RHD Gene Analysis of A Blood Donor with Del Phenotype / 中国实验血液学杂志

OBJECTIVE@#To analyze the RHD genotype of a blood donor with Del phenotype in Yunnan.@*METHODS@#Rh serological phenotype was identified. RHD gene was detected by PCR-SSP typing, and its 10 exons were sequenced. Exon 9 was amplified for sequencing and analysis. RHD zygosity was detected.@*RESULTS@#The Rh phenotype of this specimen was CcDelee. Genomic DNA exhibited a 1 003 bp deletion spanning from intron 8, across exon 9 into intron 9. The deletion breakpoints occurred between two 7-bp short tandem repeat sequences. There was no variation in the sequences of the remaining exons. The Rh hybridization box test showed that there was one RHD negative allele.@*CONCLUSION@#This specimen is Del type caused by deletion of RHD exon 9.

Síndrome da deleção 2q37.3 ­ A primeira descrição de achados orais, relato de caso / 2q37.3 delection syndrome ­ The first description of oral phenotype, a case repor

Introdução: A síndrome de deleção 2q37.3 é uma desordem causada pela microdeleção de uma subbanda no cromossomo 2, cuja epidemiologia é de aproximadamente 150 casos no mundo, e a incidência de 1:10000. As características da síndrome são: atraso no desenvolvimento, dismorfia facial, anormalidades musculoesqueléticas e aumento ao risco de doenças congênitas. O objetivo deste relato de caso é apresentar, pela primeira vez na literatura, os achados clínicos orais e dentários associados à síndrome. Relato de caso: As principais características físicas da síndrome foram identificadas na paciente durante o exame físico: lábio superior fino, columela proeminente e dobras epicânticas. No exame intraoral, relatou-se as características odontológicas, que podem estar associadas à síndrome: acúmulo de cálculo dental, hipomineralização e hipoplasia, mordida cruzada, mordida aberta, agenesias, taurodontia e cisto dentígero. O tratamento dental consistiu na aplicação de técnicas de manejo de comportamento, instrução de higiene oral e de dieta, profilaxia, raspagem supragengival, aplicação tópica de flúor, além da extração do segundo molar decíduo inferior. Este é o primeiro relato de características orais da síndrome de deleção 2q37.3 dando importância às características observadas nesta paciente. Tais achados também serão úteis na caracterização da síndrome, que é extremamente rara, podendo contribuir para o diagnóstico clínico desses pacientes, além de subsidiar o tratamento odontológico de outros indivíduos. Conclusão: Apresentamos os achados clínicos orais como hipomineralização e hipoplasia, mordida cruzada e aberta, agenesia, taurodontia e cisto dentígero presentes na paciente com a síndrome e sugerimos que o gene envolvido ou sua deleção possam ser responsáveis por tais achados.

Introduction: The 2q37.3 deletion syndrome is a disorder caused by the microdeletion of a subband on chromo-some 2, whose epidemiology is approximately 150 cases worldwide, and the prevalence of 1:10000. Its charac-teristics are developmental delay, facial dysmorphia, musculoskeletal abnormalities, and increased risk of congenital diseases. The purpose is to present, for the first time in the literature, the oral and dental clinical findings associated with the syndrome. Case report: The main physical characteristics of the syndrome were identified during the physical examination: thin upper lip, prominent columella, and epicanthic folds. In the intraoral examination, dental characteristics were reported, which may be associated with the syndrome: accumulation of dental calculus, hypomineralization and hypoplasia, crossbite, open bite, agenesis, taurodontism, and dentigerous cyst. The dental treatment consisted of the application of behavior management techniques, oral hygiene and diet instruction, prophylaxis, supragingival scraping, topical fluoride application, and extraction of the lower second molar. This is the first report of oral characteristics of the 2q37.3 deletion syndrome, giving importance to the characteristics observed in this patient. Such findings will be useful in the characterization of the syndrome, which is extremely rare, and may contribute to the clinical diagnosis of these patients, in addition to subsidizing the dental treatment of other individu-als. Conclusion: We present oral clinical findings such as hypomineralization and hypoplasia, crossed and an open bite, agenesis, taurodontism, and dentigerous cyst present in the patient with the syndrome and we suggest that the involved gene or its deletion may be responsible for such findings.

