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Background X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder characterized by progressive demyelination of the nervous system, adrenocortical insufficiency and increase of very long chain fatty acids (VLCFAs) in the plasma and tissues. Methods A total of 131 individuals from 30 Chinese pedigrees were involved in this study, including 42 symptomatic patients, 44 female carriers, and 15 high-risk fetuses from 13 families. The mutation was first pinpointed through long distance RT-PCR-based RNA approach and confirmed through peripheral blood DNA approach. Results A total of 28 mutations were identified, of which 19 were missense, 3 nonsense and 6 frame-shift mutations. Thirteen mutations were novel, i.e. p.R280L, p.P580L, p.G343V, p.S108X, p.R259W, p.P534R, p.fs A246, p.L576P, p.K602X, p.A314P, p.N148D, p.H283R, and p.fs R89. Two mutations occurred de novo, for they were not found in somatic cells of their parents. Three females from the same family developed AMN-like symptoms and they were heterozygous for the p.H283R mutation. Four asymptomatic boys were diagnosed as X-ALD patients and prenatal molecular diagnosis were provided for 13 X-ALD-stricken families. Conclusions Our work extended the spectrum of mutations in X-ALD and benefited genetic counseling through reliable identification of heterozygous females and asymptomatic males.

To identify ABCD1 gene mutation in a Chinese family with three heterozygous female patients.Four fragments covering the entire coding sequence of the ABCD1 gene from one of the female patients were amplified by reverse transcription-PCR. The PCR products were directly sequenced. The result of sequencing was confirmed by restriction enzyme digestion of PCR products from genomic DNA. Human ABCD1 gene and ALD protein were aligned with those of rat, monkey, mouse and cattle by Clustal X 1.83. Softwares of Motif Scan, TMpred and ESYpred3D were used to predict the effect of the mutation on the structure of the ALD protein.A novel missense mutation, CAC to CGC, was found at codon 283 of the ABCD1 gene from the patient, resulting in the replacement of histidine by arginine. This mutation abolished an Msl I site in the gene. Her son was free from this mutation. The mutated amino acid residue (283H) was highly conservative in evolution, and the mutation caused a dramatic change in the structure of the ALD protein.Three female patients heterozygous for ABCD1 gene mutation were first reported in China, and a novel mutation, p.H283R, was identified in this X-ALD family.

To identify asymptomatic males with X-linked adrenoleukodystrophy (X-ALD) from Chinese pedigrees by molecular genetic testing.Genomic DNA was extracted from peripheral blood of the asymptomatic individuals from X-ALD families, and fragments spanning the proband's mutation were amplified. PCR-RFLP, direct sequencing and denaturing high performance liquid chromatography (DHPLC) were used to detect the PCR products.Four asymptomatic male subjects from three Chinese X-ALD pedigrees were found to carry the same mutation with the probands. In Pedigree 1, by restriction analysis with endonuclease Eco47 I, the digestion pattern of the proband's elder brother (Subject 1) was same with the proband, which indicated that both carried the same mutation. In Pedigree 2 and Pedigree 3, the PCR products were analysed by DHPLC, and the patterns of elution peaks of the Subjects 2-4 and the heterozygous mothers were similar, which indicated the presence of sequence alterations in the ABCD1 gene. DNA sequencing of the corresponding PCR products confirmed the mutations.Molecular testing was an effective way to determine the genotype of family members of X-ALD before they develop any symptoms. Early and preferable pre-symptomatic identification of hemizygotes is of great benefit to affected individuals and their families.

To perform molecular diagnosis for a Chinese pedigree with osteogenesis imperfecta type I.Thirty pairs of primers were designed to amplify all the 52 exons, exon boundaries and promoter region of the COL1A1 gene from genomic DNA of peripheral blood cells of the family members. The PCR products were purified and directly sequenced. To check the mutation in normal controls, the genomic DNA from peripheral blood cells of the index patient, his mother and 60 normal controls were analyzed by amplification refractory mutation system.A missense mutation of GATCAT was identified at codon 1441 of the COL1A1 gene from the family, which resulted in the replacement of aspartic acid by histidine (D1441H). This mutation was not found in a group of 60 normal controls.The method for molecular diagnosis of osteogenesis imperfecta was established and a novel COL1A1 gene mutation, D1441H, was identified in the Chinese pedigree with osteogenesis imperfecta type I.

To investigate the effect of multiplex ligation-dependent probe amplification (MLPA) in molecular diagnosis of spinal muscular atrophy (SMA).Peripheral blood samples were collected from 13 SMA patients, 31 parents of SMA patients, 50 healthy individuals without family history of SMA, and 10 specimens of amniotic fluid from these families were collected too. Genomic DNA was analyzed by MLPA, conventional PCR-RFLP, and allele-specific PCR.In complete agreement with the results of conventional PCR-RFLP and allele-specific PCR, MLPA analysis showed that all of the 13 patients had homozygous deletion of the survival of motor neuron 1 (SMN1) gene, and there was significant difference between the SMA severity (type I to type III) and SMN2 copy number (P0.05). Of the 31 parents 29 (93.5%) had 1 copy of SMN1, 2 (6.5%) had 2 copies of SMN1. Of the 50 healthy individuals, 1 (2.0%) had 1 copy of SMN1, 48 (96.0%) had 2 copies of SMN1, and 1 (2.0%) had 3 copies. The SMN1 copy number of the parents was significantly higher than that of the healthy individuals (P0.01). Two of the 10 fetuses had homozygous deletion of SMN1.The MLPA technique has proved to be an accurate and reliable tool for the molecular diagnosis of SMA, both in patients and in healthy carriers.

To investigate methods for prenatal molecular diagnosis of fetuses at high risk for X-linked adrenoleukodystrophy (X-ALD).The amniotic fluid was obtained and genomic DNA was isolated from amniotic fluid cells. Maternal contamination was evaluated by paternity test. PCR-RFLP, sequencing and denaturing high performance liquid chromatography (DHPLC) were used to detect the ABCD1 gene of fetal genome.In the pedigree 1, the PCR product (799 bp) of the fetus 1 and her father (normal control) could be digested with BcnI. No P560L mutation, which was present in the index patient, was detected in the ABCD1 gene from the genomic DNA of the fetus 1 using direct sequencing. In the pedigree 2, the PCR product (232 bp) of the fetus 2 and her father could not be digested with MaeI and no Q177X mutation, which was present in the propositus, was detected in the ABCD1 gene from the genomic DNA of the fetus 2 using direct sequencing. In the pedigree 3, the PCR product (271 bp) was digested with AciI, the pattern of the fetus 3 and the propositus being the same, and the R617C mutation was found in the ABCD1 gene from the genomic DNA of the fetus 3 using direct sequencing. In the pedigree 4, the PCR product (269 bp) was analyzed with the DHPLC, and the pattern of elution peaks of the fetus 4 and her father was similar, but different from that of the propositus. No K276E mutation was detectable in the ABCD1 gene from the genomic DNA of the fetus 4 by using direct sequencing. Judging from the sex of the fetuses, fetuses 1 and 2 were normal homozygotes, fetus 3 was an ALD hemizygote, and fetus 4 was a normal hemizygote.A new protocol for X-ALD prenatal molecular diagnosis is proposed, which would ensure the accuracy of prenatal diagnosis.