Early genetic diagnosis of clarithromycin resistance in Helicobacter pylori

BACKGROUND The drug resistance rate of clinical Helicobacter pylori(H.pylori)isolates has increased.However,the mechanism of drug resistance remains unclear.In this study,drug-resistant H.pylori strains were isolated from different areas and different populations of Chinese for genomic analysis.AIM To investigate drug-resistant genes in H.pylori and find the genes for the early diagnosis of clarithromycin resistance.METHODS Three drug-resistant H.pylori strains were isolated from patients with gastritis in Bama County,China.Minimal inhibitory concentrations of clarithromycin,metronidazole,and levofloxacin were determined and complete genome sequencing was performed with annotation.Hp1181 and hp1184 genes were found in these strains and then detected by reverse transcription polymerase chain reaction.The relationships between hp1181 or hp1184 and clarithromycin resistance were ascertained with gene mutant and drug-resistant strains.The homology of the strains with hp26695 was assessed through complete genome detection and identification.Differences in genome sequences,gene quantity,and gene characteristics were detected amongst the three strains.Prediction and analysis of the function of drug-resistant genes indicated that the RNA expression of hp1181 and hp1184 increased in the three strains,which was the same in the artificially induced clarithromycin-resistant bacteria.After gene knockout,the drug sensitivity of the strains was assessed.RESULTS The strains showing a high degree of homology with hp26695,hp1181,and hp1184 genes were found in these strains;the expression of the genes hp1184 and hp1181 was associated with clarithromycin resistance.CONCLUSION Hp1181 and hp1184 mutations may be the earliest and most persistent response to clarithromycin resistance,and they may be the potential target genes for the diagnosis,prevention,and treatment of clarithromycin resistance.CONCLUSION Hp1181 and hp1184 mutations may be the earliest and most persistent response to clarithromycin resistance,and they may be the potential target genes for the diagnosis,prevention,and treatment of clarithromycin resistance.

Preimplantation genetic diagnosis for Down syndrome pregnancy

Objective: To evaluate the effect of preimplantation genetic diagnosis (PGD) conducted for women who had Down syndrome pregnancy previously. Methods: Trisomy 21 was diagnosed by using fluorescence in site hybridization (FISH) before embryo transfer in two women who had Down syndrome pregnancies. Each received one or two PGD cycles respectively. Results: Case 1: one PGD cycle was conducted, two oocytes were fertilized and biopsied. One embryo is of trisomy 21 and the other of monosomy 21. No embryo was transferred. Case 2: two PGD cycles were conducted, in total, sixteen oocytes were fertilized and biopsied. Four embryos were tested to be normal, six of trisomy 21, and one of monosomy 21. Five had no signal. Four normal embryos were transferred but no pregnancy resulted. Conclusion: For couples who had pregnancies with Down syndrome pre-viously, PGD can be considered, and has been shown to be an effective strategy.

Establishment of a Simple and Useful Way for Preimplantation Genetic Diagnosis of Chromosomal Diseases

In order to establish a simple and useful way for preimplantation genetic diagnosis (PGD) of chromosomal diseases in general IVF laboratory, the methods that are most commonly used in the embryo biopsy, fixation of blastomere and fluorescence in situ hybridization were compared. The three aspects of PGD were analyzed respectively. There was no significant difference in further de- velopment capacity of embryos between mechanical (79.7%) and chemical biopsy group (78.6%) (P>0.05). In this study, more cells were successfully fixed with the Tween/HCL method (93.8%) than with the methanol/acetic acid method (80.5%, P<0.05). There was no significant difference in cyto- plasm remains between methanol/acetic acid method and Tween/HCL method (P>0.05). The hy- bridization efficiency of fluorescence in situ hybridization was 89.5% in successive denaturation method and 90.9% in codenaturation method with the difference being not significant (P>0.05). In conclusion, the mechanical or chemical method, Tween/HCL fixation method and codenaturation fluorescence in situ hybridization method can constitute a simple and useful way for PGD of chro- mosomal diseases.

