[Show abstract][Hide abstract] ABSTRACT: To provide genetic diagnosis and counseling for a 2-year-old girl with typical Rett syndrome through analyzing the methyl-CpG binding protein 2 (MECP2) gene.
Potential mutation of the MECP2 gene was screened by DNA sequencing and multiplex ligation-dependent probe amplification (MLPA) analysis of members of the family as well as normal controls. Lymphocyte culture for karyotype analysis was carried out for the patient to exclude chromosomal abnormalities.
The karyotype of the girl was normal. No variation of the MECP2 gene was detected in the patient by direct sequencing. A heterozygosis variation, c.1072G>A in exon 4 of the MECP2 gene was detected in a normal female control, which was not found in other controls. The son and daughter of the female control were respectively heterozygous and homozygous carriers of the same mutation. By MLPA analysis, a heterozygosis deletion of exon 3 and part of exon 4 was detected in the patient. cDNA amplification and sequencing confirmed the presence of a 1176 bp deletion (c.27-1202del1176). The same deletion was not detected in the parents.
A large deletion in MECP2 gene was detected with MLPA in a patient featuring typical Rett syndrome. The same deletion was missed by sequencing analysis. With cDNA sequencing, the breakage point of the mutation can be mapped precisely.
Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics 12/2011; 28(6):625-9. DOI:10.3760/cma.j.issn.1003-9406.2011.06.006
[Show abstract][Hide abstract] ABSTRACT: Multiple osteochondromas (MO), an inherited autosomal dominant disorder, is characterized by the presence of multiple exostoses on the long bones. MO is caused by mutations in the EXT1 or EXT2 genes which encode glycosyltransferases implicated in heparin sulfate biosynthesis.
In this study, efforts were made to identify the underlying disease-causing mutations in patients from two MO families in China.
Two novel EXT1 gene mutations were identified and no mutation was found in EXT2 gene. The mutation c.497T > A in exon 1 of the EXT1 gene was cosegregated with the disease phenotype in family 1 and formed a stop codon at amino acid site 166. The fetus of the proband was diagnosed negative. In family 2, the mutation c.1430-1431delCC in exon 6 of the EXT1 gene would cause frameshift and introduce a premature stop codon after the reading frame being open for 42 amino acids. The fetus of this family inherited this mutation from the father.
Mutation analysis of two MO families in this study demonstrates its further application in MO genetic counseling and prenatal diagnosis.
Chinese medical journal 10/2011; 124(19):3054-7. DOI:10.3760/cma.j.issn.0366-6999.2011.19.018 · 1.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To investigate the clinical utility of multiplex ligation-dependent probe amplification (MLPA) for detecting 22q11 deletion and duplication in congenital heart disease (CHD) cases and to study the incidence of 22q11 deletion and duplicaton in different kinds of CHD.
Forty eight probes of which 25 located in 22q11 low copy number region (LCR 22s A-H), 7 in 22q11 surrounding region (CES, 22q13) and 16 in chromosomes 4, 8, 10 and 17 were selected to detect 22q11 deletion and duplication in 181 preoperative children with CHD and 14 fetuses with serious CHD or CHD with multiple malformations. In these cases, karyotype analysis was also performed.
MLPA demonstrated that 7 cases had 22q11 deletion [6 cases from CLTCL1 to LZTR1(LCR A-D) and 1 case from CLTCL1 to PCQAP (LCR A-C)] and that 1 case had 22q11 duplication,spanning from ZNF74 to LZTR1(LCR B-D). The phenotypes of heart defect included ventricular septal defect, atrioventricular septal defect, pulmonary stenosis and tetralogy of Fallot. Karyotype analysis showed that 1 case had 21q deletion [46, XY, 21q], 1 case had mosaic trisomy 8 [47,XY, +8/46, XY(1:2)] and 4 cases had trisomy 21. One of the 4 cases with trisomy 21 had concurrent 22q11 duplication.
MLPA is a rapid, sensitive, site specific and relatively inexpensive method for diagnosis of 22q11 deletion and duplication in CHD. 22q11 deletion and duplication may cause various kinds of CHD, suggesting that genetic detection should be performed routinely in CHD patients.
Zhongguo dang dai er ke za zhi = Chinese journal of contemporary pediatrics 11/2009; 11(11):892-6.
