ABCMdb

PMID: 22923419

Reddy MV, Iatan I, Weissglas-Volkov D, Nikkola E, Haas BE, Ruel MJ, Sinsheimer JS, Genest J, Pajukanta P
Exome sequencing identifies 2 rare variants for low high-density lipoprotein cholesterol in an extended family.
Circ Cardiovasc Genet. 2012 Oct 1;5(5):538-46. doi: 10.1161/CIRCGENETICS.112.963264. Epub 2012 Aug 25., [PubMed]

Sentences

No. Mutations Sentence Comment

38 ABCA1 p.Gly616Val ABCA1 p.Asn1800His ABCA1 p.Arg909* ABCA1 p.Gln2210His ABCA1 p.Arg1851* ABCA1 p.Ser1731Cys ABCA1 p.Lys776Asn Genotype by sex interaction We included the extended family together with 10 additional families with previously identified mutations in ABCA16-8 in a gene-sex interaction analysis, comprising 200 individuals and 9 different mutations in ABCA1 (DelED1893, G616V, K776N, N1800H, Q2210H, R1851X, R2084X, R909X and S1731C). Login to comment 41 ABCA1 p.Gly616Val ABCA1 p.Asn1800His ABCA1 p.Arg909* ABCA1 p.Gln2210His ABCA1 p.Arg1851* ABCA1 p.Ser1731Cys ABCA1 p.Lys776Asn Genotype by Sex Interaction We included the extended family together with 10 additional families with previously identified mutations in ABCA16 -8 in a gene-sex interaction analysis, comprising 200 individuals and 9 different mutations in ABCA1 (DelED1893, G616V, K776N, N1800H, Q2210H, R1851X, R2084X, R909X, and S1731C). Login to comment 46 ABCA1 p.Gln2210His The binary HDL-C affection was tested because the variance of HDL-C levels in these ascertained families is reduced and thus limited for effective quantitative analysis.1 H, Q2210H, R1851111X,XXX d by tttthhehh SSSSOLOLOLOLARARAA pppprrror g nce-component analysis for discrete traits. Login to comment 48 ABCA1 p.Ser1731Cys We compared mmmodooo els with and witho nteere aaaca tion termm wwwhiili eee kekeepepepe inggg ttht eee ABABBCCA11 ggenonotytytytypepepp s iniin botthhh thhheee nulllll aaanddd iiiintereerac assumemememedddd a dodod iimiminana tntnt geneteteticicicic inhhhherererititititananana cee, lclcllasassisisiifyfyfyfyinining cacacaarrrrieiei rsrs offff aaaa mmmutaatititionon aass 111 and a multipplicatiive iiinteraction term,,, m llultiiipllpllyiyinggg thehhh gggenotypypype score byy sex ((me 0)0) WW lal dod ded e iinte ctiio te ii hhiichh dd st ll Cell culture Human skin fibroblasts were obtained from 3.0-mm punch biopsies of the forearm of a healthy control subject and the affected proband homozygous for the ABCA1 S1731C variant. Login to comment 53 ABCA1 p.Ser1731Cys Because the affection status is adjusted for sex, the inclusion of the main effect of sex in the model was no longer necessary. The binary HDL-C affection was tested because the variance of HDL-C levels in these ascertained families is reduced and thus limited for effective quantitative analysis.1 Cell Culture Human skin fibroblasts were obtained from 3.0-mm punch biopsies of the forearm of a healthy control subject and the affected proband homozygous for the ABCA1 S1731C variant. The fibroblasts were cultured in Dulbecco`s modified Eagle`s medium (DMEM) supplemented with 0.1% nonessential amino acids, penicillin (100 U/mL), streptomycin (100 bc;g/mL), and 10% fetal bovine serum. Login to comment 63 ABCA1 p.Ser1731Cys Rare missense variant in ABCA1 The ABCA1 (S1731C) variant is not present in dbSNP13214 or The 1000 Genomes Project14 data and is located in exon 38. Login to comment 64 ABCA1 p.Ser1731Cys ABCA1 p.Ser1731Cys This previously reported rare variant is changing a conserved amino acid from serine to cysteine and is known to result in decreased cholesterol efflux.7,24-26 In order to further determine the effect of the S1731C variant on cholesterol efflux, we used human fibroblasts from the affected proband homozygous for the S1731C variant, and compared these cells to a normal control. Login to comment 72 ABCA1 p.Ser1731Cys ABCA1 p.Ser1731Cys As the lipid levels