Phenotypic and genetic analysis of an inv dup(15) case with a BP3:BP3 rearrangement / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze a case of supernumerary marker chromosome (SMC) with combined genetic techniques and explore its correlation with the clinical phenotype.</p><p><b>METHODS</b>The SMC was analyzed with G-banded karyotyping, multiplex ligation dependent probe amplification (MLPA), fluorescence in situ hybridization (FISH), and single nucleotide polymorphism array (SNP-array).</p><p><b>RESULTS</b>G-banding analysis indicated that the patient has a karyotype of 47,XX,+mar. MLPA showed that there were duplications of proximal 15q. FISH assay using D15Z4 probes indicated that the SMC was a pseudodicentric chromosome derived from chromosome 15. And SNP-array revealed that there were two extra copies of 15q11-13 region spanning from locus 20 161 372 to 29 071 810.</p><p><b>CONCLUSION</b>The duplication of Prader-Willi/Angelman syndrome critical region probably underlies the abnormal phenotype of the inv dup(15) case with a BP3:BP3 rearrangement.</p>

Analysis of AR gene mutation in a family affected with complete androgen insensitivity syndrome using long chain RT-PCR / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify potential mutation of androgen receptor (AR) gene in a patient with complete androgen insensitivity syndrome (CAIS) and his family members.</p><p><b>METHODS</b>Total RNA and genomic DNA were extracted from the peripheral blood samples derived from the proband and her family members. Sequences of 7 exons of the AR gene were amplified with reverse transcriptase PCR(RT-PCR) and subjected to direct sequencing. Suspected mutation was also analyzed with PCR-restriction fragment length polymorphism (PCR-RFLP) and direct sequencing.</p><p><b>RESULTS</b>DNA sequencing has revealed a nucleotide change (2880A>G) in the pedigree, which resulted in a missense mutation (R840H).</p><p><b>CONCLUSION</b>A prenatal diagnostic method was established for detecting mutation of the AR gene in the pedigree. Long chain RT-PCR was first used for the detection of AR gene mutations.</p>

Cytogenetic and molecular genetic analysis of a case with mosaic marker chromosomes / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the source of small supernumerary marker chromosome in a case.</p><p><b>METHODS</b>G-banded karyotyping, fluorescence in situ hybridization, multiple sequence tagged sites (STS) of the Y chromosome, and Illumima Human Cyto SNP-12 Beadchip analysis were carried out.</p><p><b>RESULTS</b>The karyotype was mos 46,X,+mar1[21]/46,X,+mar2[78]. Y chromosome STS analysis has displayed the presence of sy84, sY86, USP9Y and DDX3Y genes from the AZFa region, and sY1227 of the AZFb region, while sY1228, sY1015, sY127, sY134 from the AZFb region, and sY254 and sY255 from the AZFc region were missing. FISH analysis has verified both of the marker chromosomes to be Y chromosome fragments. Mar1 was ish.idic(Y)(q11.2)(SRY++,DXZ1+,DYZ3++,DYZ1-), while mar2 was ish.del(Y)(q11.2)(SRY+,DXZ1+,DYZ3+,DYZ1-). Single nucleotide polymorphism (SNP) microarray analysis showed that the Yq11.2-Yq12 has lost a 10.81 Mb fragment.</p><p><b>CONCLUSION</b>The marker chromosomes were verified to be aberrant Y chromosomes, with the breakage and recombination occurring in Yq11.2. Mar 1 was an isodicentric Y chromosome (idic(Y)pter to q11.2::q11.2 to pter), and mar2 was del(Y)(q11.2). The karyotype was mos 46,X,ish idic(Y)(q11.2)(DYZ3++,SRY++,DXZ1+,DYZ1-)[21]/46,X,ish del(Y)(q11.2)(DYZ3+,SRY+,DXZ1+,DYZ1-)[78]. Combined FISH, Y chromosome STS analysis, SNP microarray analysis and other technologies can facilitate determination of the nature of marker chromosomes.</p>

Genetic diagnosis and analysis of related genes for a pedigree with 2p25 and 12p13 cryptic rearrangements / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze chromosome aberration in a child with mental retardation and abnormalities and its parents.</p><p><b>METHODS</b>Chromosome G banding, multiplex ligation-dependent probe amplification, fluorescence in situ hybridization and single nucleotide polymorphisms array were employed for analysis.</p><p><b>RESULTS</b>Karyotype analysis revealed that the child was 46,XX and the father was 46,XY, while the mother was 46,XX, add (12)(p13). Subtelomeric region analysis with MLPA displayed that the child has reduced ACP1 gene copy number in 2p25 region and increased SLC6A12,KDM5A gene copy numbers in 12p11 region. SNP-array has fine mapped the duplication to 12p13.33-p12.3, a 15.142 Mb region, and a deletion to 2p25.3 for 3.194 Mb, which resulted in duplication of 9 genes including SLC6A12 as well as deletion of 11 genes including SNTG2, respectively. FISH analysis revealed that the child was 46,XX,ish,der(2),t(2;12)(p25;p13)mat, or partial monosomy 2p25 and partial trisomy 12p13. In addition,the mother was a carrier with cryptic balanced translocation chromosome, 46,XX,isht(2;12) (p25;p13). Mental abnormalities and retardation of the child may be attributed to heterozygous deletion of SNTG2, MYT1L genes and duplication of SLC6A12 gene.</p><p><b>CONCLUSION</b>Combined use of MLPA, FISH and SNP-array can facilitate accurate diagnosis of cryptic rearrangement at genomic level.</p>

