Effect of MSX1 on the cellular function of cardiomyocytes.

MSX1 (Muscle Segment Homeobox 1) has pleiotropic effects in various tissues, including cardiomyocytes, while the effect of MSX1 on cardiomyocyte cellular function was not well known. In this study, we used AC16 cell culture, real-time fluorescence quantitative PCR (qPCR), protein blotting (Western blot), flow cytometry apoptosis assay and lactate dehydrogenase (LDH) ELISA (Enzyme-Linked Immunosorbnent Assay) to investigate the effect of the MSX1 gene on cardiomyocyte function. The results showed that MSX1 plays a protective role against hypoxia of cardiomyocytes. However, further studies are required to fully understand the role of MSX1 in the regulation of LDH expression in different cell types and under different conditions.

The use of high-resolution SNP arrays to detect congenital cardiac defects.

OBJECTIVE: Copy number variations (CNVs) detected by high-resolution single nucleotide polymorphism microarrays (SNP arrays) have been associated with congenital heart defects (CHDs). The genetic mechanism underlying the development of CHDs remains unclear. METHODS: High-resolution SNP arrays were used to detect CNVs and traditional chromosomal analyses, respectively, were carried out on 60 and 249 fetuses from gestational 12-37 weeks old, having isolated or complex CHDs that were diagnosed using prenatal ultrasound. RESULTS: Twenty of the 60 fetuses (33.5%) had abnormalities, of which 23 CNVs (12 pathogenic, five probable pathogenic and six of undetermined significance) were detected by SNP arrays, and two distinct CNVs were present in three of these fetuses. In addition, in 39 patients with isolated congenital heart disease who had normal karyotypes, abnormal CNVs were present in 28.2% (11/39), and in patients with complex coronary artery disease, 19.0% (4/21) had abnormal karyotypes and 42.9% (9/21) had abnormal CNVs. In patients with complex coronary artery disease, 19.0% (4/21) had abnormal karyotypes and 42.9% (9/21) had abnormal CNVs. CONCLUSIONS: In conclusion, genome-wide high-resolution SNP array can improve the diagnostic rate and uncover additional pathogenic CNVs. The submicroscopic deletions and duplications of Online Mendelian Inheritance in Man (OMIM) genes found in this study have haploinsufficient (deletion) or triplosensitive (duplication) traits, which further clarify the etiology and inheritance of CHDs.

Generation of two induced pluripotent stem cell lines, GZHMCi009-A and GZHMCi010-A, derived from peripheral blood mononuclear cells of two SCA3 patients with 14/74 CAG repeats of the ATXN3 mutation.

Spinal cerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease, is the result of abnormal repeat amplification of CAG of the ATXN3 gene. It is one of the main types of autosomal dominant ataxia, with motor symptoms of cerebellar ataxia, mainly accompanied by non-motor symptoms, such as ocular symptoms, psychiatric symptoms, and nutritional disorders. Currently, no effective treatment is available for patients with SCA3. The construction of induced pluripotent stem cells (iPSCs) from two SCA3 patients (14/74 CAG repeats) will be an excellent tool for studying SCA3 disease mechanisms and for drug screening.

Epilepsy Combined With Multiple Gene Heterozygous Mutation.

The fast pace of gene discovery has resulted in groundbreaking advances in the field of epilepsy genetics. Clinical testing using comprehensive gene panels, exomes, or genomes is now increasingly available and has significantly increased the diagnostic yield for early-onset epilepsies and enabled precision medicine approaches. In this paper, we report a case of epilepsy in a pedigree. The proband had heterozygous mutations in KCNC1 (NM_001112741.1:c.959G>A, p. Arg320His), CAPN3 (NM_000070.2:c.526G>A, p. Val176Met), and NEFH (NM_021076.3:c. 2595 delC, p. Lys866Argfs*51). Sanger sequencing verification was consistent with the results of whole-exome sequencing. The KCNC1 mutation was a de novo mutation, and the CAPN3 and NEFH mutations were inherited from their father and mother, respectively. Based on the American College of Medical Genetics and Genomics (ACMG) guidelines, a heterozygous mutation was found for APOB (NM_000384.2: c.10579C > T, p. Arg3527Trp). The heterozygous mutation at this site was inherent in the pedigree. Coexpression analysis indicated that heterozygous mutations of KCNC1, CAPN3, NEFH, and APOB were closely related to the clinical phenotypes of the patient, and the clinical phenotypic heterogeneity of the disease may be the result of the interaction of multiple genes.