Prenatal diagnosis and genetic analysis of a female fetus with 46, XX and positive SRY gene / 中华围产医学杂志

We report a case of a pregnant woman with 46,XX karyotype and positive sex-determining region on the Y chromosome ( SRY) gene and her female fetus. Ultrasound examination at 12 +6 gestational weeks indicated a thickened fetal nuchal translucency, and 46, XX with a positive SRY gene was detected in the fetus through quantitative fluorescent-polymerase chain reaction and amniotic fluid karyotype. However, the ultrasound showed that the gender of the fetus was female, which was inconsistent with the phenotype of male syndrome with 46, XX combining positive SRY gene. The fluorescent in situ hybridization (FISH) revealed that the short arm of the Y chromosome translocated to the long arm of one of the X chromosomes, namely Yp11.3-Xq28. In addition, a copy number variation at Yp11.31p11.2 copy (about 1 MB) was found by chromosomal microarray analysis, which validated the result of FISH and was consistent with the mother. After genetic counseling, the parents chose to continue the pregnancy to full term, and no abnormalities were found in the infant during the follow-up.

Optimization of a microarray for fission yeast

Bar-code (tag) microarrays of yeast gene-deletion collections facilitate the systematic identification of genes required for growth in any condition of interest. Anti-sense strands of amplified bar-codes hybridize with ~10,000 (5,000 each for up- and down-tags) different kinds of sense-strand probes on an array. In this study, we optimized the hybridization processes of an array for fission yeast. Compared to the first version of the array (11 µm, 100K) consisting of three sectors with probe pairs (perfect match and mismatch), the second version (11 µm, 48K) could represent ~10,000 up-/down-tags in quadruplicate along with 1,508 negative controls in quadruplicate and a single set of 1,000 unique negative controls at random dispersed positions without mismatch pairs. For PCR, the optimal annealing temperature (maximizing yield and minimizing extra bands) was 58℃ for both tags. Intriguingly, up-tags required 3× higher amounts of blocking oligonucleotides than down-tags. A 1:1 mix ratio between up- and down-tags was satisfactory. A lower temperature (25℃) was optimal for cultivation instead of a normal temperature (30℃) because of extra temperature-sensitive mutants in a subset of the deletion library. Activation of frozen pooled cells for >1 day showed better resolution of intensity than no activation. A tag intensity analysis showed that tag(s) of 4,316 of the 4,526 strains tested were represented at least once; 3,706 strains were represented by both tags, 4,072 strains by up-tags only, and 3,950 strains by down-tags only. The results indicate that this microarray will be a powerful analytical platform for elucidating currently unknown gene functions.

Application of low-depth whole-genome sequencing for copy number variations in genetic diagnosis of X-linked ichthyosis due to STS gene deletion / 中华皮肤科杂志

Objective@#To evaluate the application value and significance of low-depth whole-genome sequencing for copy number variations (CNV-Seq) in the genetic diagnosis and prenatal diagnosis of X-linked ichthyosis (XLI) due to STS gene deletion.@*Methods@#Clinical data were collected from 3 616 subjects who received CNV-Seq, and single-gene test results were collected from 7 patients or pedigrees with ichthyosis in The First Affiliated Hospital of Zhengzhou University in 2018. The 3 616 samples included 2 891 prenatal samples from pregnant women (most were amniotic fluid samples, some fetal villus samples, very few umbilical blood samples) and 725 peripheral blood samples from other subjects. Genomic DNA was extracted from amniocytes or peripheral blood, and then subjected to CNV-Seq. Quantitative PCR (qPCR) and single nucleotide polymorphism (SNP) -comparative genomic hybridization (CGH) array were performed to verify the detected CNVs. Pathogenicity of the CNVs was analyzed according to the database of genomic variants (DGV) , database of genomic variation and phenotype in humans using ensembl resources (DECIPHER) , clinical genome resource (ClinGen) and online Mendelian inheritance in man (OMIM) .@*Results@#Of the 3 616 subjects receiving CNV-Seq, Xp22.31 deletion was identified in prenatal samples from 6 pregnant women, including 5 male and 1 female fetuses. The deleted fragment of Xp22.31 covered the XLI region containing the major gene STS. The parental CNV-Seq showed that the Xp22.31 deletion was spontaneous mutation in 2 of the 6 fetuses, and inherited from the parents in the other 4 fetuses. qPCR confirmed that the female fetus was a carrier of a complete heterozygous deletion of the STS gene, and there was a complete deletion of the STS gene in the other 5 male fetuses. SNP-CGH array also confirmed that the female fetus was heterozygous Xp22.31 deletion carrier, which was consistent with the CNV-Seq results. Ichthyosis gene panel sequencing in the 7 patients with ichthyosis showed 1 with harlequin ichthyosis, 2 with ichthyosis vulgaris, 3 with XLI, and no causative mutation in 1. CNV-Seq confirmed that Xp22.31 deletion existed in the above 2 patients with XLI due to STS gene deletion. Moreover, Xp22.31 duplication was found in 16 out of 3 616 subjects receiving CNV-Seq, but they were all individuals or fetuses with normal phenotype.@*Conclusions@#CNV-Seq is a stable and reliable method for screening whole-genome CNVs, and can be applied to genetic diagnosis and prenatal diagnosis of XLI due to STS gene deletion. The deletion of Xp22.31 fragment containing the STS gene can cause XLI, and the duplication of the same region is highly likely to be the polymorphic variation.

