Study subjects. The main clinical and biochemical characteristics of both studied groups (162 FCHL subjects and 165 normolipemic controls) are presented in Table 1. FCHL subjects showed higher predominance of males (60.5%) and were significantly older than normolipemic subjects (P = 0.022 and P < 0.001, respectively). Compared with normolipemic controls, FCHL subjects had significantly higher values of BMI, TC, TG, LDLc, apoB and lipoprotein(a) (P < 0.001, P < 0.001, P < 0.001, P < 0.001, P < 0.001 and P = 0.003, respectively). FCHL subjects presented higher prevalence of hypertension, type 2 diabetes and CVD than normolipemic subjects (P = 0.009, P = 0.001 and P = 0.016, respectively). The APOE genotype distribution was homogenous between both cohorts, being E3/3 genotype the most frequent in both groups, although E3/2 genotype had a lower frequency in FCHL subjects (5.56%) in contrast to normolipemic subjects (15.2%).
Table 1
Clinical and biochemical characteristics in FCHL subjects and normolipemic controls.
|
FCHL subjects
n = 162
|
Normolipemic controls
n = 165
|
p
|
Men, n (%)
|
98 (60.5)
|
78 (47.3)
|
0.022
|
Age, years
|
50.4 ± 11.4
|
38.5 ± 14.7
|
< 0.001
|
Body Mass Index, Kg/m2
|
25.6 (24.2–26.5)
|
23.6 (21.4–26.6)
|
< 0.001
|
Total Cholesterol, mg/dL
|
312 ± 36.1
|
170 ± 21.0
|
< 0.001
|
Triglycerides, mg/dL
|
277 (232–373)
|
64.0 (49.0–93.0)
|
< 0.001
|
LDL cholesterol, mg/dL
|
204 (183–230)
|
108 (91.8–117)
|
< 0.001
|
HDL cholesterol, mg/dL
|
48.5 ± 12.0
|
55.7 ± 11.4
|
0.015
|
Apolipoprotein A1, mg/dL
|
147 ± 25.0
|
147 ± 27.4
|
0.930
|
Apolipoprotein B, mg/dL
|
167 (165–190)
|
83.0 (72.0–91.0
|
< 0.001
|
Lipoprotein(a), mg/dL
|
39.1 (10.3–80.8)
|
16.2 (7.79–44.5)
|
0.003
|
Glucose, mg/dL
|
93.0 (86.0-103)
|
85.0 (80.0–92.0)
|
< 0.001
|
HbA1c, %
|
5.50 (5.30–5.80)
|
5.20 (5.00-5.40)
|
< 0.001
|
Type 2 diabetes, n (%)
|
13 (8.02)
|
2 (1.21)
|
0.009
|
Hypertension, n (%)
|
30 (18.5)
|
10 (6.06)
|
0.001
|
Cardiovascular disease, n (%)
|
7 (4.32)
|
0
|
0.016
|
Tobacco,
n (%)
|
Non smoker
|
51 (31.5)
|
96 (58.1)
|
< 0.001
|
Smoker
|
70 (43.2)
|
31 (18.8)
|
Former smoker
|
40 (24.7)
|
28 (16.7)
|
Apolipoprotein E genotype,
n (%)
|
E3/3
|
113 (69.8)
|
109 (66.1)
|
0.035
|
E3/2
|
9 (5.56)
|
25 (15.2)
|
E2/2
|
0
|
0
|
E3/4
|
31 (19.1)
|
25 (15.2)
|
E4/4
|
6 (3.70)
|
2 (1.21)
|
E2/4
|
3
|
4
|
ANGPTL3 genetic variants. Table 2 shows all variants in the ANGPTL3 gene identified in both groups. A total of 16 genetic variants, four of them not previously described, were identified by sequencing analysis. Only four of them (c.607-109T > C, c.607 − 47_607-46delGT, c.835 + 41C > A and c.*52_*60del) presented significantly different allele frequency in normolipemic group than in FCHL subjects (P = 0.020, P = 0.031, P = 0.043 and P < 0.001, respectively). Out of the 16 variants, seven variants were located in the coding region (c.379 C > T, c.565T > C, c.961T > A, c.1003T > C, c.1028A > G, c.1089T > G and c.1122G > A), three of them were missense variants: p. (Leu127Phe), p.(Tyr321Asn) and p.(His343Arg), but only p.(Leu127Phe) was described as deleterious by bioinformatics analysis. The other four variants located in the coding region, p.(Leu189Leu), p.(Leu335Leu), p.(Val363Val) and p.(Pro374Pro) were synonymous variants. Seven variants were located in the intronic region, c.496-88T > G, c.607-120A > G, c.607-109T > C, c.607 − 47_607-46delGT, c.835 + 41C > A, c.1198 + 111G > A and c.1198 + 140T > C. All of them were described as benign or not splicing change affected by the bioinformatics analysis. Nevertheless, three of them, c.607-109T > C, c.607 − 47_607-46delGT and c.835 + 41C > A, presented significantly higher allele frequency in FCHL subjects than in the normolipemic group. Finally, two variants were located in the 3’UTR, c.*52_*60del and c.*76 T > G. One of them, c.*52_*60del, showed significantly higher allele frequency in the normolipemic group than in FCHL subjects.
