1、Medical GeneticsMedical Genetics Genetic diseases and congenital malformations occur in approximately 2 to 5%of all live births,account for up to 30%of paediatric admissions to hospital,and are an important cause of death under the age of 15 years.Medical Genetics Furthermore,the psychological stres
2、s on families with children with serious genetic disorders is incalculable.Until gene therapy becomes a practical reality,the only option available for the control of genetic disease is prenatal diagnosis.Medical GeneticsThe use of prenatal diagnosis is determined by balancing the risk of the birth
3、of an abnormal child against the risk of an investigative procedure.Medical GeneticsThe general indications of prenatal diagnosis include maternal age and the results of noninvasive serum biochemical screening.Medical GeneticsSpecific indications include a positive family history and the birth of a
4、previous child affected by a particular genetic disease.Medical GeneticsTo perform prenatal diagnosis,fetal-derived tissues must first be obtained.All of the commonly used methods that yield fetal tissues are invasive.Medical GeneticsA.Amniocentesis Amniocentesis is the withdrawal of amniotic fluid
5、from the amniotic sac surrounding the fetus.For over two decades this has been the primary technique utilised for the diagnosis of fetal genetic disorders.Medical Genetics Traditionally amniocentesis has been performed around 15 to 16 weeks gestation.At this time the uterus is easily accessible to a
6、 transabdominal approach,and a sufficient volume of amniotic fluid(approximately 200 ml)exists to permit 20 to 30 ml to be withdrawn safely.Medical Genetics Amniocentesis is normally performed as an outpatient facility.An ultrasound examination is normally done immediately before the procedure to ev
7、aluate fetal number and viability,perform fetal biometric measurements,establish placental location,and estimate amniotic fluid volume.Medical GeneticsAmniocentesis performed concurrently with ultrasound scanning.Medical GeneticsSafety of amniocentesis Any procedure that involves passing a device in
8、to an organ,especially the pregnant uterus,carries with it risks;amniocentesis is no exception.Medical Genetics Amniocentesis involves potential danger to both mother and fetus.Serious maternal risks are quite low but include amnionitis which can lead to fetal loss,haemorrhage or injury to an intra-
9、abdominal viscus and leakage of amniotic fluid.Medical Genetics Fetal risks include spontaneous abortion,needle puncture injuries,placental abruption,chorioamnionitis,preterm labour,and amniotic band formation.Medical Genetics Several controlled studies have been done to evaluate the risks of amnioc
10、entesis.The data indicate that the risk of pregnancy loss attributable to amniocentesis may be as high as 0.5%.Medical Genetics In general,risks will depend on(1)the experience of the obstetrician performing the procedure;(2)clinical characteristics of a particular case(e.g.,presence or absence of b
11、iochemical markers of fetal abnormality);(3)the quality of the high-resolution ultrasound utilised.Medical GeneticsEarly amniocentesis With development of higher resolution ultrasound equipment,some centres have begun offering amniocentesis before 15 weeks gestation,usually between 10 and 14 weeks.T
12、he majority of procedures have been performed during the 13th and 14th weeks of gestation.Medical Genetics There is evidence that early amniocentesis is associated with a higher fetal loss rate and a more frequent occurrence of certain congenital abnormalities.Medical GeneticsB.Chorionic villus samp
13、ling Chorionic villus sampling(CVS)is the only tested method for first-trimester fetal genetic diagnosis that is currently in clinical use and is usually performed between 9 and 11 weeks.Medical Genetics The procedure involves the passing of a sampling instrument into the chorion(developing placenta
14、).A good procedure yields from 10 to 25 mg of tissue which is adequate for cytogenetic,enzymatic or DNA analysis.Medical Genetics The main advantage of CVS over amniocentesis is the applicability of CVS earlier in gestation.This results in considerably reduced social,emotional and psychological stre
15、ss for the couple.Medical GeneticsSafety of CVS Maternal complications include bleeding and infection.Fetal loss following CVS has been reported to be around 2%.There are also reports of limb reduction defects in infants born to mothers who have had CVS between 56 and 66 days of gestation.Medical Ge
16、neticsC.Fetal blood sampling(FBS)Fetal blood can be safely and directly sampled from approximately 18 weeks gestation onwards.FBS can be used for both diagnostic and therapeutic purposes.Medical Genetics Indications for fetal blood samplingDiagnosticDiagnosticRapid karyotypingFetal anomaly on ultras
