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1、Single-Gene DisordersXiaoshu Chen Single-Gene DisordersWhen a certain gene is known to cause a disease, we refer to it as a single gene disorder.To date, more than 10,000 single-gene traits and disorders have been identified. Most of these are individually rare, but together they affect between 1% a

2、nd 2% of the general population. Single-Gene DisordersIn this chapter, three of the more common and important single-gene disorders are described.Huntington Disease (neuronal degeneration )Duchenne Muscular Dystrophy (muscular weakness )Hemophilia (bleeding disorder)Each one will be describedclinica

3、l features (What is it?)genetic principles (Why does it cause?)clinical applications (How do we deal with it?)From From Peter D. Turnpenny, Emerys Elements of medical genetics. From Huntington DiseaseHuntington Disease (HD)HD derives its title from Dr. George Huntington in 1872.His paper on The Medi

4、cal and Surgical Reporter gave a description of HD asthe neurological disabilitythe most feared hereditary disorders in manGeorge Huntington (April 9, 1850 March 3, 1916)From Huntington Disease (HD)The natural history is characterized by slowly progressive selective cell death in the central nervous

5、 systemThe prevalence is about 1: 10,000.The onset is between 30 and 50 years old.The mean duration of the illness is 15 to 20 years.There is no effective treatment or cure. From Clinical Features of HDThe usual pattern of disease is characterized bya slowly progressive movement disorderChorea is th

6、e most common movement abnormality.This takes the form of subtle involuntary movements, such as twitching of the face and limbsfolding of the arms and crossing of the legsAs the disease progresses the walking es very unsteady speech es unclearClinical Features of HDThe usual pattern of disease is ch

7、aracterized byimpairment of intellectual function withmemory impairment and poor concentrationprogressive psychiatric disturbance eventual dementiaFrom Juvenile HD5% of HD cases present before the age of 20.The average duration of the illness is 10 to 15 years.Clinical Features are: does not bend ea

8、sily, with slowing of voluntary movementinstead of chorea a severe progressive dementiaassociation with epileptic seizuresFrom Genetics of HDMapping the HD GeneTraditionally HD has been said to show autosomal dominant inheritanceclose to complete penetranceCarrying a deleterious gene, all individual

9、s express the associated trait with a very low mutation ratewith a phenomenon, anticipationin succeeding generations, the onset is at a younger age, the disease is on a more severe levelFrom Genetics of HDMapping the HD GeneIn 1983， by collection of blood samples from over 100 affected subjectsby li

10、nkage analysis using polymorphic DNA markers G8HD was one of the first disorders to be mapped.HD was found to show close linkage on the short arm of chr4. HD homozygotes are no more severely affected than heterozygotes.explain the complete penetranceFrom Genetics of HDIsolation of the HD GeneIn 1993

11、The mRNA codes for a protein of 350 kDa, known as huntingtin (also IT15). Huntingtin is expressed in nervous tissue as well as other tissues.It was found to contain a highly polymorphic CAG repeat sequence located in the 5region.Its function remains unclear.CAGCAGCAG CAGCAGCAGFrom A Brief Review of

12、HD MechanismsLarge CAG repeats result in intracellular accumulation of huntingtin.Huntingtin are cleaved by caspase.Caspases are a family of protease enzymes.Huntingtin fragments form a toxic product that causes cell death in nervous tissue.Selective cell death in the central nervous system.From Fro

13、m Genetics of HDThe Mutation in HDHD genes are categorized under four headings on the basis of CAG repeat length.Normal 26 Mutable 2735Reduced penetrance 3639Fully penetrant 40Genetics of HDThe Mutation in HDHD genes are categorized under four headings on the basis of CAG repeat length.Normal 26 do

14、not cause diseaseare stable in meiosisGenetics of HDThe Mutation in HDHD genes are categorized under four headings on the basis of CAG repeat length.Mutable 2735do not cause diseaseshow meiotic instability with a potential to increase or decrease in sizeexpansion are associated with a particular hap

15、lotypehaplotype is a group of genes that are inherited together from a single parentGenetics of HDThe Mutation in HDHD genes are categorized under four headings on the basis of CAG repeat length.Reduced penetrance 3639do not cause disease, or cause late-onset disease Genetics of HDThe Mutation in HD

16、HD genes are categorized under four headings on the basis of CAG repeat length.Fully penetrant 40associated with diseasea negative relationship between length of repeat and disease expressionFor repeat sizes of 40, 45, and 50, (increase)the age of onset is 57, 37, and 26. (decrease)explain anticipat

