done at 25 weeks in mice; whole body, liver, perirenal and gonadal fat weight were recorded (see online supplementary table S7 http://jmg.bmj.com/content/52/4/248/suppl/DC1). PD mice had significant increases in total body weight (p=0.018), liver weight with respect to total body weight (p=0.002) and total fat (p=0.015) compared with WT.
Total protein levels were measured in mice at 25 weeks (Table 2). PD mice had increased concentration compared with WT, although it did not reach statistical significance after Bonferroni's correction.
Bilirubin levels were also studied at 4.5 and 25 weeks in mice with no significant differences in absolute values between groups of animals. WT mice had almost the same TB values at both time points, although a relative increase of DB and decrease of IB was observed at 25 weeks, resulting in increased DB to TB ratio (DB/TB) (41.9%±12.1% vs 61.2%±22.3%). In the case of DD mice, TB decreased at 25 weeks maintaining a similar DB/TB ratio at both time points (31.7%±10.8% vs 38.4%±16.6%). For PD mice, all values increased at 25 weeks except the DB/TB ratio that was similar at both time points (44.2%±14.3% vs 41.9%±12.1%).
In order to test for a putative differential compensatory regulation of the Ugt1a1 gene in the different mice, we measured its relative expression in RNA isolated from mice liver by qRT-PCR. No differences were found with respect to Ugt1a1 expression levels between the four mouse genotypes studied (see online supplementary figure S5 http://jmg.bmj.com/content/52/4/248/suppl/DC1).
Results
Metabolic Disturbances in Individuals With WBS
Significant differences with respect to the expected values were observed for the following biochemical and hormonal parameters: TSH, glucose, triglyceride, cholesterol, TB, direct bilirubin (DB), indirect bilirubin, transferrin, and total protein and albumin levels (Table 1 and see online supplementary table S1 http://jmg.bmj.com/content/52/4/248/suppl/DC1).
Subclinical Hypothyroidism. Subclinical hypothyroidism (defined by mild TSH elevation with normal T3/T4 levels) was present in 31.3% of patients (26/83), with no gender differences. The values followed a bimodal distribution, with a second peak at the 97.5th centile (see online supplementary figure S2 http://jmg.bmj.com/content/52/4/248/suppl/DC1). There were 20 patients with values above 2.5 SDs. When compared by age, these patients were significantly younger (9.35±7.22 years) than the rest of the cohort with TSH values available (15.02±9.94 years) (p=0.021). There was a significant correlation between age and TSH values (r(81)=−0.356, p=0.001) and z-score value (r(81)=−0.356, p=0.001). Increased TSH levels occurred significantly more frequently in children than in adults with WBS (see online supplementary table S3 http://jmg.bmj.com/content/52/4/248/suppl/DC1). Three patients with congenital hypothyroidism and one with autoimmune thyroiditis were excluded from the analysis.
Hyperglycaemia. Basal glucose plasma levels were increased above the 97.5th centile in 7.3% of patients (11/151), 9 children (8%) and 2 adults (5.3%) (Table 1). Only one of these patients had a diagnosis of diabetes mellitus type 1. The histogram of glucose centiles followed a normal distribution, with a small peak at 95–97.5th centiles (see online supplementary figure S3 http://jmg.bmj.com/content/52/4/248/suppl/DC1).
Hypotriglyceridemia and lipid profile. Triglyceride plasma levels were decreased in individuals with WBS; the values were arranged in a bimodal distribution with a first peak at the 2.5–5th centile and a second at the 25–50th centile (figure 1A). Specifically, 18.3% (21/115) of patients with WBS had levels below the 5th centile. There was no relationship between triglyceride levels and body mass index. Only 2.6% of patients (3/115) had hypertriglyceridemia and two of them presented associated hypothyroidism. The mean z-score value for triglyceride levels in our cohort of patients with WBS was below the reference interval mean (−0.146±0.887) (Table 1). The distribution of the z-score values followed a normal distribution with a shift towards the left (figure 1B).
(Enlarge Image)
Figure 1.
(A) Histogram of triglyceride plasma levels in centiles in individuals with Williams–Beuren syndrome. (B) Histogram of the distribution of triglyceride z-score values.
