Objective To make molecular diagnosis for puerile spinal muscular atrophy(SMA).Methods Genomic DNA was extracted directly from the blood of both the case group(10 children with SMA) and the control group(including 19 parents of SMA patients and 20 healthy individuals).Two methods,polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) and allele-specific PCR,were used to analyze exon 7 of SMN gene from genomic DNA,and consequent electrophoresis of PCR products on agarose gel was performed.Results Genotyping results obtained by both methods were in complete agreement for all of the samples analyzed.In conventional PCR-RFLP,part of the PCR products(189bp) from genomic DNA of all 39 members in the control group remained intact after digestion with Dra I,while the PCR products from genomic DNA of all 10 SMA children in the case group was completely digested by Dra I.In allele-specific PCR,exon 7 of both SMN1 and SMN2 could be seen when genomic DNA of all 39 members in the control group was used,while only SMN2's exon 7 could be seen when genomic DNA of all 10 SMA children in the case group was used.Conclusion Homozygous deletion of SMN1 was present in all 10 SMA children in the case group,while homozygous deletion of SMN1 was not detected in all 39 members in the control group.The combination of PCR-RFLP and allele-specific PCR,both their results can be references for each other,offers efficient and accurate methodology for molecular diagnosis of SMA.

T),one dinucleotide deletion(1801-02 del AG) and one base insertion(1125 ins GCCATCG),which resulted in eight missense mutations,two nonsense mutations and two frame shift mutations,namely P534R,G343V,R259W,A141T,R401Q,K276E,Y174C,A314P,S108X,Q177X,fs E471 and fs A247.Conclusion The combined DHPLC and sequencing approach may act as a rapid and efficient method for ABCD1 gene mutation analysis in patients and carriers of X-linked adrenoleukodystrophy families.There exist different ABCD1 gene mutations in different pedigrees,and no obvious correlation between the genotype and phenotype has been found.

Objective To investigate methods for prenatal molecular diagnosis of fetuses at high risk for X-linked adrenoleukodystrophy(X-ALD).Methods The amniotic fluid was obtained and genomic DNA was isolated from amniotic fluid cells.Maternal contamination was evaluated by paternity test.PCRRFLP,sequencing and denaturing high performance liquid chromatography(DHPLC)were used to detect the ABCD1 gene of fetal genome.Results 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.Conclusion A new protocol for X-ALD prenatal molecular diagnosis is proposed,which would ensure the accuracy of prenatal diagnosis.