[Show abstract][Hide abstract] ABSTRACT: To analyze the pathogenic mutation of sporadic patients in Duchenne/ Becker muscular dystrophy (DMD/BMD) families and to perform prenatal diagnosis, identify the female carriers and evaluate the ratio of de novo mutation in these pedigrees.
A total of 30 sporadic DMD/BMD families were included. Traditional multiplex PCR was used to detect the 18 exons in the deletion hot area of DMD gene. MLPA was used to detect the deletion or duplication mutations of DMD gene for 30 patients and 28 females from 23 families. Prenatal diagnosis was performed in 19 families.
Deletion mutation was detected in 19 patients through mPCR. Twenty-one deletions and 3 duplication mutations were detected by MLPA. Among 21 mothers, 10 mothers were carriers of deletion mutation and two duplication mutation carriers. The other 9 mothers were non-carriers, the mutations in these families were de novo and the ratio was 37.5%. Among seven sisters, five were carriers and two non-carriers. In the prenatal diagnosis families, 2 of 8 female fetuses were carriers and 5 of 12 male fetus patients.
The MLPA technique has proved to be an accurate and reliable tool not only for the deletion and duplication mutations of DMD patients, but also for the prenatal diagnosis and the female carriers in these families. Prenatal diagnosis;
Zhonghua yi xue za zhi 07/2009; 89(25):1753-6. DOI:10.3760/cma.j.issn.0376-2491.2009.25.009
[Show abstract][Hide abstract] ABSTRACT: To analyze the pathogenic mutation of an X-linked ichthyosis (XLI) family, and identify the genetic diagnosis of three probable female carriers in this family. To evaluate the availability of different detect methods for steroid sulfatase (STS) gene mutation.
Peripheral blood samples were collected from the family, including the proband, proband's mother, younger sister, and younger female cousin, and 10 males and 10 females as controls. Ordinary PCR was used to detect whether there was STS gene deletion in the male proband. Then, multiplex quantitative fluorescent PCR (QF-PCR) was used to detect the STS gene in the proband and his 3 female family members. Fluorescence in situ hybridization (FISH) was used to authenticate the results of multiplex QF-PCR method.
No amplified product of the exons 1-10 of STS gene deletion was detected by ordinary PCR in the proband. The proband's mother was diagnosed as a carrier, but his sister and cousin were diagnosed as normal females by multiplex QF-PCR. FISH confirmed the results of multiplex QF-PCR.
Both multiplex QF-PCR and FISH are effective to detect the complete deletion mutation of STS gene and identify the female carrier, and multiplex QF-PCR is more convenient and automatic compared with FISH.
[Show abstract][Hide abstract] ABSTRACT: To explore the genetic prenatal diagnosis method for achondroplasia (ACH).
During May to November 2007, three ACH pedigrees were diagnosed at the Prenatal Diagnosis Center, Department of Obstetrics and Gynecology, Affiliated Drum Tower Hospital of Medical College, Nanjing University. In family 1, there was a 6-month-old male ACH infant. In family 2, the expectant mother, with 18 weeks of pregnancy, was an ACH patient. Amniocentesis was performed for prenatal diagnosis. The fetus of family 3 was diagnosed as ACH by ultrasound examination on the 39th week of gestation. Umbilical cord blood of this fetus was collected for examination. Totally, three methods, restriction enzyme (SfcI and MspI) digestion analysis, denaturing high performance liquid chromatography (DHPLC) and sequencing analysis were performed simultaneously to detect the pathogenic mutation of fibroblastic growth factor receptor 3 (FGFR3) for the three ACH families.
(1) The DHPLC detection: heteroduplex was detected in the patient of family 1; both the patient and the fetus of family 2 showed heteroduplex results; the result of the fetus of family 3 was also heteroduplex. (2) The enzyme digestion analysis for the PCR products of 10 exon of FGFR3: after SfcI digestion, the PCR products of patients and the fetus of family 1 and 2 showed not only the band of 247 bp, but also bands of 162 bp and 85 bp. But their PCR products could not be digested by MspI, and it only showed the band of 247 bp. For the fetus of family 3, the PCR products could not be digested by SfcI, while after digestion by MspI, bands of 162 bp and 85 bp were shown up. The PCR products of the normal control could be digested by neither SfcI nor MspI. (3) The sequencing results: the heterozygote mutation of 1138 G-->A was confirmed in the patient of family 1. The pregnant woman and her fetus in family 2 showed the same result. The heterozygote mutation of G-->C was confirmed in the fetus of family 3. The site of 1138 was G homozygote in the normal control. The three detection results of the fetus in family 2 were the same as that of the mother, which means that the fetus inherited the same pathogenic mutation from his or her mother.