Generation of induced pluripotent stem cell GZHMCi002-A from peripheral blood mononuclear cells with APOB mutation.

Apolipoprotein (apo) B is a large, amphipathic glycoprotein which plays an important role in human lipoprotein metabolism. The 43-kb APOB gene located on the short arm of human chromosome 2 and consisted of 29 exons, mutations in the APOB gene can give rise to either hypo- or hypercholesterolemia. We used peripheral blood mononuclear cells (PBMCs) from a volunteer carrying the APOB mutation (c.10579C>T, p.Arg3527Trp) located in exon 9 to establish induced pluripotent stem cells (iPSC), which will be an effective means to reveal the key biologically relevant metabolic mechanisms, a powerful tool for medicine selection and related research.

Generation of induced pluripotent stem cell GZHMCi001-A and GZHMCi001-B derived from peripheral blood mononuclear cells of epileptic patients with KCNC1 mutation.

Myoclonus Epilepsy and Ataxia due to Potassium channel mutation (MEAK) is a rare epilepsy caused by changes in the structure and function of potassium channels due to mutations in the potassium voltage-gated channel subfamily C member 1 (KCNC1) gene. MEAK is one of the progressive myoclonus epilepsy (PME), and there are few studies on MEAK pathogenesis and targeted drugs. Here, we used peripheral blood from MEAK patients with KCNC1 (c.959G > A) gene mutation to establish induced pluripotent stem cells (iPSC). The iPSC of KCNC1 mutation established by us is a powerful tool for related research.

Generation of four induced pluripotent stem cell lines, GZWWTi001-A, GZWTZi001-A, GZWXYi001-A, and GZWXDi001-A, derived from peripheral blood mononuclear cells from a family with asparagine synthetase deficiency.

Asparagine synthetase (ASNS) deficiency (ASNSD; MIM #615574) is a rare neurodevelopmental disorder caused by mutations in the ASNS gene. The ASNS gene maps to cytogenetic band 7q21.3 and is 35 kb long. ASNSD is characterised by congenital microcephaly, severely delayed psychomotor development, seizures, and hyperekplexic activity. Here, we reported a family with compound heterozygous mutations in ASNS (NM_001178076:c.551C>T; c. 944A>C) and established induced pluripotent stem cells (iPSCs) from blood samples. To date, limited functional data have been reported to explain the underlying pathophysiology of ASNSD; therefore, iPSCs from these patients may be powerful tools for studying disease mechanisms.

CRISPR/Cas9 gene correction of HbH-CS thalassemia-induced pluripotent stem cells.

Haemoglobin (Hb) H-constant spring (CS) alpha thalassaemia (- -/-αCS) is the most common type of nondeletional Hb H disease in southern China. The CRISPR/Cas9-based gene correction of patient-specific induced pluripotent stem cells (iPSCs) and cell transplantation now represent a therapeutic solution for this genetic disease. We designed primers for the target sites using CRISPR/Cas9 to specifically edit the HBA2 gene with an Hb-CS mutation. After applying a correction-specific PCR assay to purify the corrected clones followed by sequencing to confirm the mutation correction, we verified that the purified clones retained full pluripotency and exhibited a normal karyotype. This strategy may be promising in the future, although it is far from representing a solution for the treatment of HbH-CS thalassemia now.

Generation of GZKHQi001-A and GZWWTi001-A, two induced pluripotent stem cell lines derived from peripheral blood mononuclear cells of Duchenne muscular dystrophy patients.

Duchenne muscular dystrophy (DMD) is an X-linked disease caused by mutations in the DMD gene, which spans ~2.4Mb of genomic sequence at locus Xp21. This mutation results in the loss of the protein dystrophin. DMD patients die in their second or third decade due to either respiratory failure or cardiomyopathy, as the absence of dystrophin leads to myofiber membrane fragility and necrosis, eventually resulting in muscle atrophy and contractures. Currently, there is no effective treatment for DMD, therefore induced pluripotent stem cells from DMD patients would be a powerful tool for studying disease mechanisms.

Copy number variations independently induce autism spectrum disorder.