Identification of embryonic chromosomal abnormality using FISH-based preimplantaion genetic diagnosis

Objective: Embryonic chromosomal abnormality is one of the main reasons for in vitro fertilization (IVF) failure. This study aimed at evaluating the value of Fluorescence in-situ Hybridization (FISH)-based Preimplantation Genetic Diagnosis (PGD) in screening for embryonic chromosomal abnormality to increase the successful rate of IVF. Method: Ten couples, four with high risk of chromosomal abnormality and six infertile couples, underwent FISH-based PGD during IVF procedure. At day 3, one or two blastomeres were aspirated from each embryo. Biopsied blastomeres were examined using FISH analysis to screen out embryos with chromosomal abnormalities. At day 4, embryos without detectable chromosomal abnormality were transferred to the mother bodies as in regular IVF. Results: Among 54 embryos screened using FISH-based PGD, 30 embryos were detected to have chromosomal abnormalities. The 24 healthy embryos were implanted, resulting in four clinical pregnancies, two of which led to successful normal birth of two healthy babies; one to ongoing pregnancy during the writing of this article; and one to ectopic pregnancy. Conclusion: FISH-based PGD is an effective method for detecting embryonic chromosomal abnormality, which is one of the common causes of spontaneous miscarriages and chromosomally unbalanced offsprings.

Atelosteogenesis Type 2/Diastrophic Dysplasia Phenotypic Spectrum: From Prenatal to Preimplantation Genetic Diagnosis

Atelosteogenesis type II (AO2) and diastrophic dysplasia (DTD) are two recessively inherited, severe skeletal dysplasias caused by mutations in the SLC26A2 gene. AO2 is an invariably lethal condition, while DTD patients may reach adult life, although both diseases have overlapping diagnostic features. Here we report a patient with an intermediate phenotype between AO2 and DTD and present the successful application of preimplantation genetic diagnosis (PGD) in this situation. Sequencing of SLC26A2 alleles in the infant identified two compound heterozygous mutations, p.Arg178Ter and p.Arg279Trp, of paternal and maternal origin, respectively. At request from the parents, PGD was developed by haplotype mapping of parental SLC26A2 alleles in eleven five-day embryos. Transference to the mother was attempted twice, finally resulting in pregnancy and delivery of a healthy baby. This exemplifies the utility of PGD for inherited lethal conditions with a significant risk of recurrence, and highlights the importance of accurate diagnosis of skeletal dysplasias with prenatal manifestation.

The Performance of Whole Genome Amplification Methods and Next-Generation Sequencing for Pre-Implantation Genetic Diagnosis of Chromosomal Abnormalities

Reliable and accurate pre-implantation genetic diagnosis （PGD） of patient＇s embryos by next-generation sequencing （NGS） is dependent on efficient whole genome amplification （WGA） of a representative biopsy sample. However, the performance of the current state of the art WGA methods has not been evaluated for sequencing. Using low template DNA （15 pg） and single cells, we showed that the two PCR-based WGA systems SurePlex and MALBAC are superior to the REPLI-g WGA multiple displacement amplification （MDA） system in terms of consistent and reproducible genome coverage and sequence bias across the 24 chromosomes, allowing better normalization of test to reference sequencing data. When copy number variation sequencing （CNV-Seq） was applied to single cell WGA products derived by either SurePlex or MALBAC amplification, we showed that known disease CNVs in the range of 3-15 Mb could be reliably and accurately detected at the correct genomic positions. These findings indicate that our CNV-Seq pipeline incorporating either SurePlex or MALBAC as the key initial WGA step is a powerful methodology for clinical PGD to identify euploid embryos in a patient＇s cohort for uterine transplantation,

Clinical applications of MARSALA for preimplantation genetic diagnosis of spinal muscular atrophy

Conventional PCR methods combined with linkage analysis based on short tandem repeats （STRs） or Karyomapping with single nucleotide polymorphism （SNP） arrays, have been applied to preimplantation genetic diagnosis （PGD） for spinal muscular atrophy （SMA）, an autosome recessive disorder. However, it has limitations in SMA diagnosis by Karyomapping, and these methods are unable to distinguish wild- type embryos with carriers effectively. Mutated allele revealed by sequencing with aneuploidy and linkage analyses （MARSALA） is a new method allowing embryo selection by a one-step next-generation sequencing （NGS） procedure, which has been applied in PGD for both autosome dominant and X-linked diseases in our group previously. In this study, we carried out PGD based on MARSALA for two carrier families with SMA affected children. As a result, one of the couples has given birth to a healthy baby free of mutations in SMA-causing gene. It is the first time that MARSALA was applied to PGD for SMA, and we can distinguish the embryos with heterozygous deletion （carriers） from the wild-type （normal） ones accurately through this NGS-based method. In addition, direct mutation detection allows us to identify the affected embryos （homozygous deletion）, which can be regarded as probands for linkage analysis, in case that the affected family member is absent, In the future, the NGS-based MARSALA method is expected to be used in PGD for all monogenetic disorders with known pathogenic gene mutation.

