Analysis of a patient with early-onset retinitis pigmentosa due to novel variants of CRB1 gene / 中华医学遗传学杂志

OBJECTIVE@#To explore the genetic basis for a patient with early-onset retinitis pigmentosa (RP).@*METHODS@#A patient who had presented at the West China Hospital of Sichuan University on March 10, 2020 was selected as the study subject. The patient and his parents were subjected to whole exome sequencing (WES). Candidate variants were verified by Sanger sequencing and in silico analysis.@*RESULTS@#The patient has featured substantial loss of binocular vision field. Funduscopy revealed characteristic bone spicule-type pigment deposits, as well as attenuated retinal arterioles and pale-appearing optic discs. WES revealed that he has harbored compound missense variants of a RP-associated CRB1 gene, including c.2969T>C (p.Leu990Ser) and c.1816T>C (p.Cys606Arg), which were respectively inherited from his father and mother. Homozygous c.1816T>C (p.Cys606Arg) variant has been identified among RP patients, whilst the c.2969T>C (p.Leu990Ser) variant was unreported previously. Both variants were predicted as likely pathogenic based on the guidelines from the American College of Medical Genetics and Genomics (ACMG).@*CONCLUSION@#The novel compound heterozygous variants of the CRB1 gene probably underlay the early-onset RP in this patient. Above finding has enriched the mutational spectrum of the CRB1 gene.

A novel splicing acceptor variant of the FBN2 gene contributes to a case of congenital contractural arachnodactyly / 中华医学遗传学杂志

OBJECTIVE@#To identify the pathogenic variants from a patient with suspected congenital contractural arachnodactyly, and to explore the possible molecular genetic pathogenesis, so as to provide evidence for clinical diagnosis.@*METHODS@#Whole exome sequencing was performed for the patient. The splicing site variation of candidate pathogenic genes was verified by Sanger sequencing, and the new transcript sequence was determined by RT-PCR and TA-cloning sequencing.@*RESULTS@#The patient carried a heterozygous c.533-1G>C variant of FBN2 gene, which was not reported. The sequencing of mRNA showed that the variant leaded to the disappearance of the canonical splice acceptor site of FBN2 gene and the activation of a cryptic splice acceptor site at c.533-71, resulting in the insertion of 70 bp sequence in the new transcript. It was speculated that the polypeptide encoded by the new transcript changed from valine (Val) to serine (Ser) at amino acid 179, and prematurely terminated after 26 aminoacids. According to the guidelines of American College of Medical Genetics and Genomics, the variant of FBN2 gene c. 533-1G>C was determined as pathogenic (PVS1+PM2+PP3 ).@*CONCLUSION@#A novel splicing variant of FBN2 gene (c.533-1G>C) was identified, which can lead to congenital contractural arachnodactyly.

Genetic analysis of a recurrent abnormal pregnancy case caused by a cryptic reciprocal autosomal translocation / 中华医学遗传学杂志

OBJECTIVE@#To provide genetic counseling for a couple with recurrent detection of fetal structural abnormality during second trimester pregnancy.@*METHODS@#The fetal tissue and peripheral blood samples of the couple were subjected to G banded chromosomal analysis, copy number variation sequencing (CNV-seq) and fluorescence in situ hybridization (FISH) assays.@*RESULTS@#CNV-seq has detected a 6.59 Mb duplication at 7p22.3-p22.1 and a 3.81 Mb deletion at 4p16.3 in the fetal tissue, though conventional karyotyping results of both parents were normal. FISH has confirmed that the father has harbored a cryptic translocation of t(4;7)(7p+,4q+,4p+,7q+).@*CONCLUSION@#The ultrasonographic abnormality of the fetuses may be attributed to the 7p microduplication and 4p microdeletion derived from the cryptic translocation carried by the father. Reciprocal translocation of tiny chromosomal segments should be suspected for couples with recurrent adverse pregnancies but apparently normal karyotypes.