The examination of copy number variation (CNV) is critical to understand the etiology of the CNV-related autism spectrum disorders (ASD). DNA samples were obtained from 64 ASD probands, which were genotyped on an Affymetrix CytoScan HD platform. qPCR or FISH were used as a validation for some novel recurrent CNVs. We further compared the clinical phenotypes of the genes in the Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources (DECIPHER) database with these overlapping genes. Using vast, readily available databases with previously reported clinically relevant CNVs from human populations, the genes were evaluated using Enrichment Analysis and GO Slim Classification. By using the Ploysearch2 software, we identified the interaction relationship between significant genes and known ASD genes. A total of 29 CNVs, overlapping with 520 genes, including 315 OMIM genes, were identified. Additionally, myocyte enhancer factor 2 family (MEF2C) with two cases of CNV overlapping were also identified. Enrichment analysis showed that the 520 genes are most likely to be related to membrane components with protein-binding functions involved in metabolic processes. In the interaction network of those genes, the known ASD genes are mostly at the core position and the significant genes found in our samples are closely related to the known ASD genes. CNVs should be an independent factor to induce autism. With the strategy of our study, we could find the ASDs candidate genes by CNV data and review certain pathogenesis of this disorder. Those CNVs were associated with ASD and they may contribute to ASD by affecting the ASD-related genes.

Chromosomal uniparental disomy 16 and fetal intrauterine growth restriction.

BACKGROUND: There is a well-documented association between prenatally diagnosed chromosomal uniparental disomy and poor pregnancy outcome. METHODS AND RESULT: In this study, we identified an intrauterine growth restricted fetus carrying a maternal UPD 16 with segmental hetero- and isodisomy using the Affymetrix CytoScan HD SNP-array and the UPDtool. We also performed FISH to exclude trisomy mosaicism of chr.16. We then explored the genetic mechanisms of how imprinted genes cause clinical abnormalities. Additionally, we reviewed the mUPD16 literature, compared the clinical phenotypes of our patient with other reported cases, and assessed the loss of autosomal-recessive genes in the regions of homozygosity. CONCLUSIONS: Considering UPD mechanism of potential impact on the function of the placenta, the genetic composition of chromosome 16, and the information previous literature reports, we have reason to believe that UPD16 correlates with IUGR.

Application of single nucleotide polymorphism-based array analysis for prenatal diagnosis of a fetus with de novo derivative chromosome / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze a fetus with increased nuchal translucency and nuchal fold, and to assess the recurrence risk for her family and provide a basis for prenatal diagnosis.</p><p><b>METHODS</b>G-banded karyotyping and single nucleotide polymorphism-based array (SNP-Array) analysis were used to analyze the fetus and her parents.</p><p><b>RESULTS</b>SNP-Array analysis has detected a 41.04 Mb duplication at Xp22.33p11.4 and a 30.51 Mb duplication at 13q31.3q34 in the fetus. G-banding karyotyping indicated that the fetus had a karyotype of 46,X,der(X)(13qter-13q31::Xp11.4-Xp22.3::Xp22.3-Xqter). Her parents had normal results for both G-banding karyotyping and SNP-Array analysis, suggesting that the fetus has carried a de novo derivative chromosome X.</p><p><b>CONCLUSION</b>SNP-Array combined with G-banding karyotyping is helpful to confirm the composition and connection type of de novo derivative chromosome, which can improve the accuracy of diagnosis and is valuable for the evaluation of recurrence risk.</p>

Prenatal diagnosis of 1p36.3 microdeletion in a fetus with complex heart defect / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze a fetus presenting with complex heart defect and assess the recurrence risk.</p><p><b>METHODS</b>Conventional karyotyping, fluorescence in situ hybridization (FISH) and single nucleotide polymorphism-based array (SNP-array) were used to analyze the fetus and his parents.</p><p><b>RESULTS</b>SNP-array has detected a 6.9 Mb microdeletion at 1p36.33-p36.23 in the fetus. Chromosomal and FISH analyses indicated that the father of the fetus had a karyotype of 46,XY,t(1;14)(p36.3;p12), and that the fetus has inherited an abnormal chromosome 1 derived from the paternal translocation.</p><p><b>CONCLUSION</b>SNP-array combined with GTG banding and FISH can help to detect cryptic translocation, microdeletion or microduplication of chromosomes and is valuable to assess the recurrence risk for the affected family.</p>

Microduplication of chromosome Xq25 encompassing STAG2 gene in a boy with intellectual disability.

Whole-genome microarray analysis is proven to be useful in the identification of submicroscopic copy number imbalances in families with intellectual disabilities. The first case of Xq25 duplication was identified using genome-wide array comparative genomic hybridization (array-CGH) in a 24-year-old patient with a syndromic intellectual disability. We report a 4-year-old boy with a de novo 591 kb duplication at Xq25. The duplication was first detected by a CytoScan HD array platform (Affymetrix, USA) and was confirmed by real-time quantitative PCR (qPCR) of the STAG2 gene, and by fluorescence in situ hybridization (FISH). The patient had clinical features partially consistent with published cases, including an intellectual disability and speech delay. The identification of this additional patient and a detailed analysis of duplications identified in other patient cohorts and absent in normal individuals support the existence of a rare pathological microduplication at Xq25 that encompasses STAG2.