Application of low-depth whole-genome sequencing for copy number variations in genetic diagnosis of X-linked ichthyosis due to STS gene deletion / 中华皮肤科杂志

Objective To evaluate the application value and significance of low-depth whole-genome sequencing for copy number variations(CNV-Seq)in the genetic diagnosis and prenatal diagnosis of X-linked ichthyosis(XLI)due to STS gene deletion. Methods Clinical data were collected from 3616 subjects who received CNV-Seq, and single-gene test results were collected from 7 patients or pedigrees with ichthyosis in The First Affiliated Hospital of Zhengzhou University in 2018. The 3616 samples included 2891 prenatal samples from pregnant women(most were amniotic fluid samples, some fetal villus samples, very few umbilical blood samples)and 725 peripheral blood samples from other subjects. Genomic DNA was extracted from amniocytes or peripheral blood, and then subjected to CNV-Seq. Quantitative PCR(qPCR)and single nucleotide polymorphism(SNP)-comparative genomic hybridization(CGH)array were performed to verify the detected CNVs. Pathogenicity of the CNVs was analyzed according to the database of genomic variants(DGV), database of genomic variation and phenotype in humans using ensembl resources (DECIPHER), clinical genome resource (ClinGen) and online Mendelian inheritance in man (OMIM). Results Of the 3616 subjects receiving CNV-Seq, Xp22.31 deletion was identified in prenatal samples from 6 pregnant women, including 5 male and 1 female fetuses. The deleted fragment of Xp22.31 covered the XLI region containing the major gene STS. The parental CNV-Seq showed that the Xp22.31 deletion was spontaneous mutation in 2 of the 6 fetuses, and inherited from the parents in the other 4 fetuses. qPCR confirmed that the female fetus was a carrier of a complete heterozygous deletion of the STS gene, and there was a complete deletion of the STS gene in the other 5 male fetuses. SNP-CGH array also confirmed that the female fetus was heterozygous Xp22.31 deletion carrier, which was consistent with the CNV-Seq results. Ichthyosis gene panel sequencing in the 7 patients with ichthyosis showed 1 with harlequin ichthyosis, 2 with ichthyosis vulgaris, 3 with XLI, and no causative mutation in 1. CNV-Seq confirmed that Xp22.31 deletion existed in the above 2 patients with XLI due to STS gene deletion. Moreover, Xp22.31 duplication was found in 16 out of 3616 subjects receiving CNV-Seq, but they were all individuals or fetuses with normal phenotype. Conclusions CNV-Seq is a stable and reliable method for screening whole-genome CNVs, and can be applied to genetic diagnosis and prenatal diagnosis of XLI due to STS gene deletion. The deletion of Xp22.31 fragment containing the STS gene can cause XLI, and the duplication of the same region is highly likely to be the polymorphic variation.