Comprehensive genetic diagnosis of patients with Duchenne/Becker muscular dystrophy(DMD/BMD) and pathogenicity analysis of splice site variants in the DMD gene

Duchenne muscular dystrophy(DMD)and Becker muscular dystrophy(BMD)are caused by mutations in the DMD gene.The aim of this study is to identify pathogenic DMD variants in probands and reduce the risk of recurrence of the disease in affected families.Variations in 100 unrelated DMD/BMD patients were detected by multiplex ligation-dependent probe amplification(MLPA)and next-generation sequencing(NGS).Pathogenic variants in DMD were successfully identified in all cases,and 11 of them were novel.The most common mutations were intragenic deletions(69%),with two hotspots located in the 5'end(exons 2–19)and the central of the DMD gene(exons 45–55),while point mutations were observed in 22%patients.Further,c.1149+1G>A and c.1150?2A>G were confirmed by hybrid minigene splicing assay(HMSA).This two splice site mutations would lead to two aberrant DMD isoforms which give rise to severely truncated protein.Therefore,the clinical use of MLPA,NGS,and HMSA is an effective strategy to identify variants.Importantly,eight embryos were terminated pregnancies according to prenatal diagnosis and a healthy boy was successfully delivered by preimplantation genetic diagnosis(PGD).Early and accurate genetic diagnosis is essential for prenatal diagnosis/PGD to reduce the risk of recurrence of DMD in affected families.

Review:Whole genome amplification in preimplantation genetic diagnosis

Preimplantation genetic diagnosis (PGD) refers to a procedure for genetically analyzing embryos prior to implantation,improving the chance of conception for patients at high risk of transmitting specific inherited disorders.This method has been widely used for a large number of genetic disorders since the first successful application in the early 1990s.Polymerase chain reaction (PCR) and fluorescent in situ hybridization (FISH) are the two main methods in PGD,but there are some inevitable shortcomings limiting the scope of genetic diagnosis.Fortunately,different whole genome amplification (WGA) techniques have been developed to overcome these problems.Sufficient DNA can be amplified and multiple tasks which need abundant DNA can be performed.Moreover,WGA products can be analyzed as a template for multi-loci and multi-gene during the subsequent DNA analysis.In this review,we will focus on the currently available WGA techniques and their applications,as well as the new technical trends from WGA products.

Advances in preimplantation genetic diagnosis/screening

Preimplantation genetic diagnosis(PGD)gives couples who have a high risk of transmitting genetic disorders to their baby the chance to have a healthy offspring through embryo genetic analysis and selection.Preimplantation genetic screening(PGS)is an effective method to select euploid embryos that may prevent repeated implantation failure or miscarriage.However,how and to whom PGS should be provided is a controversial topic.The first successful case of PGD of a human being was reported in 1990,and there have been tremendous improvements in this technology since then.Both embryo biopsy and genetic technologies have been improved dramatically,which increase the accuracy and expand the indications of PGD/PGS.

Study on Genetic Diagnosis of Spinal Muscular Atrophy

An improved method of mismatching polymerase chain reactionrestrictive fragment length polymorphisms(PCR-RFLP) was performed in our lab for genetic diagnosis of spinal muscular atrophy(SMA). PCR amplification and restriction endonuclease digestion of exons 7 and 8 permit distinction of the telomeric survival motor neuron (SMN) gene and its centromeric copy. Lack of a PCR product from either exon and from either gene is indicative of homozygous deletion of that sequence, and a high correlation with clinical SMA. Our data showed: 9 cases in 10 presumed SMA children were positive, i.e. deletion of telomeric SMN gene. One case was negative. 20 cases of normal familial members and 20 cases of normal health persons were all negative. Our results matched the criteria and reports of foreign countries. The method we used is highly specific, sensitive and reliable and is suitable for genetic diagnosis of SMA and its prenatal diagnosis.