Prader-Willi syndrome with a long-contiguous stretch of homozygosity not covering the critical region.

Prader-Willi syndrome is a common and complex disorder affecting multiple systems. Its main manifestations are infantile hypotonia with a poor sucking reflex, a characteristic facial appearance, mild mental retardation, hypogonadism and early-onset obesity. Prader-Willi syndrome is due to the absence of paternally expressed imprinted genes at 15q11.2-13, and 3 main mechanisms are known to be involved in its pathogenesis: paternal microdeletions, maternal uniparental disomy events, and imprinting defects. DNA methylation analysis can detect almost all individuals with Prader-Willi syndrome but is unable to distinguish between the molecular classes of the disease. Thus, additional methods are necessary to identify the molecular classes. Here, we employed chromosomal microarray analysis-single nucleotide polymorphism for diagnosis and detected a long-contiguous stretch of homozygosity on chromosome 15, which is highly predictive of maternal uniparental disomy on chromosome 15. Other methods, including fluorescence in situ hybridization, chromosomal microarray analysis-comparative genomic hybridization, genotyping and family linkage analysis, were performed for further validation. In conclusion, our study highlights the use of long-contiguous stretch of homozygosity detection for the diagnosis of Prader-Willi syndrome.

Effect of Cdc42 gene inhibited on proliferation, migration and invasion in human hepatocellular carcinoma cells / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the effect of Cdc42-shRNA plasmid to proliferation, migration, invasion and other malignant biological behavior in hepatoma SMMC-7721 cells.</p><p><b>METHODS</b>Cdc42-shRNA interfering vector transfected to SMMC-7721 cells with liposome method. The growth curve of transfected cells SMMC-7721, U6-control, Cdc42-shRNA2 was detected by MTT. The cells mobility was detected by wound healing experiment. Transwell chamber experiments to observe the cell migration and invasion. Detected AFP and PCNA expression level by Western blot.Human hepatoma SMMC-7721 transplanted subcutaneously in nude mouse, detected the expression of Cdc42 of the tumor by immunohistochemistry.t test was used to analyze the data between two groups.</p><p><b>RESULTS</b>The doubling time of Cdc42-shRNA2, U6-control and SMMC7721 was 42.7 h, 34.9 h and 35.1 h. The relative migration distance of Cdc42-shRNA2 and U6-control on 36 h was (47.1 ± 4.1)% and (86.6 ± 5.3)% (t=-10.21, P<0.05). Transwell chamber experimental methods showed the numbers of permeating cells were 18.2 ± 2.1(Cdc42-shRNA2) and 41.0 ± 3.5 (U6-control) (t=-9.67, P<0.05) on 24 h. The AFP and PCNA expression of hepatoma cells is significantly inhibited after the Cdc42-shRNA2 was transfected compared with U6-control group.The tumor average weight of group Cdc42-shRNA2 was (335.1 ± 178.2) mg, which was much lighter than that of SMMC-7721 group ((925.3 ± 241.4) mg) and U6-control group ((910.5 ± 225.6) mg) (t=-4.47, -4.39; P<0.05) and the Cdc42 expression was also weak positive.</p><p><b>CONCLUSION</b>Cdc42 interfere with plasmid significant changes in human malignant biological behavior of hepatocellular carcinoma cells, and reduces liver cancer cell growth, invasion and metastasis of capacity.</p>

Prenatal diagnosis of five cases of monochorionic-diamniotic twins discordant for karyotype analysis / 中华医学遗传学杂志

OBJECTIVE To explore the mechanism and diagnostic method for monochorionic-diamniotic twins discordant for karyotype analysis. METHODS Dual amniocentesis was performed on five pairs of monochorionic-diamniotic twins, which all consisted of a normal twin and one with multiple malformations revealed by ultrasound. Karyotype analysis was performed on amniocytes derived from each of the twins. Zygosity was also determined with DNA extracted from amniocytes with 16 polymorphic microsatellite markers. RESULTS Three cases of 45,X, one case of 47,XX,+9 and one case of 47,XY,+18 were detected among the abnormal twins, while the normal fetuses all had a normal karyotype. DNA analysis suggested that, in all cases, the twins have shared the 16 polymorphic microsatellite markers, which confirmed their monozygosity. CONCLUSION Monochorionic-diamniotic twins may be discordant for karyotyping, for which anaphase lagging, chromosomal non-disjunction and trisomy rescue may be the underlying reasons. As a simple method, dual amniocentesis can be used to obtain amniotic fluid samples for karyotype analysis and determination of zygosity for such twins.

