Analysis Of Gene Mutation And Protein Expression In X-linked Adrenoleukodystrophy

Objective: This study aimed to retrospectively investigate the clinical characteristics in patients with X-linked adrenoleukodystrophy(X-ALD)including members of large pedigrees,explore the law of development of the disease,analyze ABCD1 gene mutations,find the hotspot mutation,to expand gene mutation spectrum of Chinese patients,to predict the effect of 4 novel missense mutations in ABCD1 on structure and function of ALDP,investigate the effects of 4 novel missense mutations of ABCD1 gene on ALDP expression and to explore the correlation between genotype and phenotype as well.Methods: Thirty male patients diagnosed as X-ALD between 2009 to 2015 were enrolled in this study:(1)The clinical characteristics of patients were retrospectively summarized and analyzed by reviewing medical records or telephone consultation,and thereby explore the law of development of the disease.(2)ABCD1 gene mutations profile was analyzed by Sanger sequencing in 30 patients and some members of large pedigrees.(3)The genetic origins of novel gene variants were confirmed and polymorphisms were excluded by directly sequencing of parents and normal controls.(4)The pathogenicity of novel missense variants was analyzed using Poly Phen,SIFT,Align-GVGD,and Swiss-PDB Viewer 4.04 software,respectively.(5)We constructed expression vectors of 4 novel missense mutations(p.Gly298 Arg,p.Trp339 Cys,p.Ser515 Tyr and p.Leu580Pro)by site-directed mutagenesis.(6)Wild type and four mutant plasmids were transfected into 293 T cells.(7)The expression of wide-type and mutant ALDP were tested by western bolt.(8)Combined with the clinical data and gene mutation analysis in patients,the relationship between genotype and clinical phenotype was discussed.Results:(1)Childhood cerebral form adrenoleukodystrophy(CCALD)is the most common phenotype(66.7%)in 30 patients with X-ALD.The initial symptoms included decline in academic performance,memory loss and behavioral problems.The progressive deterioration of neurological development and cognitive functions(intellectual impairment,speech delay,exercise intolerance,encephalopathy,visual impairment,etc)were main clinical features.All patients had the brain white matter demyelination and elevated very long chain fatty acids in plasma.Different phenotypes are presented among patients and family members.(2)Twenty seven different mutations in ABCD1 gene were identified in 30 Chinese patients.Of the mutations,67.8% are missense mutations and 35.7% located in exon1.No mutation was found in exon 10.(3)9 novel mutations(p.Trp339 Cys,p.Gly298 Arg,p.Gln178*,p.Cys476*,p.Ser515 Tyr,p.Ala395Leufs*15,p.Leu142Serfs*56,p.Ile588 His and p.Leu580Pro)were confirmed,including 4 novel missense mutations(p.Gly298Arg?p.Trp339Cys?p.Ser515 Tyr and p.Leu580Pro).Then,polymorphisms were excluded.(4)The amino acid residues of four novel missense mutations(p.Gly298 Arg,p.Trp339 Cys,p.Ser515 Tyr and p.Leu580Pro)in different species were highly conserved,and were predicted to be “probably” damaging to ALDP protein function by software.(5)The recombinant expression vectors with four missense mutations(p.Gly298 Arg,p.Trp339 Cys,p.Ser515 Tyr and p.Leu580Pro)were constructed successfully.(6)The recombinant expression vectors including wide type and four mutations were respectively transfected into 293 T cells by liposome-mediated method.(7)The levels of ALDP expression were significantly lower in four kinds of mutant protein(p.Gly298 Arg,p.Trp339 Cys,p.Ser515 Tyr and p.Leu580Pro)than that in wild type(P<0.05).(8)The patients with four missense mutations(p.Gly298 Arg,p.Trp339 Cys,p.Ser515 Tyr and p.Leu580Pro)were all severe phenotypes.The same mutation could cause different phenotypes.Conclusions:(1)Childhood cerebral form adrenoleukodystrophy(CCALD)is the most common phenotype(66.7%)of X-ALD.The progressive deterioration of neurological development,the brain white matter demyelination and elevated very long chain fatty acids in plasma are essentials of diagnosis.Different phenotypes were presented within the same pedigree.(2)Twenty seven different mutations in ABCD1 gene were found in 30 Chinese patients.Nine novel mutations were identified.We did not find the hotspot mutation.No mutation in exon 10 was found.(3)The brain MRI and very long chain fatty acids in plasma determination should be advised to perform for all patients who present progressive deterioration of neurological development or primary adrenal insufficiency for the purpose of early detection of X-ALD.(4)Four missense mutation of ABCD1(p.Gly298 Arg,p.Trp339 Cys,p.Ser515 Tyr and p.Leu580Pro)were all pathogenic mutations and significantly reduced the amount of ALDP expression.(5)There was no correlation between genotype and phenotype in our patients.