Clinical and molecular cytogenetic analyses of four patients with imbalanced translocations.

BACKGROUND: Chromosomal abnormalities that result in genomic imbalances are main causes of congenital and developmental anomalies including intellectual disability and multiple congenital malformations. In this report we describe four patients from three families with imbalanced translocations. Only a small percentage of imbalanced translocation individuals can be born to live, most of them were aborted in embryonic period. It is of great significances to precisely analysis the chromosome variation to study the relationship between genotype and phenotype. RESULTS: Four patients showed common clinical manifestations including delayed growth, intellectual disability, language barrier and facial dysmorphisms. In addition to the above features, lower limbs dysplasia and both foot eversion were found in patient 1, brachydactylic hand, cerebellar ataxia and congenital heart defects were also found in patient 4. Conventional karyotype analysis revealed abnormal karyotypes 46, XX, der (6) t (6: 10) (p23; q24), 46, XX, der (20) t (3; 20) (p23; p13) and 46, XX, der (22) t (3; 22) (q27; q13.3) in the four patients, respectively. Array-CGH analyses confirmed 23.6 Mb duplication on 10q25.1-q26.3 and 0.9 Mb deletions on 6p25.3, 19.9 Mb duplication on 3p24.3-p26.3 and 0.25 Mb deletion on 20p13 and 12.5 Mb duplication on 3q27.2-q29 and 1.9 Mb deletions on 22q13.2-q13.33 in the four patients, respectively. CONCLUSION: Parents with balanced translocation are passed the imbalanced chromosome to patient, and the partial monosomy and partial trisomy lead to multiple congenital malformations of four patients.

[Detection and prenatal diagnosis for RS1 gene mutations in two Chinese families with X-linked juvenile retinoschisis].

OBJECTIVE: To identify potential mutations of retinoschisis 1 (RS1) gene responsible for X-linked retinoschisis (XLRS) in two Chinese families. METHODS: The 6 exons and flanking intronic regions were analyzed with PCR and direct sequencing. RESULTS: Two RS1 mutations were identified in the two families, which included 1 frameshift mutation (c.573delG, p.Pro192fs) and 1 missense mutation (c.626G>A, p.Arg209His). CONCLUSION: Two RS1 mutations have been identified, among which Pro192fs mutation is discovered for the first time in Chinese population. Above results may enrich our understanding of the clinical manifestations of XLRS and facilitated early diagnosis and genetic counseling for the disease.

[Significance of detecting free DNA from maternal plasma for the diagnosis of fetal chromosomal aneuploidies].

OBJECTIVE: To determine the feasibility and accuracy of detecting numerical chromosomal abnormalities by high-flux sequencing analysis of free fetal DNA from maternal plasma. METHODS: High-flux sequencing was applied to analyze fetal chromosome sequence copy numbers in 153 pregnant women. Fetal karyotyping was also carried out on amniocentesis samples. RESULTS: Six cases were detected with fetal chromosomal abnormalities by high-flux sequencing analysis, among which five were confirmed by karyotyping to be chromosomal aneuploidies (47,XYY; 45,X; 47,XY,+18; 47,XY,+21 and 47,XY,+13), 1 case was confirmed to be structural rearrangement, i.e., 46,XY,der(13;21)(q10;q10),+21. Furthermore, 3 chromosomal polymorphisms (one 46,XY,21p+ and two 46,XY,Yqh-) were identified. The two methods yielded similar results on fetal chromosome copy number detection. CONCLUSION: High-flux sequencing analysis of free DNA derived from maternal plasma is efficient for detecting fetal chromosomal aneuploidies, and is non-invasive, highly sensitive and specific. It therefore has a broad application in antenatal diagnosis.

Rapid screening for chromosomal aneuploidies using array-MLPA.

BACKGROUND: Chromosome abnormalities, especially trisomy of chromosome 21, 13, or 18 as well as sex chromosome aneuploidy, are a well-established cause of pregnancy loss. Cultured cell karyotype analysis and FISH have been considered reliable detectors of fetal abnormality. However, results are usually not available for 3-4 days or more. Multiplex ligation-dependent probe amplification (MLPA) has emerged as an alternative rapid technique for detection of chromosome aneuploidies. However, conventional MLPA does not allow for relative quantification of more than 50 different target sequences in one reaction and does not detect mosaic trisomy. A multiplexed MLPA with more sensitive detection would be useful for fetal genetic screening. METHODS: We developed a method of array-based MLPA to rapidly screen for common aneuploidies. We designed 116 universal tag-probes covering chromosomes 13, 18, 21, X, and Y, and 8 control autosomal genes. We performed MLPA and hybridized the products on a 4-well flow-through microarray system. We determined chromosome copy numbers by analyzing the relative signals of the chromosome-specific probes. RESULTS: In a blind study of 161 peripheral blood and 12 amniotic fluid samples previously karyotyped, 169 of 173 (97.7%) including all the amniotic fluid samples were correctly identified by array-MLPA. Furthermore, we detected two chromosome X monosomy mosaic cases in which the mosaism rates estimated by array-MLPA were basically consistent with the results from karyotyping. Additionally, we identified five Y chromosome abnormalities in which G-banding could not distinguish their origins for four of the five cases. CONCLUSIONS: Our study demonstrates the successful application and strong potential of array-MLPA in clinical diagnosis and prenatal testing for rapid and sensitive chromosomal aneuploidy screening. Furthermore, we have developed a simple and rapid procedure for screening copy numbers on chromosomes 13, 18, 21, X, and Y using array-MLPA.