of the ABCA1 S1731C variant carriers suggested a possible gene-gender effect (Tables 3-4), we further investigated whether exposure to -estradiol steroid hormone endogenously expressed in females, possibly corrects the cholesterol efflux defect in fibroblasts from the S1731C male ABCA1 carrier during the 22OH/9CRA ABCA1 stimulation phase of 17 hours (Figure 2B). Login to comment 73 ABCA1 p.Ser1731Cys ABCA1 p.Ser1731Cys Interestingly, after adjusting for basal cholesterol diffusion, we observed that upon treatment with elevated doses of estradiol (>20 nM), efflux in the S1731C proband significantly increased (P=7.2x10-6 , r=0.78 using a non-parametric Spearman trend test), while that in the wildtype control remained constant (P=0.2, r=0.25) (Figure 2B). Login to comment 74 ABCA1 p.Ser1731Cys ABCA1 p.Ser1731Cys ABCA1 p.Ser1731Cys Taken together, these results support a genotype-sex interaction effect, as hormonal regulation with 17 -estradiol partially restored the low efflux observed in the S1731C male proband but had no significant effect on the efflux of a wild-type control. Login to comment 79 ABCA1 p.Ser1731Cys Because the lipid levels of the ABCA1 S1731C variant carriers suggested a possible gene-sex effect (Tables 3 and 4), we further investigated whether exposure to 17b2;-estradiol steroid hormone endogenously expressed in females, possibly Table 3.ߓ The Lipid Levels and Other Clinical Characteristics of the 3 Individuals That Were Exome Sequenced IND ID ABCA1 LPL TC TG HDL-C HDL % BMI LDL-C AGE SEX Ind5 C/C C/T 4.61 1.3 0.67 <5 22.02 3.36 66 Male Ind14 G/C C/T 4.6 6.1 0.62 <5 25.81 NA 55 Male Ind19 G/C C/T 2.43 2.4 0.62 <5 27.22 0.7 21 Male LPL indicates lipoprotein lipase; TG, triglyceride; HDL-C, high-density lipoprotein cholesterol; BMI, body mass index; LDL, low-density lipoprotein; and ABCA1, ATP-binding cassette, subfamily A (ABC1), member 1 and TC, total cholesterol. Login to comment 81 ABCA1 p.Ser1731Cys ABCA1 p.Ser1731Cys ABCA1 p.Ser1731Cys By stratifying individuals by their HDL percentiles, we can see that all the affected family members with HDL-C<5th percentile carry a risk allele for either one or both of the variants (3 P234L, 11 S1731C, and 8 P234L/S1731C), except in one separate branch of the extended family in which the low HDL-C traits appears to be inherited from the affected spouse`s side (Figure 1). Login to comment 83 ABCA1 p.Ser1731Cys ߓߓCirc Cardiovasc GenetߓߓOctober 2012 corrects the cholesterol efflux defect in fibroblasts from the S1731C maleABCA1 carrier during the 22OH/9CRAABCA1 stimulation phase of 17 hours (Figure 2B). Login to comment 84 ABCA1 p.Ser1731Cys Interestingly, after adjusting for basal cholesterol diffusion, we observed that upon treatment with elevated doses of estradiol (>20 nM), efflux in the S1731C proband significantly increased (P=7.2&#d7;10-6 , r=0.78 using a nonparametric Spearman trend test), whereas that in the wild-type control remained constant (P=0.2, r=0.25) (Figure 2B). Login to comment 85 ABCA1 p.Ser1731Cys Taken together, these results support a genotype-sex interaction effect, as hormonal regulation with 17b2;-estradiol partially restored the low efflux observed in the S1731C male proband but had no significant effect on the efflux of a wild-type control. Login to comment 91 ABCA1 p.Ser1731Cys ABCA1 p.Ser1731Cys By stratifying individuals by their HDL percentiles, we can see that all the affected family members with HDL-C less than the fifth percentile carry a risk allele for either 1 or both of the variants (3 P234L, 11 S1731C, and 8 P234L/ S1731C), except in 1 separate branch of the extended family in which the low HDL-C traits appears to be inherited from the affected spouse`s side (Figure 1). Login to comment 105 ABCA1 p.Ser1731Cys Genotype by sex interaction The effect of the ABCA1 S1731C variant on low