Two novel TSC2 frameshift mutations in tuberous sclerosis complex / 中国当代儿科杂志

High-throughput sequencing was performed for the peripheral blood DNA from two probands in the family with tuberous sclerosis complex (TSC) to determine the sequences of TSC-related genes TSC1 and TSC2 and their splicing regions and identify mutation sites. Amplification primers were designed for the mutation sites and polymerase chain reaction and Sanger sequencing were used to verify the sequences of peripheral blood DNA from the probands and their parents. The two probands had c.3981-3982 insA (p.Asp1327AspfsX87) and c.4013-4014 delCA (p.Ser1338Cysfs) heterozygous mutations, respectively, in the TSC2 gene. The parents of proband 1 had no abnormalities at these two loci; the mother of proband 2 had c.4013-4014 delCA heterozygous mutation in the TSC2 gene, while the father and the grandparents of proband 2 had no abnormalities. c.3981-3982 insA mutation may cause early coding termination of amino acid sequence at the 1413th site, and c.4013-4014 delCA mutation may cause early coding termination of amino acid sequence at the 1412th site. These two mutations are the pathogenic mutations for families 1 and 2, respectively, and both of them are novel frameshift mutations, but their association with the disease needs to be further verified by mutant protein function cell model and animal model.

Gene mutation analysis and prenatal diagnosis of four pedigrees with methymalonic aciduria / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study gene mutations in four pedigrees with methymalonic aciduria, as well as the feasibility of prenatal diagnosis of methymalonic aciduria.</p><p><b>METHODS</b>High-throughput sequencing was performed for related genes in the peripheral blood of children or parents who were diagnosed with methymalonic aciduria to identify the loci with mutations. Then amplification primers were designed for each locus, and PCR and direct sequencing were performed to validate the sequencing in the first generation in the four pedigrees. Whether the mutations were pathogenic were determined with reference to literature review and medical history. In the pedigrees 1, 3, and 4, ultrasound-guided chorionic villi biopsy was performed at weeks 11-13 of pregnancy to perform early prenatal diagnosis.</p><p><b>RESULTS</b>In pedigree 1, c.656A>T and c.729-730insTT heterozygous mutations in the MUT gene were detected in the proband's father and mother, respectively. Early prenatal diagnosis showed c.656A>T and c.729-730insTT double heterozygous mutations in the fetus. The couple decided to terminate pregnancy. In pedigree 2, c.1106G>A and c.755-756insA double heterozygous mutations in the MUT gene were detected in the proband. c.1106G>A came from the father and c.755-756insA came from the mother. In pedigree 3, c.217C>T and c.609G>A double heterozygous mutations in the MMACHC gene were detected in the proband. c.217C>T came from the father and c.609G>A came from the mother. Prenatal diagnosis showed c.609G>A heterozygous mutation in the fetus. The baby was successfully delivered, and the result of umbilical cord blood testing was consistent with the prenatal diagnosis. In pedigree 4, c.609G>A and c.567dupT double heterozygous mutations in the MMACHC gene were detected in the proband. c.609G>A came from the father and c.567dupT came from the mother. Prenatal diagnosis showed c.567dupT heterozygous mutation in the fetus. The baby was successfully delivered, and the result of umbilical cord blood testing was consistent with the prenatal diagnosis.</p><p><b>CONCLUSIONS</b>Identification of gene mutations helps with prenatal diagnosis in pedigrees with methymalonic aciduria.</p>

Genetic and prenatal diagnosis for four families with Williams-Beuren syndrome / 中国当代儿科杂志

Williams-Beuren syndrome is a common chromosome microdeletion syndrome. Early diagnosis and treatment are very helpful for patients and their families. This study identified the chromosome karyotype in one fetus with ultrasonography abnormalities and three children with developmental disorders from four families. This provided guidance for subsequent pregnancy and prenatal diagnosis by using routine G-banding chromosome karyotyping analysis, multiplex ligation-dependent probe amplification (MLPA) and array comparative genomic hybridization (array-CGH). In one amniotic fluid sample from a pregnant woman with fetal abnormalities on an ultrasound screen and three peripheral blood samples from three children with developmental disorders, the decreased signal of ELN gene probes at 7q11.23 and heterozygous deletions at 7q11.23 were detected by MLPA and array-CGH analysis. The laboratory genetic tests of amniotic fluid samples were normal when the mothers from the four families became pregnant again. It was concluded that MLPA and array-CGH are rapid and accurate tools for the diagnosis of Williams-Beuren syndrome and can provide more information for clinical genetic counseling.

