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.

Novel SIL1 nonstop mutation in a Chinese consanguineous family with Marinesco-Sjögren syndrome and Dandy-Walker syndrome.

Marinesco-Sjögren syndrome (MSS) is a rare autosomal recessive disorder, which is characterized by congenital cataracts, cerebellar ataxia, progressive muscle weakness, and delayed psychomotor development. SIL1, which is located at 5q31.2, is the only gene known to cause MSS. Dandy-Walker syndrome (DWS) is defined by hypoplasia, upward rotation of the cerebellar vermis, and cystic dilation of the fourth ventricle; however, its genetic pathogeny remains unclear. Here, we report a Chinese consanguineous family with MSS and DWS. Whole exome sequencing identified a novel nonstop mutation in SIL1. Sanger sequencing revealed that the mutation was segregated in this family according to a recessive mode of inheritance. We found that the mutation changed a stop codon (TGA) to an arginine codon (CGA), and no in-frame termination codon in the 3' untranslated region (UTR) of SIL1 could be found. The mRNA levels of SIL1 were decreased by 56.6% and 37.5% in immortalized lymphoblasts of the patients respectively; the protein levels of SIL1 were substantially decreased. This case study is the first report on Chinese MSS patients, MSS complicated by DWS, and a nonstop mutation in SIL1. Our findings imply the pathogenetic association between DWS and MSS.

Molecular medicine of fragile X syndrome: based on known molecular mechanisms.

BACKGROUND: Extensive research on fragile X mental retardation gene knockout mice and mutant Drosophila models has largely expanded our knowledge on mechanism-based treatment of fragile X syndrome (FXS). In light of these findings, several clinical trials are now underway for therapeutic translation to humans. DATA SOURCES: Electronic literature searches were conducted using the PubMed database and ClinicalTrials.gov. The search terms included "fragile X syndrome", "FXS and medication", "FXS and therapeutics" and "FXS and treatment". Based on the publications identified in this search, we reviewed the neuroanatomical abnormalities in FXS patients and the potential pathogenic mechanisms to monitor the progress of FXS research, from basic studies to clinical trials. RESULTS: The pathological mechanisms of FXS were categorized on the basis of neuroanatomy, synaptic structure, synaptic transmission and fragile X mental retardation protein (FMRP) loss of function. The neuroanatomical abnormalities in FXS were described to motivate extensive research into the region-specific pathologies in the brain responsible for FXS behavioural manifestations. Mechanism-directed molecular medicines were classified according to their target pathological mechanisms, and the most recent progress in clinical trials was discussed. CONCLUSIONS: Current mechanism-based studies and clinical trials have greatly contributed to the development of FXS pharmacological therapeutics. Research examining the extent to which these treatments provided a rescue effect or FMRP compensation for the developmental impairments in FXS patients may help to improve the efficacy of treatments.

[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.

First report of HGD mutations in a Chinese with alkaptonuria.

Alkaptonuria (AKU) is one of the first prototypic inborn errors in metabolism and the first human disease found to be transmitted via Mendelian autosomal recessive inheritance. It is caused by HGD mutations, which leads to a deficiency in homogentisate 1,2-dioxygenase (HGD) activity. To date, several HGD mutations have been identified as the cause of the prototypic disease across different ethnic populations worldwide. However, in Asia, the HGD mutation is very rarely reported. For the Chinese population, no literature on HGD mutation screening is available to date. In this paper, we describe two novel HGD mutations in a Chinese AKU family, the splicing mutation of IVS7+1G>C, a donor splice site of exon 7, and a missense mutation of F329C in exon 12. The predicted new splicing site of the mutated exon 7 sequence demonstrated a 303bp extension after the mutation site. The F329C mutation most probably disturbed the stability of the conformation of the two loops critical to the Fe(2+) active site of the HGD enzyme.

[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.

[Cornelia de Lange syndrome: report of a case and the review of literature on 17 cases].

