Single-cell RNA sequencing reveals distinct gene expression patterns in glucose metabolism of human preimplantation embryos.

Precise regulation of glucose metabolism-related genes is essential for early embryonic development. Although previous research has yielded detailed information on the biochemical processes, little is yet known of the dynamic gene expression profiles in glucose metabolism of preimplantation embryos at a single-cell resolution. In the present study, we performed integrated analysis of single-cell RNA sequencing (scRNA-seq) data of human preimplantation embryos that had been cultured in sequential medium. Different cells in the same embryo have similar gene expression patterns in glucose metabolism. During the switch from the cleavage to morula stage, the expression of glycolysis-related genes, such as glucose transporter genes (solute carrier family 2 (facilitated glucose transporter), member 1 (SLC2A1) and solute carrier family 2 (facilitated glucose transporter), member 3 (SLC2A3) and genes encoding hexokinase, phosphofructokinase, pyruvate kinase and lactate dehydrogenase, is increased. The genes involved in the pentose phosphate pathway are highly expressed at the cleavage stage, generating the reducing power to balance oxidative stress derived from biosynthesis. Expression of the genes involved in the biosynthesis of glycerophospholipids is increased after the morula stage. Nevertheless, the expression of tricarboxylic acid-related genes remains relatively unchanged during the preimplantation stages. In conclusion, we discovered that the gene expression profiles are dynamic according to glucose utilisation in the embryos at different stages, which contributes to our understanding of regulatory mechanisms of glucose metabolism-related genes in human preimplantation embryos.

[Research advances on medical genetics in China in 2015].

Steady progress has been achieved in the medical genetics in China in 2015, as numerous original researches were published in the world's leading journals. Chinese scientists have made significant contributions to various fields of medical genetics, such as pathogenicity of rare diseases, predisposition of common diseases, somatic mutations of cancer, new technologies and methods, disease-related microRNAs (miRNAs), disease-related long non-coding RNAs (lncRNAs), disease-related competing endogenous RNAs (ceRNAs), disease-related RNA splicing and molecular evolution. In these fields, Chinese scientists have gradually formed the tendency, from common variants to rare variants, from single omic analyses to multipleomics integration analyses, from genetic discovery to functional confirmation, from basic research to clinical application. Meanwhile, the findings of Chinese scientists have been drawn great attentions of international peers. This review aims to provide an overall picture of the front in Chinese medical genetics, and highlights the important findings and their research strategy.

[Etiology and diagnosis of intellectual disability].

Intellectual disability, occurring in 1%-3% of the general population, is a common disease of the nervous system in children. Since diverse genetic and environmental factors contribute to its pathogenesis, the etiological diagnosis of intellectual disability is challenging with respect to the selection of diagnostic tests. It is important to determine the etiology of intellectual disability for the assessment of prognosis, treatment and the family plan. This paper summarizes the research progress in etiology and diagnosis for intellectual disability and introduces the recommended clinical genetics diagnostic approach from the American Academy of Pediatrics.

[Mutation analysis of EXT genes in two pedigrees with hereditary multiple exostoses].

OBJECTIVE: To detect the underlying genetic defect in two Chinese families with hereditary multiple exostoses and provide genetic counseling. METHODS: Potential mutations in EXT1 and EXT2 genes in the probands were detected by direct sequencing of PCR-amplified exons. Suspected mutations were verified in all available family members and 200 unrelated healthy controls. RESULTS: A heterozygous frameshift mutation c.346_356delinsTAT in exon 1 of EXT1 and a heterozygous deletion mutation c.2009-2012del(TCAA) in exon 10 of EXT1 were respectively detected in affected members from the two families. The same mutations were not detected in unaffected members and 200 unrelated healthy controls. No mutations in EXT2 were detected in the two families. CONCLUSION: Two novel mutations of EXT1 have been detected in association with hereditary multiple exostoses in two Chinese families. Above results have provided a basis for genetic counseling for the two families and expanded the spectrum of EXT1 mutations.