Application of multiplex ligation-dependent probe amplification for rapid diagnosis of chromosomes aneuploid abnormalities / 中华实用儿科临床杂志

Objective To explore the application value of multiplex ligation-dependent probe amplification (MLPA) for diagnosing chromosome aneuploid abnormality diseases.Methods Seven hundred and thirty cases of prenatal samples were tested with MLPA detection and by G-band karyotype analysis.If the findings from the samples were different by two methods,they were then detected by fluorescence in situ hybridization (FISH).The sensitivity,specificity and positive predictive value of MLPA were calculated.Results There were 709 normal euploidies,12 cases of Down syndrome,4 cases of Eswards syndrome,1 case of Patau syndrome,1 case of Turner syndrome,1 case of 47,XXX,1 case of 47,XYY,1 case of 46,XY/47,XY + 21.The sensitivity,specificity and positive predictive value of MLPA were respectively 95％,100％ and 100％,respectively.Conclusion MLPA is a rapid and efficient method for analyzing aneuploids and has the good value of clinical applications.

Cytogenetic and molecular analysis of idic(Yp) in 1 infertile man and 1 prenatal fetus / 中华男科学杂志

<p><b>OBJECTIVE</b>To evaluate idic(Yp) in genetic diagnosis by examining 1 infertile man and 1 prenatal fetus using cytogenetic and molecular techniques.</p><p><b>METHODS</b>Following conventional chromosome preparation, we performed G- and C-banding karyo. typing and fluorescence in situ hybridization (FISH). Then we extracted genomic DNA using standard procedures and analyzed it by array-CGH and multiplex ligation-dependent probe amplification (MLPA).</p><p><b>RESULTS</b>Both cases were diagnosed as 45, X/46, X, idic (Yp11.31) mosaicism. The man showed 2 intact copies of Yp11.31-q12 (chrY:2, 710, 250-57, 428, 567, SRY, ZFY, UTY and AZF), and the prenatal fetus exhibited similar findings except a paternal deletion in the AZFc region.</p><p><b>CONCLUSION</b>idic(Y) (p11.31) causes short stature and male infertility. Array-CGH and MLPA can improve the accuracy of the diagnosis of 45, X/46, X, idic (Y) mosaicism, which may contribute to the studies of the phenotype-genotype correlation and clinical genetic counseling.</p>

One case of 2q37 deletion syndrome: clinical and genetic diagnosis / 中华儿科杂志

<p><b>OBJECTIVE</b>To diagnose a new born baby with 2q37 deletion syndrome by comprehensive use of cytogenetic and molecular techniques and to investigate the phenotype characteristics and applicability of array-comparative genomic hybridization (array-CGH) and multiplex ligation-dependent probe amplification (MLPA) for detection of this syndrome.</p><p><b>METHOD</b>Following conventional chromosome preparation, G banded karyotyping was performed.Genomic DNA was extracted using standard procedures, which were then analyzed by array-CGH and MLPA.</p><p><b>RESULT</b>The patient presented with a typical face, special fist posture and congenital heart disease in 2q37 deletion syndrome. A 4.709 Mb deletion at 2q37.3 (chr2:237, 967, 852-242, 677, 269.NCBI36/hg18, including genes from COL6A3 toPDCD1) was detected by array-CGH. The results of MLPA and G banded karyotyping confirmed the existence of this deletion.</p><p><b>CONCLUSION</b>2q37.3 deletion was determined to be the cryptic cause of this case.2q37 deletion syndrome has some clinically recognizable characteristics. And array-CGH is a powerful technique for the accurate diagnosis and genotype-phenotype correlation study of this syndrome.</p>

Analysis of copy number variations in an infant with Cri du Chat syndrome by array-based comparative genomic hybridization / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze genomic copy number variations in an infant with Cri du Chat syndrome, and to explore the underlying genetic cause.</p><p><b>METHODS</b>G-banding analysis was carried out on cultured peripheral blood sample from the patient. Copy number variation analysis was performed using microarray comparative genomic hybridization, and the result was verified with fluorescence in situ hybridization.</p><p><b>RESULTS</b>The infant was found to have a 46, XY, der(5) (p?) karyotype. By microarray comparative genomic hybridization, a 23.263 Mb deletion was detected in 5p14.2-p15.3 region in addition to a 14.602 Mb duplication in 12p31 region. A derivative chromosome was formed by rejoining of 12p31 region with the 5p14.2 breakpoint. The patient therefore has a karyotype of arr cgh 5p15.3p14.2 (PLEKHG4B>CDH12)× 1 pat, 12p13.33p13.1 (IQSEC3>GUC Y2C)× 3 pat. Loss of distal 5p and gain of distal 12p were verified with fluorescence in situ hybridization.</p><p><b>CONCLUSION</b>The Cri du Chat syndrome manifested by the patient was caused by deletion of distal 5p from an unbalanced translocation involving chromosome 5. Microarray comparative genomic hybridization is a powerful tool for revealing genomic copy number variations for its high-resolution, high-throughput and high accuracy.</p>