Mutation Analysis of Synthetic DNA Barcodes in a Fission Yeast Gene Deletion Library by Sanger Sequencing

Incorporation of unique barcodes into fission yeast gene deletion collections has enabled the identification of gene functions by growth fitness analysis. For fine tuning, it is important to examine barcode sequences, because mutations arise during strain construction. Out of 8,708 barcodes (4,354 strains) covering 88.5% of all 4,919 open reading frames, 7,734 barcodes (88.8%) were validated as high-fidelity to be inserted at the correct positions by Sanger sequencing. Sequence examination of the 7,734 high-fidelity barcodes revealed that 1,039 barcodes (13.4%) deviated from the original design. In total, 1,284 mutations (mutation rate of 16.6%) exist within the 1,039 mutated barcodes, which is comparable to budding yeast (18%). When the type of mutation was considered, substitutions accounted for 845 mutations (10.9%), deletions accounted for 319 mutations (4.1%), and insertions accounted for 121 mutations (1.6%). Peculiarly, the frequency of substitutions (67.6%) was unexpectedly higher than in budding yeast (~28%) and well above the predicted error of Sanger sequencing (~2%), which might have arisen during the solid-phase oligonucleotide synthesis and PCR amplification of the barcodes during strain construction. When the mutation rate was analyzed by position within 20-mer barcodes using the 1,284 mutations from the 7,734 sequenced barcodes, there was no significant difference between up-tags and down-tags at a given position. The mutation frequency at a given position was similar at most positions, ranging from 0.4% (32/7,734) to 1.1% (82/7,734), except at position 1, which was highest (3.1%), as in budding yeast. Together, well-defined barcode sequences, combined with the next-generation sequencing platform, promise to make the fission yeast gene deletion library a powerful tool for understanding gene function.

Genetic analysis for a haemophilia B family with multi nucleotides deletion mutation of F9 gene / 中华检验医学杂志

Objective To conduct genetic diagnosis and prenatal diagnosis for a haemophilia B family with multi-nucleotides deletion mutation of F9 gene.Methods This is a genetic analysis.Whole exon mutation of the F9 gene was analyzed by PCR and Sanger sequencing for seven patients with the family of hemophilia B who consulted doctors in Henan Province People′s Hospital in April 2013.Suspected mutation was verified among non-hemophilia B members of the family and 100 healthy controls to rule out genetic polymorphism of the F9 gene.The above-mentioned detection results of hemophilia B gene , the pathogenic mutation of F9 gene in the family was clarified , and prenatal diagnosis was conducted for the female carriers in the family.It is recommended that the fetal gene detection should be conducted in amniotic fluid in the mid-term pregnancy of the female carriers of hemophilia , and then they can be informed of the non-hemophilia B fetus by the results of the gene detection .Results PCR and sequencing analysis has identified a deletion mutation of F9 gene c.185_188delGAGA[p.Glu62Asnfs?41]in seven hemophilia B patients.This mutation induced F9 gene frame shift mutation which led to early termination of F9 gene translation because there was a termination codon TAA at the 41th codon after the mutation site.The same mutation was not found among the non-hemophilia B members of the family and the 100 healthy controls. There were eight female carriers and nine female non-carriers in the family.Upon prenatal diagnosis , the Y chromosome sex-determining gene ( SRY ) in amniotic fluid was positive and no deletion mutation was observed in the F9 gene c.185_188.Conclusion The pathogenic mutation of F9 gene in the family was identified , which was helpful for prenatal diagnosis in female carriers .

Diagnostic distal 16p11.2 deletion in a preterm infant with facial dysmorphism

The 16p11.2 microdeletion has been reported in patients with developmental delays and intellectual disability. The distal 220-kb deletion in 16p11.2 is associated with developmental delay, autism spectrum disorder, epilepsy, and obesity at a young age. We have reported a case of distal 16p11.2 deletion syndrome in a preterm infant with unusual facial morphology and congenital heart disease. We suggest using chromosome microarray analysis to detect chromosomal abnormalities in newborns, especially preterm infants with unusual morphologies.

A case of X-linked ichthyosis complicated by Mal de Meleda: clinical features and mutation analysis of the SLURP-1 and STS genes / 中华皮肤科杂志

Objective To report a case of X-linked ichthyosis complicated by Mal de Meleda,and to identify the gene mutations.Methods Clinical data were collected from the patient,and peripheral blood samples were obtained from the patient,his parents and 100 unrelated healthy people who served as controls.Genomic DNA was extracted from these blood samples,and PCR was performed to amplify all the exons and their flanking sequences of the SLURP-1 and STS genes.All the amplification products were analyzed by agarose gel electrophoresis,and amplification products of the SLURP-1 gene were analyzed by DNA sequencing.Results The patient presented with regularly-arranged polygonal brown or black scales all over the trunk and limbs,erythematous hyperkeratotic lesions on the palms and soles,elbows and knees,inguinal and perianal regions,which extended to the dorsa of the hands and feet.Then,the patient was diagnosed with X-linked ichthyosis complicated by Mal de Meleda.Genetic testing showed complete deletion of the STS gene,and a homozygous mutation (c.286C ＞ T) at position 286 in exon 3 of the SLURP-1 gene,which led to the formation of a premature termination codon at amino acid position 96 (p.R96*).His parents were heterozygous carriers of the mutation (c.286C ＞ T).No mutation was found in the unrelated healthy controls.Conclusion The complete deletion of the STS gene and the homozygous nonsense mutation in the SLURP-1 gene may be the reason for X-linked ichthyosis complicated by Mal de Meleda in the patient.