HDL-C levels appears more profound in the males than in females in the extended family (Table 4). Login to comment 107 ABCA1 p.Ser1731Cys ABCA1 p.Ser1731Cys Although the frequency of the S1731C variant may be individually too rare for testing genetic interactions (as large sample sizes are necessary), rare variants with large phenotypic effects are collectively common in low HDL-C families.26 We hypothesized that the apparent sex effect may not be restricted to the S1731C allele, but rather it may generally extend to ABCA1 alleles with major phenotypic effects. Login to comment 111 ABCA1 p.Ser1731Cys ABCA1 p.Ser1731Cys The S1731C variant was present in 3 of these additional low HDL-C pedigrees7 and together with the exome sequenced family the association signal for the main effect of S1731C on low HDL-C status resulted in a p-value of 0.008. Login to comment 113 ABCA1 p.Ser1731Cys ABCA1 p.Ser1731Cys Althou the frequency of the S1731C vari ividually too rare for testing genetic interactions (as large sample sizes are neces t s esi ed that the apparent se effect ma not be restricted to the S1731C allele b in fefefefemamamalelelel sss iniii ttthehhh extended family (Tablllleee 4)4 . Login to comment 114 ABCA1 p.Ser1731Cys Genotype by Sex Interaction The effect of the ABCA1 S1731C variant on low HDL-C levels appears more profound in the males than in females in the extended family (Table 4). Login to comment 116 ABCA1 p.Ser1731Cys ABCA1 p.Ser1731Cys Although the frequency of the S1731C variant may be individually too rare for testing genetic interactions (as large sample sizes are necessary), rare variants with large phenotypic effects are collectively common in low HDL-C families.26 We hypothesized that the apparent sex effect may not be restricted to the S1731C allele, but rather it may generally extend to ABCA1 at Semmelweis University (Egyetem) on December 3, 2015 http://circgenetics.ahajournals.org/ Downloaded from Reddy et alߓߓ Identification of Rare Variants for HDLߓߓ alleles with major phenotypic effects. Login to comment 120 ABCA1 p.Ser1731Cys ABCA1 p.Ser1731Cys The S1731C variant was present in 3 of these additional low HDL-C pedigrees,7 and together with the exome sequenced family, the association signal for the main effect of S1731C on low HDL-C status resulted in a P value of 0.008. Login to comment 123 ABCA1 p.Ser1731Cys ABCA1 p.Ser1731Cys Mutations disrupting the normal function of ABCA1 result in little or no circulating HDL.34 Previous studies have shown that cell lines with the identified ABCA1 variant S1731C exhibit low levels of protein expression,25 and that cells transfected with the S1731C allele express abundant ABCA1 mRNA but fail to generate significant amounts of ABCA1 protein.25 rtion ooooffff ththththeeee HDHDHDHDL-L-L-L-CCCC nd heritability in an extended family We also observed a sex effect for ABCA1 a m s n ants of which one is less severe in females. Traditional linkage analysis was una nd herittitabababilititity ininn an extended family. Login to comment 124 ABCA1 p.Ser1731Cys ABCA1 p.Ser1731Cys ABCA1 p.Ser1731Cys We aalsl o observed a sexexx effect for ABCA1 allel carriers exxhihihibiiitititingnng sssigigigigninin fififificcantnn lly lllowoowerr HHDLDLDL-CCCC lelel vvvelslls ttthahahannn fefefemmmalelelees.ss FuFuFuFurtrtheheheherm unnnnafafafa fefefeectctcttedededd ffamamammililili yyy mememem mbmbmbbererrrssss hahh dddd thththheeee LPLPLPLLLL vavavavariririr anananntttt orororr bbbotototthhhh vvavaririririannanntstststs yyy.... OOOOururur stutututudydydydy s how utilizationononon oooffff exexexomomomomeee seseseseququuquenenene cicicicingnngng wwwwasass ccccrirr ticacacacalll totototo rrevevvveaeeaealll thththeee cocococomplex combin aaantntss ofof wwhihiichhch oonene iiiss lelessss ssevevereree inini fffememalalleses.. TrTTradadditititioionanall