OBJECTIVE: To explore the clinical characteristics of Cornelia de Lange Syndrome (CdLS) and to review the latest clinical research reports. METHOD: Clinical and laboratory data of one case of neonatal CdLS are reported, and literature on 17 cases of CdLS in China and the international reports of the clinical and molecular biological research on this disease were reviewed. RESULT: (1) The patient was an infant with intrauterine growth retardation and born as a term small for gestational age infant with specific facial features, bone abnormality of extremities, and patent ductus arteriosus (PDA). She also had severe feeding difficulty and slow weight gain. She was followed up till 4 months of age and showed severe developmental retardation. (2) The total number of past reported case of CdLS in China was 17 with a male to female ratio of 6:12. The average age of diagnosis was 17 months. The following specific facial features could be observed: synophrys, long and curved eyelashes, hirsutism, microcephalus, low hairline, broad depressed nasal bridge, long prominent philtrum, and high palate. Most of the patients were complicated with mental retardation, recurrent vomiting or feeding difficulty, abnormal muscle tone, cutis marmorata, hypophalangism, and genitalia anomaly. Clinical manifestations of Chinese patients were similar to those of the overseas reports. The karyotype of 15 cases was investigated and was normal. The etiology of CdLS is unknown. There is no specific treatment. The commonest causes of death are lung diseases caused by gastroesophageal reflex/aspirate related pneumonia. CONCLUSION: Typical clinical manifestations of CdLS are specific facial features (mainly synophrys, long and curved eyelashes, long prominent philtrum), complications of multi-system malformations (mainly growth and developmental retardation, esophagogastric reflex, hypophalangism), related gene mutations occurred in NIPBL, SMC1A, and SMC3 gene.

Vitreous amyloidosis in two large mainland Chinese kindreds resulting from transthyretin variant Lys35Thr and Leu55Arg.

OBJECTIVE: To describe the clinical and pathological findings of two large mainland Chinese kindreds with vitreous amyloidosis and associated transthyretin mutation. METHODS: Twenty individuals from two kindreds with vitreous amyloidosis were ascertained. The transtheretin (TTR) gene of each individual was analyzed, and a clinical examination was obtained on the index patient. RESULTS: Vitreous amyloidosis and radiculopathy were the significant findings in affected individuals. Vitrectomy was performed on the severely affected individuals, with resulting postoperative visual acuity of 20/80 to 20/25. Congo red staining demonstrated amyloid in the vitreous specimen. In Case A, DNA sequencing of exon 2 in the TTR gene revealed a base-pair substitution at codon 35, AAG > ACG (Lys35Thr). In Case B, a missense mutation of leucine-to-arginine substitution was identified at amino acid position 55 in exon 3, CTG > CGG (Leu55Arg). CONCLUSIONS: TTR Lys35Thr and Leu55Arg mutations are associated with vitreous amyloidosis. The phenotype is variable, with vitreous opacities occurring earlier, and sometimes as the sole signs of amyloidotic polyneuropathies (FAPs). Vitrectomy improves vision in some patients with vitreous amyloidosis.

[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.

[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.

[Molecular analysis of IDS gene and prenatal diagnosis in a Chinese family with mucopolysaccharidosis type II].

OBJECTIVE: Mucopolysaccharidosis type II (MPSII) is a lethal, X-linked recessive disorder caused by mutation of iduronate-2-sulfatase (IDS) gene. Up to now there is no really effective treatment for this disorder, therefore it is important to provide an accurate genetic diagnosis and prenatal diagnosis for the MPSII families. In this study, we identify the pathogenic mutation in a Chinese family with MPSII. METHOD: The 8 years old male proband from a Chinese family was clinically diagnosed with MPSII. There are other 4 patients with similar phenotypes in the family who died at 9, 11, 7 and 10 years of age, respectively. Mutation analysis was carried out by polymerase chain reaction and direct sequencing of all exons and exon/intron boundaries of IDS gene. Denaturing high performance liquid chromatography (DHPLC) analysis was performed to screen the unknown variations of IDS gene in 100 unrelated control males. RESULT: Two allelic variants of exon 5 (c.684A > G) and exon 6 (c.851C > T) and a nonsense mutation of exon 7 (c.892C > T) were detected in IDS gene of the proband. Heterozygous mutations c.684A > G, c.851C > T and c.892C > T were detected in both proband's mother and maternal grandmother. The unknown variations of c.684A > G and c.851C > T were not found in the 100 unrelated control males. The male fetus (IV11) inherited the same mutation of IDS gene as the proband. CONCLUSION: Mutation c.892C > T of IDS gene causes MPSII in this family and prenatal diagnosis in one affected fetus was achieved.

[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.

[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.

[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.