17、oundLate attending patients who require fetal karyotypingAlloimmunisationRhesusPlatelet antigensFetal infectionToxoplasmosisCytomegalovirus infectionGeneticHaemoglobinopathiesMetabolic disorders and enzyme deficienciesFetal well beingSevere intrauterine growth retardationTherapeutiTherapeutic cTrans
18、fusionRed cell alloimmunisationTransplantationStem cellsMedical Genetics FBS is contraindicated if the mother is suffering from infections that can be transmitted to the fetus by the procedure.Examples include human immunodeficiency virus and hepatitis B virus infection.Medical GeneticsSafety of FBS
19、 Maternal complications from FBS are uncommon but include amnionitis,infection,rhesus sensitisation and transplacental haemorrhage.Medical Genetics Fetal loss rates following FBS have been reported to be approximately 1%in several large series.The presence of structural abnormalities or severe growt
20、h retardation of the fetus is associated with a much increased fetal loss rate.Medical Genetics Other fetal complications include infection,premature rupture of membranes,haemorrhage,severe bradycardia and umbilical cord thrombosis.Medical GeneticsD.Fetal biopsy Although advances in molecular and bi
21、ochemical genetics have made the diagnosis of many Mendelian disorders possible by analysis of amniotic fluid cells or chorionic villi,some conditions still require direct analysis of tissues in which the disorder is manifested.Tissues which have been successfully biopsied include fetal skin,liver a
22、nd muscle.Medical GeneticsSafety of fetal biopsy Due to the relatively small numbers performed in different centres,no precise figures for the safety of fetal biopsy is available.Medical Genetics Following the acquisition of fetal tissues,these materials are then subjected to analysis using a variet
23、y of techniques.Medical GeneticsA.Cell culture and conventional cytogenetics These are the most commonly used methods for the diagnosis of chromosomal aneuploidies such as Down syndrome.Medical GeneticsB.Molecular cytogenetics using FISH FISH involves the hybridization of DNA probes representing a s
24、pecific chromosome or chromosomal region to target DNA such as metaphase chromosomes or interphase nuclei,where the probe binds to homologous sequences in the cell.Medical Genetics Using FISH,several groups have demonstrated that trisomies such as trisomy 21 and trisomy 18 can be detected in uncultu
25、red interphase nuclei as three positive hybridisation signals rather than the normal two.Medical Genetics The main advantage is speed:thus results are available in 24 to 48 hours compared with the 10 to 14 days more typical of standard culture-based cytogenetic analysis.This type of technology can b
26、e applied to fetal materials obtained following amniocentesis,CVS or fetal blood sampling.Medical GeneticsC.DNA-based techniques The main advantage of DNA-based techniques is that any nucleated fetal cell can be used.Techniques which are used include the polymerase chain reaction(PCR)and Southern bl
27、otting.Medical Genetics PCR-based techniques allow a rapid diagnosis to be made in several hours.These methods can be used for direct mutation detection or linkage analysis.The latter type of analysis is needed when the exact mutation or gene causing the disease is not known.Medical GeneticsMedical
28、Genetics Genetic diagnosis is then carried out by analysing DNA sequences within the gene itself or DNA loci closely linked to it.An analysable difference or polymorphism must exist between the disease-carrying allele and the normal allele to distinguish them.Medical GeneticsMedical GeneticsMedical
29、GeneticsD.Metabolic analysis of fetal tissues Fetal tissues or fluids can be subjected to analysis to detect the characteristic metabolic or cellular defects of an inherited metabolic disease.Medical Genetics For this type of analysis to be carried out,the specific enzyme or metabolite of interest m
30、ust be expressed in the fetal tissues sampled,and the range of normal values as well as the assay sensitivity and reproducibility must be established within the tissue of interest.Medical Genetics Although an increasing number of inherited metabolic diseases are amenable to direct DNA-based diagnosi
31、s,enzyme-based techniques are still useful in situations where the disease-causing gene has not been identified or where the precise mutation is not known.Medical GeneticsE.Microarray Analysis Much of the excitement today centers on gene expression profiling that uses a technology called microarrays
32、.Medical Genetics A DNA microarray is a thin-sized chip that has been spotted at fixed locations with thousands of single-stranded DNA fragments corresponding to various genes of interest.Medical Genetics A single microarray may contain 10,000 or more spots,each containing pieces of DNA from a diffe