Both DHPLC and restriction enzyme digestion analysis could detect the mutation of FGFR3 gene, but DHPLC is more rapid, convenient and sensitive. So DHPLC can be applied to genetic diagnosis and prenatal diagnosis for ACH patients.
[Show abstract][Hide abstract] ABSTRACT: To explore the feasibility of application of multiplex quantitative fluorescent PCR with non-polymorphic loci in prenatal diagnosis of aneuploidies.
From Mar 2006 to Nov 2007, a total of 63 samples were collected from the Department of Obstetrics and Gynecology, Affiliated Drum Tower Hospital of Medical College, Nanjing University, including 54 villous samples obtained for karyotyping because of spontaneous abortion, six amniotic fluid samples of second trimester and three umbilical cord blood samples of third trimester. Blood samples of 60 healthy adults were obtained at the same time as a control group, including 30 males and 30 females. Non-polymorphic QF-PCR was performed on both testing group and control group for the detection of aneuploidies. The Amelogenin gene (AMXY) was selected as an internal control, and dosage quotiety (DQ) of each locus was calculated according to the known formula. If DQ was between 0.7 and 1.3, the sample was considered as normal. If the figure turned out to be > 1.3 or < 0.7, a potential duplication or deletion of the corresponding gene or chromosome was indicated. If the results implied numerical abnormalities in more than one euchromosome, sex chromosome aneuploidies should be considered. Cell culture and karyotyping were carried out for every sample simultaneously. The results of non-polymorphic QF-PCR were checked with karyotypes.
(1) In the control group, all female samples presented only an AMX peak for sex chromosome while all males showed AMX and AMY amplified peaks. The AMY/AMX ratios were between 0.7 - 1.3, and SD was between 0.05 - 0.12. (2) Among 19 QF-PCR abnormal cases, 13 cases were proved by karyotyping. Of the six cases which turned out to be conflicting, one case of trisomy 18 shown by karyotyping was not completely detected by QF-PCR, a locus on chromosome 18 implied trisomy, while another turned out to be normal (DQ = 1.28). Four cases were detected by non-polymorphic QF-PCR as trisomies but showed normal female karyotype because of maternal contamination during cell culture. A karyotypingly '46, XY' case did not present an AMY peak. Thirty-six out of 44 (82%) normal results implied by non-polymorphic QF-PCR were in accordance with cytogenetic analysis. Of the other eight cases, one case which failed cytogenetic analysis was detected by QF-PCR as normal. Four cases showed multiploidy by karyotyping but normal in QF-PCR analysis, including three cases of 69, XXX, one case of 92, XXXX and one case of 45, XX, rob (13;21). The other two cases that showed normal male results turned out to be normal female karyotypes.
Prenatal aneuploidy detection by non-polymorphic QF-PCR is feasible in a clinical diagnostic setting. With the advantages of high throughput, rapidness and low cost, this method shows a good prospect in clinical application.
Zhonghua fu chan ke za zhi 11/2008; 43(11):818-23.
[Show abstract][Hide abstract] ABSTRACT: To detect the mutation of the SEDL gene in an X-linked spondyloepiphyseal dysplasia tarda (SEDL) family.
Two patients and three females of the X-SEDL family were detected using reverse transcriptase PCR (RT-PCR) and sequence analysis.
A G209A mutation of SEDL gene was detected in the cDNA sequences of the patients, which was confirmed by sequence analysis of the exon 4 of the SEDL gene. The daughter of the proband was a carrier of the mutation.
Since the SEDL gene is relatively small, sequence analysis of cDNA of the SEDL gene was possible after extraction of total RNA followed by RT-PCR. Mutations in the open reading frame can be detected y by cDNA sequencing. It was relatively more rapid and direct than amplifying and detecting the exons one by one.
Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics 09/2008; 25(4):421-3.