Genetic diagnosis for heavy typhoon rainfall attenuated by Fujian landfall

This study used the dynamic synthetic analysis method to analyze the causes of attenuated heavy rainfall from a westward moving typhoon after landfall over Fujian by focusing on the genetic diagrgnosis of the strongest 12 h rainstorms based on typhoon data obtained from the Shanghai Typhoon Institute,precipitation data from Fujian Province,and NCEP reanalysis data from the United States.The results showed that:(1)the environmental field of the westward moving typhoon benefits the long-term maintenance of convergence in coastal areas,which provides synoptic scale forcing for rainstorm intensification along the southeastern coast;(2)the southwest jet in the boundary layer transports warm water vapor from low latitudes into the eastern circulation of typhoon;the water vapor peak occurs 6 h before the strongest rainstorm and can be used as a reference index to predict heavy rainstorms;(3)the high altitude strong divergence center is located at 100-150 hPa,and the strong convergence center is located near 925-950 hPa in the boundary layer,which is higher(lower)than the 200 hPa divergence layer(850 hPa convergence layer)commonly used in professional work;(4)warm and wet advection in the boundary layer transports unstable energy and weak cold air southward,strengthens the baroclinic pressure,increases the latent heat flux on the sea surface,and plays a significant role in triggering and developing mesoscale convective clouds along the southeast coast.

Improving genetic diagnosis of Mendelian disease with RNA sequencing:a narrative review

Targeted sequencing and whole exome sequencing are the most common approaches used to detect causative variants in Mendelian diseases;however, using DNA-based sequencing techniques, the current molecular diagnostic yield is at best 50%. In recent years, RNA sequencing has been shown to be able to provide a genetic diagnosis in patients whose conditions were previously unable to be identified by DNA analysis. RNA sequencing can reveal expression outliers, aberrant splicing events, allele-specific expression, and new pathogenic variants, and as such can complement and expand on the traditional genomic methods used to diagnose Mendelian diseases. Therefore, RNA sequencing is expected to become a routine method for genetic diagnosis in the future. This article reviews the applications and challenges of RNA sequencing in the genetic diagnosis of Mendelian diseases.

A New Next-Generation Sequencing-Based Assay for Concurrent Preimplantation Genetic Diagnosis of Charcot-Marie-Tooth Disease Type 1A and Aneuploidy Screening

Charcot-Marie-Tooth （CMT） disease is the most common hereditary neuropathy, with a population prevalence of 1 in 2500. CMT disease type 1A （CMT1A）, accounting for ~70% of CMT1 cases and ~ 50% of all CMT cases, is transmitted in an autosomal dominant manner. CMT1A maps to chromo- some 17pl 1.2 and is caused, in the majority of cases, by a 1.4- Mb tandem duplication that includes the peripheral myelin protein22 （PMP22） gene （Li et al., 2013）. The disease usually presents in the first 20 years of age, causing difficulty in walking or running, distal symmetrical muscle weakness and wasting, and sensory loss （van Paassen et al., 2014）.

Preliminary Investigation on the Role of Vitrification in Pre-Implantation Genetic Diagnosis

Objective To investigate the feasibility of vitrification of blastocysts following blastomere biopsy. Methods Among patients undergoing pre-implantation genetic diagnosis （PGD）, artificial shrinkage of the blastocoelic cavity and subsequent vitrification of applicable surplus blastocysts after day-3 blastomere biopsy were performed. According to patient requirements, thawed blastocysts were transferred into patients due to pregnancy failure after fresh embryo transfer, ectopic pregnancy, ovarian hyperstimulation. Results Twenty-four PGD cycles were carried out. According to genetic diagnosis and the development of blastocysts, transfer was cancelled in 7 cycles due to absence of applicable embryos or ovarian hyperstimulation. In the remaining 17 cycles, 26 blastocysts were thawed and transferred, which resulted in 13 implanted （50.0%）. Clinical pregnancies were observed in 11 patients （64.71%）. Following transfer, 30 applicable blastocysts in 10 cycles were cryopreserved. Six patients received transfer of thawed blastocysts. All 8 thawed embryos survived and were transferred, and singleton pregnancies occurred in 5 patients. Two women delivered healthy infants and 3 pregnancies are ongoing. Conclusion Vitrification with artificial shrinkage is effective for preserving blastocysts following blastomere biopsy.