lililinknkkagagee ananalalysysisis wwasas uunana Furthermore, the cholesterol efflux of S1731C has been shown to be reduced to 12.3-68.0% of the wildtype.7,25,26 Here, we observed a ~40% cholesterol efflux reduction in the proband homozygous for the S1731C variant as compared to a normal control, in line with the earlier findings.7,25,26 In our previous paper,7 we showed that three heterozygous subjects with the S1731C variant have cholesterol efflux values of 63%, 66%, and 68% of the wildtype. Login to comment 126 ABCA1 p.Ser1731Cys Although the phenotype data suggest that the S1731C variant does not have a gene dose effect, this conclusion warrants additional functional studies in future, as there are only 4 homozygotes in the family two of which are also heterozygous for the LPL variant, and furthermore the variant has a large range (12.3-68.0% of the wildtype) in its effect on the cholesterol efflux.7,25,26 The main function of LPL is to hydrolyze triglycerides in order to deliver fatty acids to the tissue. Login to comment 129 ABCA1 p.Ser1731Cys Regarding the identified P207L variant, individuals with this mutation have reduced HDL particles compared with the control subjects.35 Previous studies have also shown that missense mutations in exon 5 of the LPL gene where the P207L variant resides are the most common cause of LPL deficiency.36,37 Importantly, Ma et al. reported that upon site-directed in vitro mutagenesis this variant produces a catalytically inactive lipoprotein lipase protein which is the cause of the lipoprotein lipase deficiency in the patients.29 Taking together these previous data, along with our PolyPhen15 and SIFT16 predictions, it is highly likely that both identified variants S1731C and P207L affect protein function. Login to comment 130 ABCA1 p.Ser1731Cys ABCA1 p.Ser1731Cys ABCA1 p.Ser1731Cys ABCA1 p.Ser1731Cys ABCA1 p.Ser1731Cys Mutations disrupting the normal function of ABCA1 result in little or no circulating HDL.33 Previous studies have shown that cell lines with the identified ABCA1 variant S1731C exhibit low levels of protein expression,25 and that cells transfected with the S1731C allele express abundant ABCA1 mRNA but fail to generate significant amounts of ABCA1 protein.25 Furthermore, the cholesterol efflux of S1731C has been shown to be reduced to 12.3% to 68.0% of the wild-type.7,25,26 Here, we observed a ࣈ40% cholesterol efflux reduction in the proband homozygous for the S1731C variant as compared with a normal control, in line with the earlier findings.7,25,26 In our previous article,7 we showed that 3 heterozygous subjects with the S1731C variant have cholesterol efflux values of 63%, 66%, and 68% of the wild type. Login to comment 131 ABCA1 p.Ser1731Cys ABCA1 p.Ser1731Cys ABCA1 p.Ser1731Cys SSSeS quqq ence variation tedd to bb iiatedd ii hth thhe iiskk fof CCHDHD TGTG dd HDHDLL CC 33 AAn ffffiiciie The two identified variants, S1731C and P207L have been reported previously in French Canadian dyslipidemic individuals but not in normal controls.7,24-29 The S1731C ABCA1 variant was present in three French Canadian dyslipidemic families with low HDL-C levels7 but not in 528 chromosomes from French Canadian subjects with normal HDL-C levels.24 It was also absent in 108 French Canadian subjects with high HDL-C.26 The P207L LPL variant was previously observed in 37 unrelated French Canadian patients with lipoprotein lipase deficiency with 54 mutant alleles present in that study sample.29 In the same study, the variant was also genotyped in 34 unrelated patients with LPL deficiency from ancestries other than French Canadian. Login to comment 136 ABCA1 p.Ser1731Cys 22OH indicates 22(R)-hydroxycholesterol; 9CRA, 9-cis-retinoic acid; and ApoA-I, apolipoprotein A-I. B, Elevated concentrations of 17b2;-estradiol improve cholesterol efflux