[Screening for chromosomal abnormalities using nuchal translucency measurement with materal serum biochemistry markers in first trimester].

OBJECTIVE: To Investigate the performance of prenatal screening for chromosomal abnormalities in first trimester. METHODS: Maternal serum were collected from 2 739 pregnant women between 11 and 14 weeks gestation. Free beta human chorionic gonadotrophin(beta-hCG), pregnancy-associated plasma protein(PAPP-A) from materal serum were measured using time resolved fluorescence immunoassay(TRFIA) and fetal nuchal translucency(NT) thickness were measured using transabdominal or transvaginal ultrasound. 22 chromosomal defects were diagnosed in 22 cases using karyotyping. The levels of three markers were analyzed among 22 cases and 870 controls. RESULTS: The level of three markers were significant difference between affected and unaffected pregnancies. In affected cases, the value or level of NT and free beta-hCG were higher, while the level of PAPP-A was lower. We found that screening for chromosomal defects using a combination of NT and serum biochemistry was associated with a detection rate of 91.67% for all types of chromosomal defects, with a false-positive rate of 11.16%. CONCLUSION: A combination of nuchal translucency measurement with materal serum biochemistry markers provides an effective method of screening for chromosomal defects.

[Establishment of permanent lymphoblastoid cell lines of 47 patients with abnormal chromosome karyotype].

To increase the efficiency of in vitro transformation of human lymphocytes by Epstein-Barr virus (EBV) and establish permanent lymphoblastoid cell lines from patients with abnormal chromosome karyotype, B lymphoid cells were prepared from cryopreserved heparinized blood samples. The lymphoid cell pellet was resuspended with 0.5 ml medium of RPMI with 20% fetal calf serum(FCS), and added 2 ml virus-containing superatant of the EB virus-producing cell lines by filtrated, and mixed. Four 25 cm2 cell culture bottles were put upright. A total of 2.5 ml of RPMI with 20% FCS was put in each of them. The blood-virus mixture was distributed among the four cell culture bottles as follows: Bottle I, Bottle II, Bottle III and Bottle IV were added with 0.3 ml, 0.6 ml, 1.2 ml and the rest respectively. The cells culture bottles were put into the cell culture incubator in an upright position. After 3 days the cells were puting new medium with 20% FCS as follows: Bottle I 3 ml, Bottle II 4 ml, Bottle III 5 ml and Bottle IV 6 ml. After one week, the medium was changed again as described above. The medium change was conducted until the cells grew very fast. The right ratio between blood cells and virus titer can not be exactly determined for every blood sample, and therefore a dilution series with four different blood/virus ratios was set up. Due to the dilution series, addition of immune inhibitors like cyclosporine, was not necessary. Forty-seven permanent lymphoblastoid cell lines of patients with abnormal chromosome karyotype. Transformed cells were found in only one or two of the four cell culture bottles. The total successive rate increased up to 97.87%. Of the four cell culture bottles, Bottle I, Bottle II, Bottle III and Bottle IV, the successive rates were 6.39%, 61.70%, 31.91% and 8.51% respectively. This method can be used for preserving large number of lymphoblastoid cell lines, and also provide enough research materials for further studies.

[Genetic polymorphisms of human short tandem repeat vWA].

We researched the genetic polymorphisms of vWA in Henan population and its usefulness in forensic science.DNA extracted from non-relative persons in Henan population with Chelex was amplified by polymerase chain reaction and was typed by nondenaturing polyacrylamide gel electrophoresis silver staining.A total of 8 alleles and 19 genotypes were found in Henan population,its heterozygosity is high and the locus can be used in forensic genetics. We obtained the allelic frequency of the locus vWA in Henan population. The results of amniotic fluid,villus,blood stain indicate vWA is a good locus for forensic study.

[Genetic polymorphism of six STR loci in the Han population in Henan province].

To investigate the allele frequencies of six short tandem repeats (STR) loci: F13A1, F13B, D8S1179, CSF1PO, D5S818 and TPOX in Han population in Henan province, DNA was extracted with chelex from EDTA-blood samples of the unrelated individuals in Henan province and amplified with PCR technique. The PCR product was analyzed with the undenatured PAGE vertical electrophoresis and silverstain. The results were obtained through genetical analysis. The authors got the number of allele of six loci, Three alleles of F13A1 could be detected in this population. The F13A1 * 7 is the most common allele with a frequency of 45.2%, followed by F13A1 * 5 and F13A1 * 4 respectively. F13B had four alleles among which F13B * 10 has the highest frequency, followed by F13B * 9, F13B * 8, F13B * 11. The 8 alleles of D8S1179 in the order from high to low frequency is D8S1179 * 14, * 15, * 12, * 11, * 10. And the most common frequencies of alleles of CSF1PO, D5S818, TPOX ore CSF1PO * 12, D5S818 * 11, TPOX * 8, respectively followed by the allele of * 11, * 14, * 10, * 13 in CSF1PO locus, and * 12, * 13, * 10, * 9, * 14, * 8, * 15, * 7 in D5S818 locus, * 11, * 9, * 12, * 10 in TPOX locus. The heterozygosities of the six loci were 0.62, 0.46, 0.83, 0.59, 0.78 and 0.65; the discrimination powers were 0.78, 0.66, 0.95, 0.79, 0.92 and 0.82. The heterozygosities of the six loci are high and the frequencies are in good agreement with Hardy-Weinberg equilibrium. The six loci are good as genetic markers. We can use these loci in dividualidentification and partenity test in forensic area.