A study on pathogenicity of TGM1 gene mutations in a collodion baby / 中华皮肤科杂志

Objective:To investigate the genetic etiology of a pedigree with autosomal recessive congenital ichthyosis.Methods:Whole-exome sequencing was performed in a collodion baby, and Sanger sequencing was conducted to verify gene mutations. The PolyPhen-2, PROVEAN and Mutation Taster softwares, as well as protein homology modeling methods, were used to predict effects of gene variants; real-time fluorescence-based quantitative PCR and Western blot analysis were performed to analyze the effect of mutations on allelic mRNA and protein expression.Results:Whole-exome sequencing and Sanger sequencing confirmed a mutation c.919C>T (p.Arg307Trp) in exon 6 and a mutation c.1019G>A (p.Gly340Glu) in exon 7 of the TGM1 gene in the infant, which were inherited from his mother and father respectively. Bioinformatics analysis suggested that both the two mutations were harmful to protein structures, which were further supported by protein homology modeling. In vitro experiments showed that there was no significant difference in the mRNA expression of the TGM1 gene between the 293T cells transfected with wild-type plasmids and those transfected with mutant plasmids containing the mutation c.919C>T or c.1019G>A ( t=1.97, 1.28, P=0.12, 0.27, respectively) , but the TGase1 protein expression significantly decreased in the 293T cells transfected with the mutant TGM1 plasmids. Conclusion:The mutations c.919C>T and c.1019G>A in the TGM1 gene may be the molecular genetic etiology of severe ichthyosis in the infant, and the missense amino acids encoded by the two mutations may affect the TGase1 protein function by destroying its structure.

Genetic analysis of two couples with a history of multiple fetal malformations / 中华医学遗传学杂志

OBJECTIVE@#To explore the genetic basis for a couple with recurrent conceptions of fetus with abnormal longbones, and another couple with a history of omphalocele.@*METHODS@#Genomic DNA was extracted from the peripheral blood samples from both couples. All exons and flanking regions were analyzed with next generation sequencing. Candidate variants were verified by Sanger sequencing.@*RESULTS@#Couple one was found to be heterozygous for, a c.997+1G>T splice-site variant and a missence c.871G>A(p.Glu291Lys) variant of the ALPL gene. Both variants were predicted to be pathogenic and may result in reduced function or loss of alkaline phosphatase. For couple two, the wife was found to harbor a novel c.637_652 delins CCC variant of the CDKN1C gene. This deletion-insertion variant resulted in frame-shift and loss of function (p.Ala213Profs*55) of the CDKN1C protein. Maternally inherited CDKN1C LOF variant has been found to underlie Beckwith-Wiedemann syndrome (BWS), which may manifest as omphalocele.@*CONCLUSION@#Dispite the lack the direct proof from the lost fetuses, the variants of ALPL and CDKN1C genes can explain the recurrence of fetal malformations for both couples.

Identification of a novel variant of F5 gene in a consanguineous pedigree affected with inherited coagulation factor V deficiency / 中华医学遗传学杂志

OBJECTIVE@#To explore the genetic basis for a consanguineous pedigree affected with inherited coagulation factor V deficiency.@*METHODS@#Genomic DNA was extracted from peripheral blood samples from the pedigree and subjected to next generation sequencing for screening variants of the F5 gene. Suspected pathogenic variant was verified by using Sanger sequencing. Pathogenicity of the variant was evaluated according to ACMG guidelines.@*RESULTS@#A homozygous frameshifting variant, c.4096delC (p.Leu1366Phefs*3), was identified in the F5 gene in the proband, which was confirmed to be derived from her consanguineous parents. This variant was absent in all databases including 10 000 in-house Chinese exome sequences. Based on the ACMG guidelines, the c.4096delC was predicted to be a pathogenic variant.@*CONCLUSION@#A novel pathogenic variant has been identified in the F5 gene in a consanguineous pedigree with inherited coagulation factor V deficiency, which has enriched the spectrum of F5 gene variants.