33、rent gene.Fluorescent-labeled probe DNA fragments are added to ask if there are any places on the microarray where the probe strands can match and bind.Complete patterns of gene activity can be captured with this technology.Medical GeneticsMedical GeneticsA.Preimplantation diagnosis Preimplantation
34、diagnosis is the performance of prenatal genetic analysis on embryos or oocytes prior to implantation.Medical Genetics This technology has the advantage that it allows prenatal diagnosis to be carried out much earlier than existing methods such as amniocentesis and CVS.Medical Genetics Furthermore,c
35、ouples who are at exceptionally high genetic risk and those who have had previous terminations for genetic indications may find preimplantation diagnosis a more acceptable form of prenatal testing.Medical GeneticsMedical Genetics In the future,preimplantation diagnosis may also be used in conjunctio
36、n with gene therapy.At present,given the expense of the procedure and the small number of centres equipped to perform this form of diagnosis,preimplantation diagnosis is unlikely to become a standard procedure in the foreseeable future.Medical GeneticsAccess to oocyte and embryonic cells Preimplanta
37、tion genetic analysis can be carried on either embryonic cells or oocytes.In the later situation,diagnosis is carried out even prior to fertilisation.Individual oocytes are aspirated and their polar body biopsied.Medical Genetics For preimplantation diagnosis carried out on embryonic cells,the embry
38、o may be fertilised in vitro and then individual blastomeres biopsied.Alternatively,the embryo may be fertilised in vivo and then the embryos are obtained by uterine lavage followed by biopsy and genetic analysis.Medical Genetics For heterozygous women carrying one mutant and one normal allele of a
39、disease-causing gene,in the absence of chromosomal crossing-over,the aspirated polar body containing a mutant allele would indicate that the primary oocyte pronucleus is carrying the normal allele.Medical Genetics In both situations,only the embryos confirmed not to possess the full disease-causing
40、genotype are then implanted back into the uterus.Medical GeneticsMedical GeneticsDiagnostic methods Preimplantation diagnosis may be achieved using PCR,FISH or by measurement of embryonic secretory products such as certain enzymes.Medical Genetics This type of analysis has been carried out successfu
41、lly for the determination of fetal sex for the avoidance of sex-linked disorders such as Duchenne muscular dystrophy and haemophilia A,and for the diagnosis of single gene disorders such as cystic fibrosis,alpha-1-antitrypsin deficiency,Tay-Sachs disease,fragile X and sickle cell anaemia.Medical Gen
42、etics Worldwide,preimplantation diagnosis of embryos has been attempted on over 1,200 in vitro fertilisation cycles in 2019,with clinical pregnancy resulted in 20%.No increase in the occurrence of abnormalities has been observed in the liveborns.Medical GeneticsB.Noninvasive prenatal diagnosis using
43、 fetal cells isolated from maternal blood Circumstantial evidence that fetal nucleated cells exist in maternal peripheral blood can be traced back to 1969.Medical Genetics However,convincing proof of the existence of these cells have to await the development of molecular biological techniques,especi
44、ally the PCR.Medical Genetics Using the PCR,investigators are able to demonstrate the presence of cells possessing fetal genetic markers circulating in the peripheral blood of pregnant women.The isolation of these cells offer the possibility of a noninvasive and safe method for prenatal diagnosis.Me
45、dical GeneticsFetal nucleated cell types in maternal blood Three populations of fetal nucleated cells are currently known to be present in maternal peripheral blood:fetal lymphocytes,trophoblasts and fetal nucleated red cells.Medical Genetics At present,the isolation of fetal nucleated red cells is
46、receiving the most attention from investigators in the field due to the availability of relatively specific monoclonal antibodies against these cells.Medical GeneticsIsolation of fetal cells and genetic analysis A combination of physical and immunological methods are used to isolate fetal nucleated
47、cells from maternal blood.Physical methods include density gradient centrifugation and micromanipulation techniques while immunological methods include the use of monoclonal antibodies.Medical Genetics Genetic analysis of these isolated fetal cells can be performed using PCR or FISH.Fetal cells in m
48、aternal blood have been successfully used on a research level to diagnose trisomy 21,trisomy 18,beta-thalassaemia and sickle cell anaemia.Actual clinical use will have to await further technological development to improve its reliability and clinical trials to assess the sensitivity and specificity of this approach.