Paternally originated Wolf-Hirschhorn syndrome detected by multiplex ligation-dependent probe amplification and microarray comparative genomic hybridization / 中华儿科杂志

<p><b>OBJECTIVE</b>To confirm the diagnosis of a Wolf-Hirschhorn syndrome by family study using both cytogenetic and molecular genetic techniques.</p><p><b>METHOD</b>G-band karyotyping was performed for all the 6 members in the family. Multiplex ligation-dependent probe amplification (MLPA) was used to detect the chromosome abnormality for the proband, his father and brother. Microarray comparative genomic hybridization (Array-CGH) was carried out to map the exact chromosomal breakpoints for the proband.</p><p><b>RESULT</b>The proband presented with a typical face, delayed growth and hypotonia in Wolf-Hirschhorn syndrome. His G-band karyotype was 46, XY, der(4)t(4;8) (p16.2; p23.1)pat. MLPA showed 4pter loss and 8pter gain. Array-CGH revealed an XY male with a 3.781 Mb deletion of 4p16.3-p16.2 and a 6.760 Mb duplication of 8p23.3-p23.1. The proband's brother has mental retardation and skeletal abnormalities. His G-band karyotype was 46, XY, der(8)t(4;8)(p16.2;p23.1)pat. MLPA showed 4pter gain and 8pter loss. The proband's father had normal phenotype with a balanced translocation of 46, XY, t(4;8)(p16.2;p23.1)pat. MLPA showed a normal result. The proband's grandfather showed a normal phenotype with a balanced translocation 46, XY, t(4;8)(p16.2;p23.1). The other members in the family showed normal phenotypes with normal karyotypes.</p><p><b>CONCLUSION</b>The proband has features of Wolf-Hirschhorn syndrome with partial monosomy 4p and partial trisomy 8p. The proband's brother has a partial trisomy 4p and partial monosomy 8p. The derived chromosomes are inherited from paternal balanced translocation t(4;8)(p16.2;p23.1).</p>

Molecular and prenatal diagnosis for a Chinese pregnant woman with a novel mutation of β thalassemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To conduct molecular and prenatal diagnosis for a couple with β thalassemia.</p><p><b>METHODS</b>Blood routine examination and hemoglobin analysis were used for screening of thalassemia. Seventeen common Chinese mutations of β thalassemia were detected for the carriers with β thalassemia using PCR/RDB. The unknown mutation of β thalassemia was identified by DNA sequencing and DHPLC analysis.</p><p><b>RESULTS</b>The husband was heterozygote of CD41/42 (-TCTT). The wife carried a mutation IVS-I-110 (G→A) of β thalassemia having not been reported in Chinese so far. The fetus was a double mutated heterozygote of IVS-I-110 (G→A) and CD41/42 (-TCTT). The pregnancy was terminated.</p><p><b>CONCLUSION</b>Mutation IVS-I-110 (G→A) of β thalassemia in Chinese is of importance to the genetic counseling and prenatal diagnosis of thalassemia.</p>

Mutation of thyroid peroxidase gene in 35 patients with congenital hypothyroidism / 中华儿科杂志

<p><b>OBJECTIVE</b>To identify thyroid peroxidase (TPO) gene mutations in 35 patients with congenital hypothyroidism.</p><p><b>METHOD</b>Genomic DNA was isolated from peripheral blood samples of 35 patients with congenital hypothyroidism. All of the 17 exons and flanking introns of TPO gene were amplified by PCR, then the PCR products were sequenced bi-directionally and were analyzed by restriction endonucleases.</p><p><b>RESULT</b>One patient had compound heterozygous mutations c.961A>G/c.2422delT, one was c.2268insT/c.1477G>A, and three was homozygous mutation c.2268insT. The TPO gene mutation c.961A>G [p. Thr321Ala] was one novel mutation.</p><p><b>CONCLUSION</b>High frequency mutation in TPO gene was detected in patients with congenital hypothyroidism.</p>

A novel mutation Glu441stop (GAA to TAA) of androgen receptor gene resulting in complete androgen insensitivity syndrome / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify the mutation of human androgen receptor gene (AR) in a patient with complete androgen insensitivity syndrome (CAIS).</p><p><b>METHODS</b>DNA sequences of 8 exons and their exon/intron boundaries of the AR gene in the patient were amplified by PCR and directly sequenced.</p><p><b>RESULTS</b>DNA sequencing revealed a nonsense mutation in exon 1, resulting in a change of codon 441 GAA (glutamic acid) to a stop codon (TAA).</p><p><b>CONCLUSION</b>A novel mutation Glu441stop (GAA to TAA) of the androgen receptor gene leading to complete androgen insensitivity syndrome was identified in this study in a Chinese patient. It may help us further understanding the pathogenesis of CAIS.</p>