Clinical and genetic analysis of a rare case with mosaic partial trisomy 5p syndrome / 中华医学遗传学杂志

OBJECTIVE@#To determine the size and origin of a small supernumerary marker chromosome (sSMC) identified in a patient featuring developmental retardation.@*METHODS@#High-throughput sequencing for copy number variation (CNV-seq) was carried out to delineate the sSMC identified upon G-banded chromosomal karyotyping. The genotype-phenotype correlation was explored by database retrieval and literature analysis.@*RESULTS@#The patient was found to have a karyotype of mos 47,XX,+mar[36]/46,XX[23]. CNV-seq has identified a 18 Mb duplication at 5p14.1-p12 (hg19: 27,399,261-46,083,784)x2.6 with a mosaicism rate of approximately 60%.@*CONCLUSION@#Patients with mosaic partial trisomy 5p may have extensive clinical manifestations, and the ratio of trisomy 5p cells is correlated with clinical severity of this syndrome.

Reflection of a case misdiagnosed as trisomy 21 syndrome by G-banded chromosomal karyotyping analysis / 中华医学遗传学杂志

Objective@#To emphasize the clinical significance of copy number variations (CNVs) detection by describing a case misdiagnosed as trisomy 21 syndrome by G-banded chromosomal karyotype analysis.@*Methods@#A girl with obesity and short stature was diagnosed as trisomy 21 syndrome by G-banded chromosomal karyotype analysis. Considering the discrepancy of her karyotype with her phenotype, genomic CNVs was detected by next-generation sequencing and the result was verified by quantitative PCR (qPCR).@*Results@#A microduplication of 16p11.2: 29 642 339-29 775 631 (133.292 kb) was detected. qPCR assay for QPRT and SPN located in the duplicated region confirmed the finding of CNVs assay. Meanwhile, her parents did not present similar duplication in 16p11.2.@*Conclusion@#The 16p11.2 microduplication was a novel genomic structural variation in the girl, though it may not be associated with her clinical manifestations. Chromosomal microarray or next-generation sequencing-based CNVs detection can accurately determine the origin of small supernumerary marker chromosome and reduce the chance of misdiagnosis.

Analysis of HINT1 gene variant in a case with neuromyotonia and axonal neuropathy / 中华医学遗传学杂志

OBJECTIVE@#To explore clinical and genetic features of a pedigree affected with autosomal recessive neuromyotonia and axonal neuropathy (NMAN).@*METHODS@#For the proband and her parents, clinical data was collected, genomic DNA was extracted from peripheral blood samples. Triplet primed-PCR was carried out to detect dynamic mutation of DMPK and ZNF9 genes, which are responsible for myotonic dystrophy, by capillary electrophoresis. High-throughput sequencing was used to screen variants of candidate genes for Mendelian disorders involving the nervous system. Candidate variants were confirmed by Sanger sequencing. The genotype of the variant was determined in the parents and 100 healthy controls. Pathogenicity of the variant was assessed by ACMG criterion.@*RESULTS@#Mutation of DMPK and ZNF9 genes was excluded. DNA sequencing has identified a homozygous missense variant (c.335C>T, p.R119W) in the HINT1 gene. Both parents were found to carry the variant. The same variant was not found among the healthy controls. According to the ACMG criterion, the missense variant was classified as a pathogenic variant.@*CONCLUSION@#The c.335C>T (p.R119W) of the HINT1 gene probably underlie the disease in this pedigree. Above finding provided further evidence for the connection between HINT1 and NMAN and enriched the mutation spectrum of HINT1 gene.

Reflection of a case misdiagnosed as trisomy 21 syndrome by G-banded chromosomal karyotyping analysis / 中华医学遗传学杂志

OBJECTIVE@#To emphasize the clinical significance of copy number variations (CNVs) detection by describing a case misdiagnosed as trisomy 21 syndrome by G-banded chromosomal karyotype analysis.@*METHODS@#A girl with obesity and short stature was diagnosed as trisomy 21 syndrome by G-banded chromosomal karyotype analysis. Considering the discrepancy of her karyotype with her phenotype, genomic CNVs was detected by next-generation sequencing and the result was verified by quantitative PCR (qPCR).@*RESULTS@#A microduplication of 16p11.2: 29 642 339-29 775 631 (133.292 kb) was detected. qPCR assay for QPRT and SPN located in the duplicated region confirmed the finding of CNVs assay. Meanwhile, her parents did not present similar duplication in 16p11.2.@*CONCLUSION@#The 16p11.2 microduplication was a novel genomic structural variation in the girl, though it may not be associated with her clinical manifestations. Chromosomal microarray or next-generation sequencing-based CNVs detection can accurately determine the origin of small supernumerary marker chromosome and reduce the chance of misdiagnosis.