Impacts of A20 gene deletion on clinicopathological features and prognosis of diffuse large B cell lymphoma and relative molecular mechanism / 重庆医学

Objective To detect the A20 gene deletion,investigate the impacts of A20 gene deletion on clinicopathological features and prognosis of DLBCL,and relationship between activation of NF-κB pathway and relative molecular pathogenesis.Methods A20 gene deletion was detected by fluorescence in situ hybridization (FISH).The expression of A20,Survivin,P65 and Ki-67 were detected by immunohistochemistry stain.Apoptosis was assayed by TUNEL.Follow-up and statistical analysis were done.Results The deletion rate of A20 gene was 21.7％.The deletion rate of A20 gene was obviously higher in ABC-like DLBCL than that in GCB-like DLBCL (30.6％ vs.8.3％,P＜0.05).It was observed that there was a negative correlation between A20 protein expression and A20 gene deletion (r=-0.259,P =0.023).The expression of P65 and Survivin protein was positively correlated with the A20 gene deletion (r=0.280,P =0.015;r =0.313,P =0.007).Apoptosis rate was significantly reduced in DLBCL patients with A20 gene deletion.The apoptosis rate was higher in cases with positive expression of A20 protein,while that was lower in cases with positive expression of p65 and Survivin protein than those with negative expression of corresponding protein.There was no statistically significant difference in apoptosis rate between ABC-like and GCB-like DLBCL patients (P＞0.05).COX regression analysis indicated that age,A20 gene deletion,types of DLBCL and Ki67 expression were independent factors associated with survival status.Log-rank test showed that there was a statistical difference in survival status between the cases with and without A20 gene deletion (P=0.015).Conclusion A20 gene deletion may associate with the attenuation of A20 protein expression.The latter weakens negative feedback regulation of A20 protein for NF-κB pathway.An up-regulated expression of Survivin and abnormal proliferation and apoptosis may be result from the abnormal activation of NF-κB.A20 gene deletion brings certain influence on clinical course and prognosis of DLBCL.

Clinical analysis of adult Philadelphia chromosome-positive acute lymphoblastic leukemia with p16 gene deletion / 中华血液学杂志

Objective@#To investigate the clinical implications of p16 gene deletion in adult Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) .@*Methods@#Retrospective analysis of clinical, immunophenotypic, cytogenetics, molecular characteristics and prognosis of 80 newly diagnosed Ph+ ALL patients with p16 deletion.@*Results@#Of 80 adult Ph+ ALL, the prevalence of p16 gene deletion was 31.3%. p16 gene deletion carriers frequently accompanied with high WBC counts (WBC≥30×109/L) and CD20 expression. The incidence of complex chromosome abnormality in p16 gene deletion group was higher than that in non-deletion group, with alternations in chromosome 7, 8, 19 and der (22) more frequently observed. There was no difference occurred between patients with or without p16 gene deletion in complete remission (CR) rate following induction chemotherapy combined with tyrosine kinase inhibitors (TKIs) . However, after three cycles of chemotherapy, the MMR and CMR rate in the p16 gene deletion group was lower than patients with wild-type p16 gene (P=0.034, P=0.036) . The p16 gene deletion patients showed no significant differences in MMR, CMR and relapse rate between Imatinib or Dasatinib plus chemotherapy (P>0.05) . Deletion of p16 gene was significantly associated with poor outcomes including worse overall survival (OS) (37.1% vs 54.1%, P=0.037) , lower disease free-survival (DFS) (12.4% vs 45.9%, P=0.026) , and increased cumulative incidence of relapse (P=0.033) . Among the 25 patients with p16 deletion, 14 underwent allo-HSCT and the median survival was 21 months, better than that of patients received chemotherapy alone (12 months) (P=0.030) .@*Conclusion@#This study indicated that deletion of p16 was associated with poor prognosis in adult Ph+ ALL, and the utility of second-generation TKI (Dasatinib) does not necessarily have an edge on efficacy over Imatinib, but allo-HSCT has the potential of elongating life expectancy. It is an important significance to define the status of p16 in Ph+ ALL for predicting prognosis and guiding therapy decision-making.