Clinical significance of large-scale screening of A1555G mutation of mitochondria DNA for neonates / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the necessity of large-scale screening of mitochondria DNA (mtDNA) A1555G mutation for prevention of aminoglycoside antibiotic induced deafness in newborns.</p><p><b>METHODS</b>One thousand blood filter samples were collected from neonates born in July 2008 in Shenzhen. DNA was extracted with Chelex-100 Resin and amplified by PCR. The mtDNA A1555G mutation was determined by denaturing high-performance liquid chromatography(DHPLC) for PCR products. The positive frequency was calculated.</p><p><b>RESULTS</b>The mitochondrial DNA A1555G mutation was detected in 2 cases of 1000 neonates. The frequency of mutation was 0.2%.</p><p><b>CONCLUSION</b>There is a high frequency of mtDNA A1555G mutation in neonates, the large-scale screening of mtDNAA1555G mutation in newborns might detect the individuals sensitive to aminoglycoside antibiotic, which is helpful to guide a rational medication for newborns and the maternal relatives at high-risk. Furthermore, it might be useful to prevent aminoglycoside antibiotic induced deafness.</p>

Analysis of fetal chromosomal karyotype and etiology in 252 cases of early spontaneous abortion / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the relationship between fetal chromosomal karyotype and early spontaneous abortion, and the effect of the environmental factors on spontaneous abortion.</p><p><b>METHODS</b>Choronic villi from 252 cases of missed abortion were sampled as patient group and 50 normal pregnancies as control group. Chorionic villi were cultured and karyotype analysis was performed by G-banding. Clinical information was collected.</p><p><b>RESULTS</b>The rate of chorion chromosome abnormality in the patient group was 58.09%, significantly higher than that in the control group (4.17%) (P<0.01). Among the 140 cases of karyotype abnormalities, 81 were trisomy, 29 were monosomy X and 17 were polyploidy, accounting for 57.86%, 20.71% and 12.14% of total abnormalities, respectively. Long time and low dose radiation exposure of the pregnant female seemed to be related with spontaneous abortion(P<0.01).</p><p><b>CONCLUSION</b>Chorion chromosome abnormality is a major reason of early spontaneous abortion and karyotype analysis of chorionic villus is of clinical importance. For pregnant women, long-term exposure to computers and television seems a risk factor for missed abortion.</p>

Genetic analysis and prenatal diagnosis for a family with megalencephalic leukoencephalopathy and subcortical cysts / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify potential mutation in the MLC1 gene in a Chinese family affected with megalencephalic leukoencephalopathy and subcortical cysts (MLC), and to provide prenatal diagnosis.</p><p><b>METHODS</b>Genomic DNA of the patients, their parents and younger sister were extracted from peripheral blood. That of the fetus was extracted from an amniotic fluid sample. A total of 12 exons and at least 100 bp flanking the intronic sequence of the MLC1 gene were amplified with PCR. MLC1 mutations were screened by sequencing. Linkage analysis was performed for the family to assure accuracy of prenatal diagnosis.</p><p><b>RESULTS</b>The two patients were both heterozygote for c.177_178delG (p.Ser60AlafsX5) mutation in exon 2 and c.598-2A>C change in intron 7. The c.177_178delG mutation was inherited from the father, and the c.598-2A>C mutation was inherited from the mother. The younger sister and the fetus have both inherited c.177_178delG from the father but did not inherit c.598-2A>C from the mother. Prenatal diagnosis suggested the fetus to be a carrier for a MLC1 mutation. Linkage analysis was consistent with the result of mutation detection. The fetus was born normal as predicted.</p><p><b>CONCLUSION</b>The c.598-2A>C is a novel splicing mutation. Prenatal diagnosis through DNA sequencing and linkage analysis were performed for the first time on Chinese patients with MLC.</p>

Effect of neoadjuvant chemotherapy on ERCC1 gene expression in breast cancer / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the expression of ERCC1 gene in breast cancer before and after neo-adjuvant chemotherapy.</p><p><b>METHODS</b>The expression of ERCC1 gene was detected by RT-PCR in 40 breast cancer patients and in 14 patients treated with neoadjuvant chemotherapy.</p><p><b>RESULTS</b>Positive expression of ERCC1 gene was detected by RT-PCR in 35.0% of the breast cancer specimens, and ERCC1 expression was not correlated to the patients' age, tumor size, axillary lymph node metastasis, pathological type, histological grade, ER, PR or HER-2 (P>0.05). ERCC1 gene expression was significantly higher in neo-adjuvant chemotherapy group than in non-chemotherapy group (P<0.05).</p><p><b>CONCLUSION</b>The expression of ERCC1 gene does not affect the clinical and pathological features of breast cancer. Neo-adjuvant chemotherapy can increase the expression of ERCC1 gene, due attention should be given to with in subsequent chemotherapy.</p>