Cholesterol levels followed a bimodal distribution when analysed by centiles or z-score values (see online supplementary figure S4 http://jmg.bmj.com/content/52/4/248/suppl/DC1). Only one patient had total cholesterol values above the 97.5th centile. Cholesterol levels were below the 5th centile in 12.4% (16/129) of patients.
Hyperbilirubinemia. TB levels were increased in 20.2% (18/89) of patients with WBS. Two previously described patients with portal hypertension were excluded from the analysis, as well as a patient diagnosed with beta-thalassaemia minor. TB levels followed a bimodal distribution, with a second peak at the 97.5th centile and a median between the 25th and 50th centiles (figure 2). There were 10 individuals with values above 3 SD (z-score range 3.5–15.78), with equal gender distribution. Indirect hyperbilirubinemia was present in 34% (18/53) of individuals with WBS and the distribution was also bimodal, while DB levels were only increased in 7.5% (4/53) of WBS cases. There were also five individuals with normal TB levels and an increase of either indirect bilirubin (IB) or DB alone. Hepatic function parameters (aspartate aminotransferase (AST), alanine transaminase (ALT), gamma glutamyl transpeptidase (gGT)) were normal in all cases with hyperbilirubinemia but one, who had a slight increase in AST and ALT but normal gGT levels. Out of the 14 individuals with hyperbilirubinemia and fractionated bilirubin levels available, unconjugated hyperbilirubinemia (DB/TB ratio <30%) was present in 28.6% (4/14) while mixed hyperbilirubinemia (DB/TB ratio 30–70%) was found in the remaining 71.4% (10/14). None presented a conjugated hyperbilirubinemia (DB/TB ratio >70%). Individuals with hyperbilirubinemia were significantly older than those with normal bilirubin levels (20.21±10.31 years vs 11.82±8.40 years, respectively. p=0.001). TB and IB levels were more frequently increased in adults compared with children (see online supplementary table S3 http://jmg.bmj.com/content/52/4/248/suppl/DC1).
(Enlarge Image)
Figure 2.
(A) Histogram of total bilirubin plasma levels in centiles in individuals with Williams–Beuren syndrome. (B) Histogram of total bilirubin z-score values.
Abnormal Protein and Iron Levels. A high proportion of individuals with WBS displayed elevated total protein (27.3%) and elevated albumin levels (11%). Only 7 of the 22 cases with hyperproteinemia and albumin levels available had an elevation of this parameter. Total protein levels followed a bimodal distribution with a second peak at 97.5th centile.
We also found alterations in iron metabolism, with 21.4% (15/70) of individuals showing iron concentrations above the 97.5th centile. Haemoglobin and haematocrit levels were normal in all cases. Iron concentration had a bimodal distribution with a first peak at the 25–50th centile and a second at >p97.5th centile. Iron z-score values followed a normal distribution with a shift towards the right.
Correlations Between Metabolic Disturbances
TSH z-score values correlated significantly with TB z-score values in an inverse manner (r(66)=−0.328, p=0.006). Triglyceride z-score value correlated significantly with TB z-score (r(78)=−0.220, p=0.050) and with total protein z-score value (r(79)=−0.321, p=0.003) in an inverse manner. Cholesterol z-score values also correlated with DB z-score values (r(51)=−0.436, p=0.001). As expected, total protein z-score values correlated significantly with albumin z-score values (r(71)=0.666, p<0.001). We did not find any correlation of the biochemical alterations detected and molecular data at 7q11.23, either the size or the parental origin of the deletion.
Genetic Modifiers of the Lipid and Bilirubin Disturbances
In order to test whether genetic variants at the non-deleted allele might be responsible for the variation in the lipid profile of individuals with WBS, we genotyped an SNP of the MLXIPL gene previously associated with hypotriglyceridemia. We compared allelic frequencies of individuals with WBS with triglyceride values below the 5th centile (n=21) with those with triglyceride values above the 50th centile that were considered controls (n=24). No significant differences were found between cases and controls (see online supplementary table S4 http://jmg.bmj.com/content/52/4/248/suppl/DC1).