[Analysis of four fetuses with de novo chromosomal rearrangments using single nucleotide polymorphism microarray chips].

OBJECTIVE: To assess the value of single nucleotide polymophism (SNP) microarray for delineation of de novo chromosomal rearrangements detected upon prenatal diagnosis. METHODS: SNP microarray analysis was carried out for 4 fetuses with de novo sSMCs or balanced reciprocal translocations. Genomic DNA was extracted from cord blood samples, and amplified, tagged and hybridized following the manufacturer's protocol. Data were collected and analyzed. RESULTS: No pathogenic CNVs were detected in fetus A, whose sSMCs was verified to be heterochromatin. Fetus B, who had a de novo mosaic sSMCs, was found to have a 9 Mb duplication in 4p12-q13 which is associated with speech delay and mental retardation. No pathogenic CNVs were detected in fetus C who has 2 translocation chromosomes inherited from its mother and 2 chromosomes derived from a de novo translocation. Fetus D, who had a de novo "balanced" reciprocal translocation, was found to have a 25 Mb duplication in 1q25 and a 17 Mb deletion in 9p22. Cases A and C had normal physical and mental evaluation after birth. CONCLUSION: For its ability to detect cryptic imbalance in de novo sSMCs or balanced reciprocal translocations, SNP-array has provided a powerful aid to conventional karyotype analysis during prenatal diagnosis.

[Deletion and mutation analysis to FOXL2 in blepharophimosis-ptosis-epicanthus inversus syndrome].

OBJECTIVE: To perform genetic analysis in 5 patients with blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) and refine the genotype-phenotype correlation. METHODS: G-band karyotyping, fluorescent in situ hybridization (FISH), SNP array, PCR and sequencing techniques were performed to one patient with BPES and mental retardation and 4 only with BPES. RESULTS: Patient 1 with mental retardation carried a 9.4 Mb heterozygous deletion in chromosome 3q22.1-q23 including FOXL2 gene; Both patient 2 and 3 carried a c.704delG heterozygous mutation of FOXL2, while they were assigned to the different clinical type from those reported previously. Patient 3 was assigned to type II BPES; No mutation of FOXL2 was detected in patient 4 and 5. CONCLUSIONS: There might be the gene(s) responsible for mental retardation within chromosome 3q22.1-q23. It was indicated that the mutation c.704delG in FOXL2 led to a truncated protein is associated with both type I and II of BPES.

[Genetic diagnosis and prenatal diagnosis of Angelman syndrome].

OBJECTIVE: To evaluate the conventional cytogenetic methods in genetic diagnosis and prenatal diagnosis in the family with a proband of Angelman syndrome (AS). METHODS: High-resolution G-banding karyotyping and fluorescence in situ hybridization (FISH) on metaphase chromosomes were performed. RESULTS: Two AS patients and 1 normal fetus in the family were successfully detected by FISH. CONCLUSION: Our result demonstrated that patient with type I AS could be detected by combining the techniques of high-resolution G-banding and FISH with clinical observation, which would offer accurate genetic counseling information to the geneticists and provide the prenatal diagnosis for the AS family.

[Mutational screening of the SLC26A4 gene in patients with nonsyndromic hearing loss by denaturing high-performance liquid chromatography].

OBJECTIVE: To study the SLC26A4 gene mutations in patients with nonsyndromic hearing loss (NSHL) and provide the clinical guidance of gene diagnosis. METHODS: PCR and denaturing high-performance liquid chromatography (DHPLC) were used to screen the 21 exons and their flanking regions of the SLC26A4 gene. Samples with abnormal DHPLC wave patterns were sequenced to identify the variations. RESULTS: Among the 30 unrelated NSHL patients in whom no deafness-causing mutations of the GJB2 gene were identified, 10 types of variations were detected, including 7 known mutations, 2 novel mutations (F572L and D87Y), and 1 known polymorphism (Ivs11+47T>C). The Ivs7-2A>G is the most common type of variation, accounting for 40% of all the mutations. CONCLUSION: SLC26A4 mutation is a major cause of NSHL, just next to the GJB2 mutations. For NSHL patients without deafness-causing GJB2 mutations, the SLC26A4 mutation rate was 23.3%, and the Ivs7-2A>G was the most common mutation.