Meta-analysis of insertion/deletion genetic variation of ACE gene and onset riskof type 2 diabetic nephropathy in Chinese population / 重庆医学

Objective To systematically assess the relation between angiotensin-I converting enzyme(ACE) gene insertion/deletion (I/D) variation and type 2 diabetic nephropathy (T2DN) onset risk among Chinese population.Methods The related literatures were retrieved from the China National Knowledge Infrastructure (CNKI) and Wanfang Data until June 1st,2016.The RevMan 5.0 was used to conduct the statistical analysis.The merge OR value and corresponding 95% confidence interval(95%CI) were used to assess ACE gene I/D polymorphism and T2DN onset risk.Results Totally 29 papers with 4 357 subjects were included according to the inclusion and exclusion standard,including 2 208 cases of DN and 2 149 cases of T2DM without DN.Meta analysis showed that compared with ACE gene I/D polymorphism I allele,D allele could significantly increase the risk of T2DM patients suffering from DN,the OR value and corresponding 95%CI were 1.44(1.25,1.66);the gene analysis showed that ACE gene I/D polymorphism loci were significantly correlated with DN onset risk in the Asian population.The corresponding relative onset risk OR and 95%CI were 1.42(1.15,1.76) and 1.75(1.46,2.10) in the dominant and recessive genetic model.The Begg′s test showed that the included data had no obvious publication bias existence.Conclusion ACE gene I/D polymorphism is closely correlated with the onset risk of T2DN,and D allele might be a risk genetic factor for DN occurrence in the patients with T2DM.

Analysis of clinical and electrophysiological features in patients with hereditary neuropathy with liability to pressure palsy diagnosed by gene analysis / 中华神经科杂志

Objective To study the clinical and electrophysiological features of the patients with hereditary neuropathy with liability to pressure palsy ( HNPP) diagnosed by gene analysis.Methods Seven patients from two HNPP families were assessed on medical history, physical examination, electrophysiology findings and gene analysis.Results A clinical manifestation of acute, painless, recurrent peripheral nerve palsies was typical for HNPP.Median, ulnar and peroneal nerves were usually affected.Electrophysiology study revealed that prolonged distal motor latency and slowing nerve conduction velocity were prominent.Gene studies exhibited a deletion of the peripheral myelination protein 22 gene in all the seven patients.Conclusions HNPP usually affects areas where nerves are subject to entrapment, and many episodes are preceded by minor compression on the affected nerve.As a reliable screening tool in detecting HNPP, the electrophysiological study shows that segmental demyelination is most commonly seen at common nerve entrapment sites.

SMN1 gene deletion analysis using mid-trimester amniotic fluid cells by real-time PCR / 中华检验医学杂志

Objectives To investigate the prenatal diagnosis method of spinal muscular atrophy with amniotic fluid sample.Methods Totally 1 064 amniotic fluid samples from mid-trimester pregnant women were enrolled during January 2015 and January 2016 in 4 hospitals.Genetic analysis was performed for detecting potential contamination of maternal tissue by a genetic technique based on short tandem repeat ( STR) markers.Deletion of SMN1 gene was detected in 1 062 uncontaminated amniotic fluid samples by real-time PCR and multiplex ligation-dependent probe amplification ( MLPA) respectively.Results Two contaminated amniotic fluid samples were detected within 1 064 mid-trimester pregnant women by STR genotyping.The other 1 062 uncontaminated amniotic fluid samples were tested by real-time PCR.There were 37 samples with heterozygous deletion of Exon 7 of SMN1 gene ( 3.67%) , 34 samples with heterozygous deletion of Exon 8 of SMN1 gene (3.2%) and two samples with homozygous deletion of Exon 7 and Exon8 of SMN1 gene ( 0.19%) respectively , while other samples observed with no deletion of Exon 7 and Exon8 in SMN1 gene.Totally 41 samples with heterozygous or homozygous deletion of SMN 1 gene and 55 samples with undetected deletion of SMN 1 gene were confirmed by MLPA and the results showed 100%consistence with that of real-time PCR.Conclusions Both real-time PCR and MLPA are suitable for detecting the deletion of SMN 1 gene with amniotic fluid sample . Real-time PCR exhibits less sample requirement and time compared with MLPA .