Identification of proteins interacting with the circadian clock protein PER1 in tumors using bacterial two-hybrid system technique / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify protein-protein interaction partners of PER1 (period circadian protein homolog 1), key component of the molecular oscillation system of the circadian rhythm in tumors using bacterial two-hybrid system technique.</p><p><b>METHODS</b>Human cervical carcinoma cell Hela library was adopted. Recombinant bait plasmid pBT-PER1 and pTRG cDNA plasmid library were cotransformed into the two-hybrid system reporter strain cultured in a special selective medium. Target clones were screened. After isolating the positive clones, the target clones were sequenced and analyzed.</p><p><b>RESULTS</b>Fourteen protein coding genes were identified, 4 of which were found to contain whole coding regions of genes, which included optic atrophy 3 protein (OPA3) associated with mitochondrial dynamics and homo sapiens cutA divalent cation tolerance homolog of E. coli (CUTA) associated with copper metabolism. There were also cellular events related proteins and proteins which are involved in biochemical reaction and signal transduction-related proteins.</p><p><b>CONCLUSION</b>Identification of potential interacting proteins with PER1 in tumors may provide us new insights into the functions of the circadian clock protein PER1 during tumorigenesis.</p>

Progress in epigenetic research on male infertility / 中华医学遗传学杂志

Male infertility is one of the major diseases that affect human health and social life, and is influenced by many genetic and environmental factors. Epigenetic modification on DNA strands in response to environmental factors plays an important role in the process of spermatogenesis. Abnormalities of epigenetic regulation may affect both the quantity and quality of sperm production and result in disorders of male reproduction. We hereby review recent progress made in research on epigenetic regulation including DNA methylation, histone modification and non-coding RNA related with male infertility.

Effects of Xi Yanping injection on electrocardiogram of Beagle dogs / 中国比较医学杂志

Objective To study the effect of Xi Yanping injection on electrocardiogram in Beagle dogs.Methods 24 Beagle dogs were divided into negative control group (0 mg/kg), low-dose group (15 mg/kg), middle-dose group (75 mg/kg), high-dose group (250 mg/kg).Each group consist of 3 animals/sex/group, all animals were administrated by intravenous drip at 20 mL/kg once a day.Administration period was 28-days with 14-days recovery phase.Animals LeadⅡelectrocardiogram ( ECG) was measured during quanrantine, administration and recovery phase.The following parameters included heart rate, P -Rinterval, Q -T interval, P, Q, RS wave amplitude and ST and T wave amplitude were determined, serum biochemical parameters were measured to evaluate the effect on main organs.Results All parameters of quarantine period were fell in the normal limit.After the first administration, heart rate decrease was found in low dose group and P-Rinterval prolongation was found in high dose group.No abnormal was found in dose metaphase,end of dose stage and recovery stage.Conclusion Xi Yanping injection had no obvious influence on ECG of Beagle dog.

Research progress of TSPY1 gene family / 中华医学遗传学杂志

TSPY1 (testis-specific protein, Y-linked 1) gene family, located in male-specific region of Y-chromosome (MSY), has the maximum number of copies organized as a long tandem repeat array in protein-coding gene families of human genome. TSPY1 is identified to be the most important candidate gene for gonadoblastoma, and its coding protein can promote the proliferation and differentiation of tumor cells. Recently, TSPY1 gene family is also proposed to play an important role in spermatogenesis. In this review, the structure characteristics of the gene family were illustrated, and the functional studies of TSPY1 in the process of tumorigenesis and spermatogenesis were discussed.