[Rapid detection of the hot spot gene mutations in Chinese patients with nonsyndromic hearing loss by polymerase chain reaction-restrictive fragment length polymorphism].

OBJECTIVE: To develop a rapid genetic diagnosis technique for the patients with hereditary hearing loss by screening hot spots of mutations, namely 235delC of the GJB2 gene, IVS7-2A>G of the SLC26A4 gene, and 1555A>G of mitochondrial 12S rRNA. METHODS: Multiple PCR amplification of the three fragments covering the expected mutations in GJB2, SLC26A4 and 12S were carried out and the amplified products were analyzed by restriction fragment length polymorphism (RFLP). RESULTS: Eighteen homozygous and 18 heterozygous 235delC, 2 homozygous and 13 heterozygous IVS7-2A>G, and 8 homogeneous 1555A>G were detected in the 200 patients with hearing loss. All the results were confirmed by sequencing. The detection rate of the three mutant alleles was 21.7% (71/400 + 8/200 = 0.217) and the genetic diagnosis rate was 14% [(18+2+8)/200 = 0.14]. CONCLUSION: It is a convenient, efficient and economical method to screen the hot spots of mutation in the patient with hereditary hearing loss by using PCR-RFLP.

[Mutation screening of 20 candidate genes located in chromo-some 5q31-5q32 for DFNA52 locus].

Previously, we mapped the DFNA52 (OMIM: 607683) locus to an 8.8 cM interval between STR D5S2056 and D5S638 on human chromosome 5q31.1-q32 in a large consanguineous Chinese family with congenital sensorineural hearing loss. Positional candidate cloning approach was applied to analyze the candidate genes in this region. We analyzed 20 genes according to cochlear expression pattern, which were also located in the DFNA52 interval as candidate genes. Sequencing of the coding and splice site regions of these genes did not reveal any potentially pathogenic mutations segregating with the disease, implying that none of these genes are likely virulence gene for DFNA52.

Efficient protein expression from the endogenous RNA polymerase I promoter using a human ribosomal DNA targeting vector.

Vector systems to deliver, integrate and express therapeutic genes in host cells are essential for gene therapy. In the present study, we investigated a novel vector system for integration and expression of a transgene. In this system, the transgene expression was driven by an endogenous RNA polymerase I (Pol I) promoter after being integrated into the ribosomal DNA (rDNA) locus. Human coagulation factor IX coding sequence (FIX), with an internal ribosome entry sites element at its leader region, was targeted into the 18S rDNA locus via homologous recombination. FIX protein expression, which was under the control of the endogenous Pol I promoter, was found to be similar to that of a moderate Pol II promoter. The average FIX expression level of the rDNA recombinants was additionally enhanced to that from a strong Pol II promoter as a result of elimination of position effects. Our data suggest the possibility of applying this system in gene therapy for hereditary diseases.

[Multiplex quantitative PCR detection for female carrier in an X-linked ichthyosis family].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Growing of human embryonic stem cells on feeders derived from themselves].

This study was carried out to determine whether mesenchymal stem cells (MSCs) derived from teratoma of human embryonic stem cells (hESCs) function as feeder cells to support hESCs growth. Approximately 5x10(6) hESCs were injected into the hind limb muscle of each SCID-beige mouse to form teratoma. After 8 weeks, the MSCs were isolated from the teratoma and cultured in Mesencult medium. Purified MSCs were then used as the feeder cells for hESCs culture. High purity MSCs derived from teratoma were isolated. The cells were morphologically similar to bone marrow MSCs (bMSCs). The teratoma-derived MSCs were negative for CD34 and CD45 but positive for CD29, CD49b, CD105, CD73, and CD90, which resembled those expressed by bMSCs. After passaged on MSCs feeder cells more than 10 passages, hESCs maintained hESC characteristics in morphology. Reverse PCR showed the expression of Oct4 and Nanog. SSEA-1 was negative and SSEA-4, TRA-1-60, and TRA-1-81 were positive. Alkaline phosphatase staining showed positive results.The karyotype remained normal. Moreover, the hECSs cultured on teratoma-derived MSCs formed teratoma in vivo and embryoid body in vitro confirmed their pluripotency. Accordingly, MSCs derived from hESCs by in vivo differentiation can be used as the feeder cells for hESCs culture.