A novel assay for detecting the mutation of nucleotide 1758-1777 deletion in core promoter region of hepatitis B virus / 解放军医学杂志

Objective Nucleotide (nt) 1758-1777 deletion in core promoter (CP) region of hepatitis B virus (HBV) has been suggested to be associated with disease progression. However, the complicated and less sensitive assay for it limited its use in clinic. The present study was aimed at setting a novel assay for its detection using single-tube nested PCR amplification and real-time PCR melting curve analysis. Methods The PCR primers were designed through analysis of HBV genomic sequences in GenBank, and detection conditions were optimized. HBV CP region from 340 serum samples of chronic hepatitis B patients were amplified and directly sequenced, and fifty samples were randomly selected for cloning and sequencing for analysis of nt 1758-1777 deletion. The wild-type and deletion-type plasmids were extracted from mono-cloning samples. Positive standard of melting curve analysis was set up in light of the results of PCR amplification of two standard plasmids and cloning samples. The new method of assay was used in 340 samples, and the data were verified by the results of pyrosequencing. Results Sixteen (4.7%) samples were positive for the deletion by direct sequencing, and no less than 15% samples in standard plasmids and cloning sequencing showed sequence deletion. The melting temperature (Tm) of deletion-type plasmid and cloning samples containing ≥15% proportion of the deletion sequence was ≥88.3°C, which was determined as positive standard of the novel assay. Forty-seven (13.8%) samples were detected positive for nt 1758-1777 deletion by the novel assay. Among them, deletion ratio was ≥1.0% in 38 samples and <1.0% in 9 samples by pyrosequencing, respectively. The deletion ratio was all <1.0% in 15 negative control samples. The deletion ratio of 1.0% was taken as positive cutoff by pyrosequencing, the novel assay had 80.9% positive consistency and 100% negative consistency, with a Kappa value of 0.671. Conclusions Comparing with direct sequencing, the novel assay significantly increased detection rate of nt 1758-1777 deletion. In combination with pyrosequencing for confirmation, the accuracy and cost-effectiveness of the detection could be significantly improved. The novel assay offers an example for detecting HBV genetic deletions.

miR-150 deletion affects breeding and hematologic parameters of mice / 中国免疫学杂志

Objective:To investigate the effect of miR-150 gene deletion on the breeding and hematologic parameters of mice. Methods:The nest litter size,wean rate and weight changes of miR-150 knock out (miR-150ko) and C57BL/6J mice were com-pared. The hematology indexes were analyzed by automated blood cell counter, the serum biochemical parameters were analyzed by automatic biochemical analyzer. Results:The nest litter size and wean rate of miR-150ko mice were significantly decreased compared with that of C57BL/6J mice. The number of total white blood cells,intermediate cells,neutrophils,and the percentage of neutrophils and intermediate cells were significantly increased in miR-150ko mice compared with that of C57BL/6J mice. However,the number and per-centage of platelets and lymphocytes decreased significantly in miR-150ko mice. In addition,the levels of serum glucose and TC were in-creased significantly in miR-150ko mice compared with that of C57BL/6J mice. Conclusion: miR-150 gene deletion impairs the breeding and has complex impact on hematologic parameters of mice.

Quantitative proteomics of CDC42 in HBx-mediated cellular transformation / 军事医学

Objective To identify the mediators of CDC42 signaling pathway involved in hepatitis B virus X protein (HBx)-mediated cellular transformation.Methods The mass defect-based pseudo-isobaric dimethyl labeling method (pIDL)was used to detect the differentially expressed proteins with a deficiency of CDC42.Furthermore,we conducted a gene ontology (GO)of differentially expressed proteins.Results and Conclusion We totally qualified 3409 proteins and found 220 differentially expressed proteins.Palladin,formin-like 1 (FMNL1)and keratin-19,which were implicated in cytoskeleton organization,were down-regulated with the deficiency of CDC42.Our results have provided candidate genes and proteins that may play an important role in HBx-mediated cellular transformation.