[Show abstract][Hide abstract] ABSTRACT: To elucidate the pathogenic genes in a pedigree with autosomal dominant ichthyosis vulgaris (IV).
Linkage analysis was performed by using STR markers in chromosome 1, and mutation detection was used to screen for FLG gene mutation.
A maximum two-point Lod score of 3.46 (theta=0) was obtained at D1S2696. Haplotype analysis placed the critical region in a 15-CM interval defined by D1S2726 and D1S305, but no mutation of FLG was found in our IV patients.
The pathologic gene of the IV family locates near D1S2696, and the FLG gene may not ruled out from the pathologic genes.
Zhongguo yi xue ke xue yuan xue bao. Acta Academiae Medicinae Sinicae 07/2007; 29(3):302-6.
[Show abstract][Hide abstract] ABSTRACT: To detect two exons of Duchenne muscular dystrophy (DMD) gene and a gender discrimination locus amelogenin gene by single cell triplex PCR, and to evaluate the possibility of this technique for preimplantation genetic diagnosis (PGD) in DMD family with DMD deletion mutation.
Single lymphocytes from a normal male, a normal female, two DMD patients (exon 8 and 47 deleted, respectively) and single blastomeres from the couples treated by the in vitro fertilization pre-embryo transfer (IVF-ET) and without family history of DMD were obtained. Exons 8 and 47 of DMD gene were amplified by a triplex PCR assay, the amelogenin gene on X and Y chromosomes were co-amplified to analyze the correlation between embryo gender and deletion status.
In the normal single lymphocytes, the amplification rate of exons 8 and 47 of DMD and amelogenin gene were 93.8%, 93.8%, and 95.3% respectively. The false positive rate was 3.3%. In the exon 8 deleted DMD patient, the amplification rate of exon 47 of DMD and amelogenin gene was 95.8%, and the false positive rate was 3.3%. In the exon 47 deleted DMD patient, the amplification rate of exon 8 of DMD and amelogenin gene was 95.8%, and the false positive rate was 0. In the single blastomeres, the amplification rate of exons 8 and 47 of DMD and amelogenin gene was 82.5%, 80.0% and 77.5%, respectively, and the false positive rate was 0.
The single cell triplex PCR protocol for the detection of DMD and amelogenin gene is highly sensitive, specific and reliable, and can be used for PGD in those DMD families with DMD deletion mutation.
Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences 05/2007; 32(2):246-51.
[Show abstract][Hide abstract] ABSTRACT: To investigate the correlation between male infertility and Y chromosome microdeletions of azoospermia factor (AZF) regions, and to establish a reliable genetic diagnosis in idiopathic infertile male patients with azoospermia or severe oligozoospermia.
Multiplex PCR amplification of 6 sequence-tagged sites in AZF regions of the Y chromosome was examined among 100 normal karyotype male patients with azoospermia or oligozoospermia.
Four patients (4%) had Y chromosome microdeletions, the microdeletions of 3 patients were idiopathic azoospermic and those of the other 1 patient were secretory azoospermia.
The PCR-based Y chromosome microdeletion screening is simple and effective in the diagnosis of patients with severe male infertility. Microdeletion of Y chromosome is one of the major causes of severe dyszooospermia.
Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences 05/2007; 32(2):241-5.
[Show abstract][Hide abstract] ABSTRACT: By using multiple polymerase reaction (mPCR) and haploid analysis of 11 short tandem repeats (STRs) in dystrophin gene locus to identify female carriers in deletional DMD/BMD (Duchenne/Becker Muscular Dystrophy) families, valuable information can be gathered for prenatal diagnosis. In this article, de novo mutations were detected in two out of the four patients, and one of the four female members was identified as an obligate DMD gene carrier based on the haplotype analysis. Multiple PCR and STRs haploid linkage analysis are rapid, accurate, objective methods to identify female member status, and well suited for routine use in clinical laboratories engaged in DMD/BMD research for counseling, gene diagnosis and prenatal diagnosis. During mPCR analysis, the amplicon of exon 45 showed different electrophoresis mobility in different kinds of gels. Polyacrylamide Gels Electrophoresis (PAGE) was accurate and rapid for analyzing the products of mPCR, but the mobility of different amplicons need to be considered in data analysis.