We also tested whether the hyperbilirubinemia observed in patients with WBS was related to known genetic susceptibility factors: a dinucleotide repeat polymorphism at the UGT1A1 promoter and an SNP at the SLCO1B1 gene. The UGT1A1 polymorphism was studied in 79 patients with WBS with bilirubin values available and 94 Spanish population controls (no bilirubin levels available). Allelic frequencies were not significantly different in individuals with WBS and controls (see online supplementary table S5 http://jmg.bmj.com/content/52/4/248/suppl/DC1). UGT1A1 genotype was significantly associated with elevated plasma concentrations (>2.5SD) of TB (p<0.001) and IB (p<0.001) in WBS (DB (p=0.055)). The penetrance of hyperbilirubinemia in individuals with WBS homozygous for the UGT1A1-promoter allele [(TA)7] was of 72.7%. Hyperbilirubinemia was present in 13.3% of [(TA)6/(TA)7] heterozygous individuals with WBS, either unconjugated or mixed hyperbilirubinemia, and 13.2% of [(TA)6] homozygous individuals with WBS, all with unconjugated hyperbilirubinemia (figure 3). Although there was a significant correlation between UGT1A1 genotypes and bilirubin levels, specifically increased bilirubin in patients homozygous for [(TA)7], there was a great variability of bilirubin levels among individuals with the same UGT1A1 genotype. However, no significant differences in the allelic frequencies of the SLCO1B1 SNP genotypes were observed in WBS with respect to bilirubin levels.
(Enlarge Image)
Figure 3.
Total bilirubin (TB), direct bilirubin (DB) and IB z-score values with respect to UGT1A1 genotype (mean±SD). Significant differences were found between genotypes with respect to TB (F(2,76)=6.412, p=0.003), DB (F(2,49)=5.497, p=0.007) and IB (F(2,49)=4.397, p=0.018).
Metabolic Disturbances in WBS Mouse Models
TSH was measured in all mouse models at 25 weeks of age (Table 2). Only PD mice had a significant increase in TSH levels compared with WT (p=0.004). Basal blood glucose level at 25 weeks and the intraperitoneal glucose tolerance test in 11-week-old mice showed no significant differences between WT animals and deletion mouse models (figure 4A). A morphological analysis of the pancreatic Langerhans islets revealed that CD mice had a higher percentage of smaller islets compared with the WT (66% in CD vs 48.8% in WT) and less bigger islets (0.51% in CD vs 6.51% in WT) (figure 4B).
(Enlarge Image)
Figure 4.
(A) Intraperitoneal glucose tolerance test. Results represent mean±SD (n=8–16). (B) Langerhans islets area analysis. Results represent mean±SD (n=3–6).
Triglyceride plasma concentration was significantly decreased only in the DD and PD mice at 4.5 weeks of age compared with WT (p=0.003 and 0.005, respectively), a decrement that was milder and non-significant at 26 weeks of age (Table 2). Total cholesterol, high-density lipoprotein and low-density lipoprotein levels were not significantly different in any group of animals at any age (Table 2 and see online supplementary table S6 http://jmg.bmj.com/content/52/4/248/suppl/DC1).
An anthropometric analysis was done at 25 weeks in mice; whole body, liver, perirenal and gonadal fat weight were recorded (see online supplementary table S7 http://jmg.bmj.com/content/52/4/248/suppl/DC1). PD mice had significant increases in total body weight (p=0.018), liver weight with respect to total body weight (p=0.002) and total fat (p=0.015) compared with WT.
Total protein levels were measured in mice at 25 weeks (Table 2). PD mice had increased concentration compared with WT, although it did not reach statistical significance after Bonferroni's correction.
Bilirubin levels were also studied at 4.5 and 25 weeks in mice with no significant differences in absolute values between groups of animals. WT mice had almost the same TB values at both time points, although a relative increase of DB and decrease of IB was observed at 25 weeks, resulting in increased DB to TB ratio (DB/TB) (41.9%±12.1% vs 61.2%±22.3%). In the case of DD mice, TB decreased at 25 weeks maintaining a similar DB/TB ratio at both time points (31.7%±10.8% vs 38.4%±16.6%). For PD mice, all values increased at 25 weeks except the DB/TB ratio that was similar at both time points (44.2%±14.3% vs 41.9%±12.1%).
In order to test for a putative differential compensatory regulation of the Ugt1a1 gene in the different mice, we measured its relative expression in RNA isolated from mice liver by qRT-PCR. No differences were found with respect to Ugt1a1 expression levels between the four mouse genotypes studied (see online supplementary figure S5 http://jmg.bmj.com/content/52/4/248/suppl/DC1).