[Mapping of pathogenic genes in two families with autosomal dominant ichthyosis vulgaris].

To localize the pathogenic genes of autosomal dominant ichthyosis vulgaris, we ascertained two ichthyosis vulgaris families from Hunan Province. Venous blood samples were collected from affected and unaffected family members and genomic DNA was extracted. We then performed genome scan and linkage analysis using microsatellite markers around known ichthyosis vulgaris loci in chromosomes 1 and 10. In family 1, the locus linked to ichthyosis vulgaris was located near D1S498 (1q21), which overlapped with known ichthyosis vulgaris loci. In family 2, however, all known loci for ichthyosis vulgaris were excluded and the new locus remains to be identified.

[Mapping of pathogenic genes in a pedigree with autosomal dominant ichthyosis vulgaris].

OBJECTIVE: To elucidate the pathogenic genes in a pedigree with autosomal dominant ichthyosis vulgaris (IV). METHODS: Linkage analysis was performed by using STR markers in chromosome 1, and mutation detection was used to screen for FLG gene mutation. RESULTS: 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. CONCLUSION: The pathologic gene of the IV family locates near D1S2696, and the FLG gene may not ruled out from the pathologic genes.

[Biological characteristics and safety evaluation of endothelial progenitor cells from the umbilical cord blood].

OBJECTIVE: To investigate the biological characteristics of endothelial progenitor cells (EPCs) from the umbilical cord blood (UCB), and to evaluate their oncogenicity after long-term culture in vitro. METHODS: The mononuclear cells (MNCs) were isolated from the UCB and cultured in MCDB131 medium supplemented with 20% FBS, VEGF and other growth factors. Morphology of the EPCs was observed, and the growth curve of the EPCs was investigated. Surface antigens of the EPCs were analyzed by the flow-cytometer. The capability of intaking the acetylated low-density lipoprotein (acLDL) of the EPCs was detected using fluoresencent chemical method. The vasoformative capability and genetic stability of EPCs were cultured in matrigel, and examined by karyotype analysis. The oncogenicity of EPCs was verified by the tumorigenesis test in athymic mouse and soft agar. RESULTS: EPCs were successfully derived from the UCB, and could be passaged to at least 42(nd) generation and had strong abilities of proliferation, acLDL intake and vasoformation, but there was not oncogenicity. They expressed endothelial cell-surface antigens and maintained normal karyotype. CONCLUSION: The EPCs with proliferative potential can be isolated from the UCB. They can be passaged in long-term cultures without oncogenicity, and can maintain normal karyotype. The EPCs can be served as a new type of cells in cell and gene therapy.

[Identification of the origin of marker chromosome by comparative genomic hybridization].

OBJECTIVE: To identify the origin of the marker chromosome in a patient with chromosome aberration, and to provide the precise genetic diagnosis. METHODS: Comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) were performed to detect the known small marker chromosome in this patient. RESULTS: The small marker chromosome originated from chromosome 13 pter->q12. CONCLUSION: CGH and FISH can be used to detect the small marker chromosome, which is convenient and quick in detecting the origin of small marker chromosome.

[Azoospermia factor microdeletion on Y chromosome in patients with idiopathic azoospermia or severe oligozoospermia].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Preimplantation genetic diagnosis of Duchenne muscular dystrophy by single cell triplex PCR].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.