Chromosomal microarray analysis for lateral ventriculomegaly in fetus / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the relationship between fetal lateral ventriculomegaly and chromosomal microarray analysis (CMA) abnormalities.</p><p><b>METHODS</b>Fifty fetuses with lateral ventriculomegaly detected by ultrasound and a normal karyotype were included. Forty four fetuses were classified as mild ventriculomegaly (MVM), in which the lateral ventricular atrium was 10-15 mm. Six had severe ventriculomegaly (SVM), with the lateral ventricularatrium being ≥ 15 mm. The fetuses were also divided into isolated (n= 21) and non-isolated groups (n= 29) based on whether they are associated with other anomalies.</p><p><b>RESULTS</b>Thirteen (26%) of the fetuses were found to be abnormal by CMA. For the 44 cases with MVM, 9 (20.9% ) were found to be abnormal, while for the 6 cases with SMV, 4 (66.7%) were found to be abnormal (P>0.05). CMA abnormalities were found in 2 (9.5%) of the 21 fetuses with isolated ventriculomegaly group and 11 (37.9%) of the 29 fetuses with non-isolated ventriculomegaly group (P<0.05).</p><p><b>CONCLUSION</b>Chromosome microdeletions and microduplications are the most common abnormalities found in fetal lateral ventriculomegaly. When ventriculomegaly is associated with other anomalies, the incidence of CMA abnormally is much higher. Prenatal diagnosis is necessary for fetuses with lateral ventriculomegaly.</p>

Misdiagnosis of mosaic tetrasomy 9p in a fetus by single nucleotide polymorphism-based array / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the reason for discordant results of karyotyping and microarray analysis in a fetus with mosaic tetrasomy 9p.</p><p><b>METHODS</b>Amniocentesis was carried out for a pregnant woman with advanced age for whom ultrasound scan has indicated fetal ventricular expansion, intrauterine growth retardation and persistent upper venous cavity. G-banded karyotyping and single nucleotide polymorphism-based arrays (SNP-array) analysis were performed at the same time.</p><p><b>RESULTS</b>Analysis of amniocytic chromosome has suggested mosaic tetrasomy 9p (47,XX,+psu idic(9)(q21)[23]/46,XX[27]). While SNP-array has detected a non-mosaic trisomy 9p with a 68.7 Mb duplication at 9p24.3q21.11. The results of the two methods were therefore discordant.</p><p><b>CONCLUSION</b>SNP-array will analyze genetic material in the form of numbers rather than morphology. For chimeras containing two types of cell lines, when the mosaic rate was close to 50% and the average amount of genetic material of the chimeras was equivalent to the amount of genetic material of non-chimeras, microarray analysis may come to the conclusion of a non-mosaic heteroploidy. Therefore, microarray results for large segment chromosome abnormalities should be combined with the results of G-banded karyotyping for genetic counseling.</p>

Confirmation of a maternal cryptal balanced translocation through analysis of a fetus using microarray / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze a fetus with heart defects and to assess the recurrence risk for her family.</p><p><b>METHODS</b>Single nucleotide polymorphism-based arrays (SNP-Array) analysis using Affymetrix Genome Wide Human SNP CytoHD was performed to analyze the fetus and her parents. Karyotype analysis was also carried out.</p><p><b>RESULTS</b>SNP-Array has detected a 14.5 Mb duplication at 9p and a 14.7 Mb deletion at 11q. Karyotype analysis indicated that the fetus' mother has a karyotype of 46, XX, t(9;11) (p23;q24). Therefore, the fetus has inherited a derivative chromosome 11 derived from the maternal translocation, and her karyotype was 46, XX, der(11) t(9;11) (p23;q24) mat.</p><p><b>CONCLUSION</b>SNP-Array combined with high resolution GTG banding has confirmed that the fetus has a derivative chromosome 11 derived from her mother's balanced translocation, resulting in partial 9p trisomy and partial 11q monosomy. This couple therefore have a high recurrence risk. SNP-Array is capable of detecting small chromosomal imbalance in abnormal fetuses and can pinpoint the breakpoints. It therefore has the advantage for the detection of unbalanced translocation which is difficult to detect with GTG banding, which is important for assessment the recurrence risk for cryptic balanced translocation carriers.</p>