Expression of Exogenous Human Hepatic Nuclear Factor-1alpha by a Lentiviral Vector and Its Interactions with Plasmodium falciparum Subtilisin-Like Protease 2

The onset, severity, and ultimate outcome of malaria infection are influenced by parasite-expressed virulence factors as well as by individual host responses to these determinants. In both humans and mice, liver injury follows parasite entry, persisting to the erythrocytic stage in the case of infection with the fatal strain of Plasmodium falciparum. Hepatic nuclear factor (HNF)-1alpha is a master regulator of not only the liver damage and adaptive responses but also diverse metabolic functions. In this study, we analyzed the expression of host HNF-1alpha in relation to malaria infection and evaluated its interaction with the 5'-untranslated region of subtilisin-like protease 2 (subtilase, Sub2). Recombinant human HNF-1alpha expressed by a lentiviral vector (LV HNF-1alpha) was introduced into mice. Interestingly, differences in the activity of the 5'-untranslated region of the Pf-Sub2 promoter were detected in 293T cells, and LV HNF-1alpha was observed to influence promoter activity, suggesting that host HNF-1alpha interacts with the Sub2 gene.

Transcriptional Activity of Plasmodium Subtilisin-like Protease 2 (Pf-Sub2) 5'Untranslated Regions and Its Interaction with Hepatocyte Growth Factor

The onset, severity, and ultimate outcome of malaria infection are influenced by parasite-expressed virulence factors and individual host responses to these determinants. In both humans and mice, liver injury is involved after parasite entry, which persists until the erythrocyte stage after infection with the fatal strain Plasmodium falciparum (Pf). Hepatocyte growth factor (HGF) has strong anti-apoptotic effects in various kinds of cells, and also has diverse metabolic functions. In this work, Pf-subtilisin-like protease 2 (Pf-Sub2) 5'untranslated region (UTR) was analyzed and its transcriptional activity was estimated by luciferase expression. Fourteen TATA boxes were observed but only one Oct-1 and c-Myb were done. In addition, host HGF interaction with Pf-Sub2 was evaluated by co-transfection of HGF- and Pf-Sub2-cloned vector. Interestingly, -1,422/+12 UTR exhibited the strongest luciferase activity but -329 to +12 UTR did not exhibit luciferase activity. Moreover, as compared with the control of unexpressed HGF, the HGF protein suppressed luciferase expression driven by the 5'untranslated region of the Pf-Sub2 promoter. Taken together, it is suggested that HGF controls and interacts with the promoter region of the Pf-Sub2 gene.

Research on genetic characteristics in the HA1 of influenza A(H1N1) viruses isolated in Taian City / 中华疾病控制杂志

Objective To analyze the data of influenza A(H1N1) viruses surveillance and genetic characteristics from Taian City during 2005-2008,so a scientific basis can be provided for the prevention and treatment of influenza.Methods The specimens from Influenza-Like Illness(ILI) were collected.The viruses were isolated with MDCK cell and identified with HAI and RT-PCR.The product of PCR were sequenced.Then the sequences were analyzed through biometric software.Results A total of 121 influenza strains were obtained from 615 specimens,and 4 of them were identified as A(H1N1) subtype.There were 3 strains mutated on several sites.Compared with strains isolated in 2005,there were 5 and 8 mutations in the amino acid sequences of virus strains isolated in 2007 and 2008 respectively.And there were a total of 22 amino acid mutations compared with A/Brisbane/59/2007(H1N1).Conclusions Influenza type A(H1N1) are detected in Taian City.There are several mutations in the amino acid sequences of virus strains isolated in Taian. The antigenic drift of virus strains is due to accumulation of amino acid substitutions

The A-204C Polymorphism in CYP7A1 Gene Affects Its Promoter Activity / 中国生物化学与分子生物学报

cholesterol 7α-hydroxylase gene ( CYP7A 1 ) plays a key role in the catabolism of cholesterol into bile acids. To investigate whether the A-204C polymorphism in CYP7A1 gene affects the gene expression,using luciferase as the reporter gene, four recombinants were constructed by inserting forward or reverse sequence with A or C allele at the polymorphism site into the promoter-less vector pGL3-basic. The constructs were then transfected into four cell lines and the luciferase activity of each expression vector was examined by dual luciferase reporter gene assay system. The results showed that activities of the forward sequence of both genotypes were higher than that of reverse sequence. Promoter activity of the recombinants with A allele was about one third lower than that with C allele. According to the analysis with TRANSFAC database, there may exist a Zic3 binding site when there is the C allele at -204. Our study indicates that the A-204 C polymorphism in CYP7A1 promoter region decreases its promoter activity and thus represses the gene expression, possibly due to the lack of a potential Zic3 binding site.