Breakpoint localization of Y-chromosome massive deletions in 49 spermatogenesis dysfunction patients / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the characteristics of azoospermia factor(AZF) deletions in Y-chromosome.</p><p><b>METHODS</b>Based on the AZF microdeletion screening on 272 cases of azoospermia and 240 cases of severe oligozoo spermia, 49 cases were investigated using 23 sequence-tagged sites (STS) in AZFa, AZFb and AZFc. For some cases, single nucleotide rarians (SNV) method was applied to identify the single nucleotide polymorphism (SNPs) in four DAZ gene copies and to determine the copy number of the DAZ gene.</p><p><b>RESULTS</b>In 6 cases with deletions of AZFb+c, there was 1 case with sY98/sY1206 deletion, 4 cases with P5/distal-P1 recombination and 1 with P4/distal-P1 recombination. In 3 cases with deletions in AZFb, 1 case showed P5/P3 deletion and 2 cases showed P5/proximal-P1 recombination with DAZ1 and DAZ2 deletions. b2/b4 recombination was observed in all the 40 cases with deletions in AZFc. A fraction of patients with AZFb and AZFb+c deletions showed oligospermia and spermatogenic failure by testicular biopsy.</p><p><b>CONCLUSION</b>Breakpoint localization of deletions in AZF regions may help elucidating the mechanisms of microdeletions, and analysis of the characteristics and quantity of deleted genes essential for normal spermatogenesis may evaluate the association of phenotype with spermatogenic failure.</p>

Expression and preliminary use of human sperm-specific lactate dehydrogenase in Escherichia coli / 中华检验医学杂志

Objective To construct a recombinant vector of sperm-specific human lactate dehydrogenase ( hLDH-C4 ), express it in Escherichia coli ( E. coli ) BL21 ( DE3 ) and utilize it in the detection of anti-sperm antibody. Methods The coding sequence of hLDH-C4 was amplified from human testis λTripIEx cDNA library, and inserted into pET-28a( + ) after restriction enzyme digestion with Hind Ⅲ and Xho Ⅰ. The resultant recombinant vector was used to transform E. coli BL21 ( DE3 ) and the His-Tag fused hLDH-C4 was expressed after induction with IPTG. Western blot was used to analyzed the recombinant protein and LDH activity of bacterial lysates was determined. An indirect ELISA method for the detection of anti-sperm antibody was established by using the recombinant hLDH-C4 as antigen matrix. Results pET-28a( + )-hLDHC was successfully established. The protein with size of 35kD could be induced by IPTG when the recombinant plasmid was transfected into E. coli BL21 ( DE3 ). Western blot showed that the recombinant protein could be specifically recognized beth by anti-His tag monoclonal antibody and by rabbit anti-human LDH-C4 antibody. In addition, the recombinant protein showed high-level LDH activity when the bacterial lysate after IPIG induction was used to check LDH activity. The recombinant hLDH-C4 was confirmed when it was used in indirect EL1SA to detect anti-hLDH-C4 antibody. Conclusions The coding sequence of hLDH-C4 is cloned into the vector pET-28a( + ) and recombinant hLDH-C4 was expressed at a high level in E. coli. The recombinant hLDH-C4 is useful in the detection of anti-sperm antibody.

Prenatal molecular diagnosis of four fetuses at high risk for X-linked adrenoleukodystrophy / 中华妇产科杂志

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.

Expression and characterization of subunit C of mouse lactate dehydrogenase in Escherichia coli / 中华男科学杂志

<p><b>OBJECTIVES</b>To construct a prokaryotic recombinant vector for mouse lactate dehydrogenase-C and to detect its expression in BL21.</p><p><b>METHODS</b>The coding sequence of mouse lactate dehydrogenase subunit C was amplified from mouse testis RNA with specific primers, and cloned into pGEX-2T after the restriction digestion with BamH I and EcoR I. GST fusion protein was expressed after induction with IPTG.</p><p><b>RESULTS</b>Sequencing and restriction digestion of the recombinant plasmid revealed the existence of coding sequence for mouse lactate dehydrogenase subunit C. A protein band of about 60,000 could be induced by IPTG in the recombinant plasmid.</p><p><b>CONCLUSIONS</b>The coding sequence of mouse lactate dehydrogenase subunit C was introduced into the pGEX-2T plasmid and a GST-fused protein could be induced at a high level.</p>

Fetal karyotyping by color Doppler ultrasound-guided percutaneous umbilical blood sampling with fetuses cardiac anomalies / 中华超声影像学杂志

Objective To perform fetal karyotyping by percutaneous umbilical blood sampling guided by color Doppler ultrasonography in fetuses with congenital cardiac anomalies. Methods Fetal blood samples obtained by color Doppler ultrasound-guided cordocentesis in 56 fetuses with cardiac abnormalities detected by fetal echocardiography were taken for karyotyping.Fetuses were monitored post operation. Results The procedures were successfully performed in all cases and no procedure-related complications occurred.Six cases with abnormal karyotypes, 1 trisomy 21,2 trisomy 18,1 trisomy 13,1 trisomy AO and 1 cases 47XYY were identified and pre-operation ultrasonography detected multiple system anomalies besides cardiac anomalies in them. Conclusions Color Doppler ultrasonography facilitated percutaneous umbilical blood sampling by clear umbilical vein imaging and shortening the operation time.Abnormal karyotypes in fetuses with cardiac anomalies seemed to be related with multiple system anomalies.

Molecular diagnosis of puerile spinal muscular atrophy (with report of 10 cases) / 解放军医学杂志

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.