in the male proband with the ABCA1 S1731C variant. Login to comment 137 ABCA1 p.Ser1731Cys ABCA1 p.Ser1731Cys These interesting sex-specific mechanisms of ABCA1 may involve hormonal regulation of ABCA1, a hypothesis supported by our efflux experiment (Figure 2B), demonstrating that exposure of fibroblasts of a male proband with the ABCA1 S1731C variant to es other than Frenchchchch the risiii kkkk alllllllel lllle. Login to comment 141 ABCA1 p.Ser1731Cys Upon exposure to increasing estradiol concentrations (>20 nM), cholesterol efflux in the S1731C proband significantly increases. Login to comment 145 ABCA1 p.Ser1731Cys 22OH indicates 22(R)-hydroxycholesterol; 9CRA, 9-cis-retinoic acid; and ApoA-I, apolipoprotein A-I. ***P=7.2&#d7;10-6 (r=0.78) using a nonparametric Spearman trend test for the dose effect on efflux in the S1731C proband; and nsP=0.2 (r=0.25) using a nonparametric Spearman trend test for the dose effect on efflux in the wild-type control. Login to comment 147 ABCA1 p.Ser1731Cys Although the phenotype data suggest that the S1731C variant does not have a gene dose effect, this conclusion warrants additional functional studies in future, because there are only 4 homozygotes in the family 2 of which are also heterozygous for the LPL variant, and furthermore the variant has a large range (12.3%-68.0% of the wild-type) in its effect on the cholesterol efflux.7,25,26 The main function of LPL is to hydrolyze TGs to deliver fatty acids to the tissue. Login to comment 150 ABCA1 p.Ser1731Cys Regarding the identified P207L variant, individuals with this mutation have reduced HDL particles compared with the control subjects.34 Previous studies have also shown that missense mutations in exon 5 of the LPL gene where the P207L variant resides are the most common cause of LPL deficiency.35,36 Importantly, Ma et al reported that upon site-directed in vitro mutagenesis this variant produces a catalytically inactive LPL protein, which is the cause of the LPL deficiency in the patients.29 Taken together, these previous data, along with our PolyPhen15 and SIFT16 predictions, show that it is highly likely that both identified variants S1731C and P207L affect protein function. Login to comment 151 ABCA1 p.Ser1731Cys ABCA1 p.Ser1731Cys The 2 identified variants, S1731C and P207L, have been reported previously in French-Canadian dyslipidemic individuals but not in normal controls.7,24 -29 The S1731C ABCA1 variant was present in 3 French-Canadian dyslipidemic families with low HDL-C levels,7 but not in 528 chromosomes from French-Canadian subjects with normal HDL-C levels.24 It was also absent in 108 French-Canadian subjects with high HDL-C.26 The P207L LPL variant was previously observed in 37 unrelated French-Canadian patients with LPL deficiency with 54 mutant alleles present in that study sample.29 In the same study, the variant was also genotyped in 34 unrelated patients with LPL deficiency from ancestries other than French-Canadian. Login to comment 158 ABCA1 p.Gly616Val ABCA1 p.Asn1800His ABCA1 p.Arg909* ABCA1 p.Gln2210His ABCA1 p.Arg1851* ABCA1 p.Ser1731Cys ABCA1 p.Lys776Asn Figure 3 shows the age-sex specific population HDL-C percentiles by ABCA1 genotypes and sex in 200 French-Canadian family members from 11 French-Canadian families with different ABCA1 mutations (DelED1893, G616V, K776N, N1800H, Q2210H, R1851X, R2084X, R909X, and S1731C). Login to comment 165 ABCA1 p.Ser1731Cys at Semmelweis University (Egyetem) on December 3, 2015 http://circgenetics.ahajournals.org/ Downloaded from Reddy et alߓߓ Identification of Rare Variants for HDLߓߓ demonstrating that exposure of fibroblasts of a male proband with the ABCA1 S1731C variant to increasing concentrations of 17b2;-estradiol led to a significantly increased efflux in the male proband with the ABCA1 variant. Login to comment