Preimplantation genetic diagnosis of hereditary hearing loss:a narrative review

Preimplantation genetic diagnosis(PGD)uses molecular biological techniques to genetically diagnose embryos beforein vitro fertilization.The information obtained through PGD can help clinicians select healthy embryos for implantation,prevent the transmission of inherited diseases and help affected families have healthy children.This paper reviews the development of PGD technology,the history of its application to hereditary hearing loss,and the general process of how PGD is applied to screen for hereditary hearing loss.The aim of this review is to demonstrate the reliability of PGD in the primary prevention of hereditary hearing loss,assist clinicians in counseling patients at risk of transmitting an inherited disease,and explore the journey from PGD toin vitro fertilization.Given that the application of PGD technology to hereditary hearing loss varies in different countries and regions,there is still a long way to go before PGD is routinely applied for the primary prevention of hereditary hearing loss.

preimplantation genetics diagnosis译为“植入前遗传诊断”为妥

1.implantation按1993年《全国自然科学名词审定委员会公布、胚胎学名》第126页,审定“implantation”译为“植入”(胚泡进入子宫内膜的过程)编号为02.078。 2.人民卫生出版社出版的《英汉医学词汇》第706页和《汉英医学词汇》均将implantation译为“植入”(胚泡在子宫内)。

Genetic analysis of Chinese families reveals a novel truncation allele of the retinitis pigmentosa GTPase regulator gene

AIMTo make comprehensive molecular diagnosis for retinitis pigmentosa (RP) patients in a consanguineous Han Chinese family using next generation sequencing based Capture-NGS screen technology.

Gene diagnosis of infantile neurofibromatosis type I:A case report

BACKGROUND Neurofibromatosis is an autosomal dominant genetic disorder with various manifestations.Systemic multiple neurofibromatosis is rare in infancy.The disease is difficult to identify in the early stage,and it is prone to misdiagnosis and missed diagnosis.In the presence of lower limb swelling with subcutaneous nodules of unknown cause,café-au-lait spots,and axillary freckles,this disease must be considered.This report presents the clinical manifestations,early detection,diagnosis and treatment,and prognosis of infantile neurofibromatosis type I(NF1).CASE SUMMARY The clinical manifestations,imaging examinations,and gene results of a 3-mo-old male infant with NF1 were analyzed retrospectively.He had“swelling of both legs”at the onset and developed café-au-lait spots,axillary freckles,and multiple neurofibromas later.He had a family history of similar conditions.Gene detection showed a heterozygous mutation of c.4537C>T in the NF1 gene,leading to a nonsense mutation of amino acids(p.R1513x),which originated from the mother of the infant.He was diagnosed with NF1.CONCLUSION Gene diagnosis plays an important role in the early diagnosis of NF1.

Chromosomal disorders and male infertility

Infertility in humans is surprisingly common occurring in approximately 15% of the population wishing to start a family. Despite this, the molecular and genetic factors underlying the cause of infertility remain largely undiscovered. Nevertheless, more and more genetic factors associated with infertility are being identified. This review will focus on our current understanding of the chromosomal basis of male infertility specifically： chromosomal aneuploidy, structural and numerical karyotype abnormalities and Y chromosomal microdeletions. Chromosomal aneuploidy is the leading cause of pregnancy loss and developmental disabilities in humans. Aneuploidy is predominantly maternal in origin, but concerns have been raised regarding the safety of intracytoplasmic sperm injection as infertile men have significantly higher levels of sperm aneuploidy compared to their fertile counterparts. Males with numerical or structural karyotype abnormalities are also at an increased risk of producing aneuploid sperm. Our current understanding of how sperm aneuploidy translates to embryo aneuploidy will be reviewed, as well as the application of preimplantation genetic diagnosis （PGD） in such cases. Clinical recommendations where possible will be made, as well as discussion of the use of emerging array technology in PGD and its potential applications in male infertility.