The effect of DLC1 and RhoA in lymphatic metastasis of breast cancer / 重庆医学

Objective Through detect the expression of DLC1 and RhoA protein in breast cancer tissues ,investigate the effect of DLC1 and RhoA in lymphatic metastasis of breast cancer .Methods Totally 25 breast cancer specimens that had occurred lymph node metastasis and 30 breast cancer specimens that had not occurred lymph node metastasis were collected ,the expression of DLC1 and RhoA were tested by immunochemistry ,and the experimental data were analyzed by statistical methods .Results The positive rate of DLC1 in the breast cancer specimens that had occurred lymph node metastasis was 44 .00% ,that had not occurred lymph node metastasis was 73 .33% (χ2 =4 .889 ,P=0 .027) ,and the positive rates of RhoA in the two specimens were 92 .00%and 50 .00% (χ2 =11 .264 ,P=0 .001) .Spearman rank correlation analysis showed the expression of DLC 1 and RhoA had a nega‐tive correlation in breast cancer tissues (r= -0 .454 ,P=0 .000) .Conclusion The breast cancer lymphatic metastasis and is closely related to the low expression of DLC1 and the high expression of RhoA .

Análise do gene PTEN por hibridização in situ fluorescente no carcinoma de células renais / Analysis of PTEN gene by fluorescent in situ hybridization in renal cell carcinoma

OBJETIVO: avaliar a frequência de deleção do gene PTEN no carcinoma de células renais e o impacto da deleção nas taxas de sobrevida global e livre de doença. MÉTODOS: foram analisados 110 pacientes portadores de carcinoma de células renais submetidos à nefrectomia radical ou parcial entre os anos de 1980 e 2007. Em 53 casos foi possível a análise do gene PTEN pelo método de hibridização in situ fluorescente através da técnica de "tissue microarray". Para a análise estatística, os pacientes foram classificados em dois grupos, de acordo com a presença ou ausência de deleção. RESULTADOS: o tempo médio de seguimento foi de 41,9 meses. Deleção hemizigótica foi identificada em 18 pacientes (33,9%), ao passo que deleção homozigótica esteve presente em três (5,6%). Em aproximadamente 40% dos casos analisados havia deleção. Monossomia e trissomia foram detectadas, respectivamente, em nove (17%) e dois pacientes (3,8%). Em 21 pacientes (39,6%), a análise por hibridização in situ do gene PTEN foi normal. Não houve diferenças estatisticamente significativas nas taxas de sobrevida global (p=0,468) e livre de doença (p=0,344) entre os pacientes portadores ou não de deleção. Foram fatores independentes para a sobrevida global: estádio clínico TNM, sintomatologia ao diagnóstico, alto grau de Fuhrmann performance status (Ecog) e recorrência tumoral. A livre de doença foi influenciada unicamente pelo estádio clínico TNM. CONCLUSÃO: deleção do gene PTEN no CCR foi detectada com frequência de aproximadamente 40% e sua presença não foi determinante de menores taxas de sobrevida, permanecendo os fatores prognósticos tradicionais como determinantes da evolução dos pacientes.

OBJECTIVE: To evaluate the frequency of deletion of the PTEN gene in renal cell carcinoma (RCC) and its impact on the rates of overall and disease-free survival. METHODS: We analyzed 110 patients with renal cell carcinoma who underwent radical or partial nephrectomy between 1980 and 2007. In 53 cases it was possible to analyse the PTEN gene by the method of fluorescent in situ hybridization using the technique of tissue microarray. For statistical analysis, patients were classified in two groups according to the presence or absence of the deletion. RESULTS: The mean follow-up time was 41.9 months. Hemizygous deletion was detected in 18 patients (33.9%), while the homozygous one was present in three (5.6%). Deletion was present in approximately 40% of the analyzed cases. Monosomy and trisomy were detected in nine (17%) and two patients (3.8%), respectively. In 21 patients (39.6%) the analysis of the PTEN gene by in situ hybridization was normal. There were no statistically significant differences in overall (p = 0.468) and disease-free (p = 0.344) survival rates between patients with or without deletion. Factors which were independent for overall survival: TNM clinical stage, symptoms at diagnosis, high Fuhrmann grade, performance status (ECoG) and tumor recurrence. Disease-free survival was influenced only by the clinical TNM stage. CONCLUSION: Deletion of the PTEN gene in RCC was detected with a frequency of approximately 40% and its presence was not determinant of lower survival rates, the traditional prognostic factors remaining as determinants of outcome.