Table 2
Frequency and bioinformatics clinical significance of identified variants in ANGPTL3 in FCHL cases and controls.
Variant
|
Location
|
Nucleotide change
|
Protein change
|
Bioinformatics analysis
|
Allele frequency in the general population
|
Allele frequency in our study
|
ACMG classification5
|
MicroRNAs6
|
PredictSNP21 (probability)
|
FruitFly2
|
GnomAD3
|
1000 Genomes Project4
|
Normolipemic subjects
|
FCHL subjects
|
p
|
rs72649573
|
Exon 1
|
c.379C > T
|
p.(Leu127Phe)
|
Deleterious (82%)
|
NA
|
0.00711
|
0.0020
|
0.000
|
0.003
|
0.313
|
Benign7
|
NR
|
-
|
Intron 1
|
c.496-88T > G
|
NA
|
Neutral (88%)
|
Not splicing change
|
-
|
-
|
0.000
|
0.003
|
0.313
|
-
|
-
|
rs111414963
|
Exon 2
|
c.565T > C
|
p.(Leu189Leu)
|
Neutral (88%)
|
Not splicing change
|
0.00025
|
0.0008
|
0.003
|
0.000
|
0.313
|
Likely benign
|
NR
|
rs531071581
|
Intron 2
|
c.607-120A > G
|
NA
|
Neutral (88%)
|
Not splicing change
|
0.00013
|
0.0006
|
0.000
|
0.003
|
0.313
|
-
|
NR
|
rs72649576
|
Intron 2
|
c.607-109T > C
|
NA
|
Neutral (88%)
|
Not splicing change
|
0.01079
|
0.0042
|
0.024
|
0.003
|
0.020
|
-
|
NR
|
rs72649577
|
Intron 2
|
c.607 − 47_607-46delGT
|
NA
|
Neutral (88%)
|
NA
|
0.02222
|
0.0136
|
0.022
|
0.003
|
0.031
|
-
|
NR
|
rs185472483
|
Intron 3
|
c.835 + 41C > A
|
NA
|
-
|
Not splicing change
|
0.00032
|
0.0006
|
0.000
|
0.012
|
0.043
|
-
|
NR
|
rs747725081
|
Exon 6
|
c.961T > A
|
p.(Tyr321Asn)
|
Neutral (88%)
|
NA
|
NR
|
NR
|
0.000
|
0.003
|
0.313
|
-
|
NR
|
rs12563308
|
Exon 6
|
c.1003T > C
|
p.(Leu335Leu)
|
Neutral (88%)
|
NA
|
0.03550
|
0.0559
|
0.003
|
0.003
|
0.989
|
VUS7
|
NR
|
rs199555921
|
Exon 6
|
c.1028A > G
|
p.(His343Arg)
|
Neutral (89%)
|
NA
|
0.00016
|
NR
|
0.003
|
0.000
|
0.321
|
-
|
NR
|
rs763259225
|
Exon 6
|
c.1089T > G
|
p.(Val363Val)
|
Neutral (96%)
|
NA
|
NR
|
NR
|
0.003
|
0.000
|
0.321
|
-
|
NR
|
rs145086916
|
Exon 6
|
c.1122G > A
|
p.(Pro374Pro)
|
Neutral (96%)
|
NA
|
0.00077
|
0.0006
|
0.003
|
0.000
|
0.321
|
-
|
NR
|
rs72651034
|
Intron 6
|
c.1198 + 111G > A
|
NA
|
-
|
Not splicing change
|
NR
|
NR
|
0.003
|
0.003
|
0.989
|
-
|
NR
|
rs908541128
|
Intron 6
|
c.1198 + 140T > C
|
NA
|
-
|
Not splicing change
|
0.000
|
0.000
|
0.003
|
0.000
|
0.321
|
-
|
NR
|
rs34483103
|
3’UTR
|
c.*52_*60del
|
NA
|
-
|
NA
|
0.33531
|
0.3484
|
0.276
|
0.102
|
< 0.001
|
-
|
hsa-miR-151a-3p
hsa-miR-7702
|
-
|
3’UTR
|
c.*76T > G
|
NA
|
-
|
NA
|
-
|
-
|
0.000
|
0.003
|
0.313
|
-
|
-
|
NR: Not reported; NA: Not applicable; VUS: Variant of uncertain significance.
1PredictSNP2 uses CADD, DANN, FATHMM y Funseq2 as predictors.
2FruitFly. New prediction score 0.87 (wild type score 0.89).
3GnomAD. https://gnomad.broadinstitute.org/
41000 Genomes Project Consortium, Abecasis GR, Auton A, Books LD et al. An integrated map of genetic variation from 1092 human genomes. Nature 2012;491:56–65.
5Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, Grody WW, Hegde M, Lyon E, Spector E, Voelkerding K, Rehm HL; ACMG Laboratory Quality Assurance Committee. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015 May;17(5):405 − 24. doi: 10.1038/gim.2015.30. Epub 2015 Mar 5. PMID: 25741868; PMCID: PMC4544753.
6PolymiRTS Database 3.0: http://compbio.uthsc.edu/miRSNP/
7Tikka A, Metso J, Jauhiainen M. ANGPTL3 serum concentration and rare genetic variants in Finnish population. Scand J Clin Lab Invest. 2017;77:601–609.