17、ionwith longer CAG repeats the onset is at a younger age the disease is on a more severe levelFrom Genetics of HDParent of Origin Effect in Disease TransmissionHD is autosomal dominant inheritance.Theoretically, the risk to offspring is 50%, regardless of whether the affected parent is male or femal

18、e.The mutant allele is transmitted by a male in most cases. Meiotic instability is greater in male than female.huntingtin is expressed in female, so that there could be selection against female, as a consequence of preferential cell death.Expansion is caused by slippage of DNA polymerase, reflecting

19、 the number of mitoses in male. Slippage of DNA polymeraseWith repeating pattern of bases, slippage can occur.Slippage and reattachment cause a bubble to form in the new strand.DNA repair mechanisms realign with the new strand.The bubble is straightened out, and the length of repeating pattern is in

20、creased. From Clinical Applications of HDPredictive genetic testing Prenatal diagnosisTherapeutic approachCaspase inhibitors have beneficial effects in preventing cell death. Fetal neuronal cell transfer into regions of the brain.From From Duchenne Muscular DystrophyDuchenne Muscular Dystrophy (DMD)

21、DMD is the most common and most severe form of muscular dystrophy. The title is derived from the French neurologist Guillaume Duchenne in 1861.A similar but milder condition, Becker muscular dystrophy (BMD), is caused by mutations in the same gene. The incidences of DMD and BMD are about 1 : 3500 ma

22、les and 1 : 20,000 males. From Guillaume-Benjamin-Amand Duchenne (de Boulogne)( September 17, 1806 September 15, 1875 )Clinical Features of DMDMales with DMD usually present between the ages of 3 and 5 with slowly progressive muscle weakness resulting in:an awkward gaitinability to run quicklydiffic

23、ulty in rising from the floorMost affected boys have to use a wheelchair at 11, because of severe leg muscle weakness.Subsequently leads to lumbar lordosis, joint contractures.Death at a mean age of 18.About one-third of boys with DMD show mild intellectual impairment.From Clinical Features of DMDOn

24、 examination, boys with DMD show an apparent increase in the size of the calf muscles, replacement of muscle fibers by fat and connective tissueFrom Peter D. Turnpenny, Emerys Elements of medical genetics. Clinical Features of BMDIn BMD the clinical picture is very similar, but the condition is mild

25、er.The mean age of onset is 11. Many patients remain able to walk until well into adult life.Overall life expectancy is only slightly reduced. A few patients with proven mutations in the DMD/BMD gene have been asymptomatic over 50.From Clinical Features comparison of DMD and BMDDMDBMDPrevalenceAge o

26、f onset Age of death Clinic features Clinical Features comparison of DMD and BMDDMDBMDPrevalence1: 3500 1: 20,000Age of onset Age of death Clinic features Clinical Features comparison of DMD and BMDDMDBMDPrevalence1: 3500 1: 20,000Age of onset 3-5 years old 11 years oldAge of death Clinic features C

27、linical Features comparison of DMD and BMDDMDBMDPrevalence1: 3500 1: 20,000Age of onset 3-5 years old 11 years oldAge of death 18 years old Life span is longerClinic features Clinical Features comparison of DMD and BMDDMDBMDPrevalence1: 3500 1: 20,000Age of onset 3-5 years old 11 years oldAge of dea

28、th 18 years old Life span is longerClinic features Severe MilderAt 3-5 years old, patients show slowly progressive muscle weakness. Most affected boys have to use a wheelchair at 11 years old. Subsequent deterioration leads to lumbar lordosis, joint contractures.Many patients remain ambulant until w

29、ell into adult life.Overall life expectancy is only slightly reduced. About one-third of boys show mild-moderate intellectual impairment.Genetics of DMDBoth DMD and BMD show X-linked recessive inheritance.Males with DMD rarely reproduce. Genetic fitness equals to zero.The mutation rate is about 1 :

30、10,000.Its one of the highest known mutation rates in humans.Genetics of DMDIsolation of the Gene for DMDIn 1981,affected females with a balanced X-autosome translocation by identification of positional cloning In 1987, affected males with visible microdeletions by identification of conserved sequen

31、ces in muscle cDNA libraries X-autosome translocationAt a common X-chromosome breakpoint Xp21autosomal segment and X chromosome segment are exchangeda derivative X chromosome and a derivative X autosomeMales only have one copy of an X chromosome One X-chromosome is inactivated/silent in femaleWith d

32、erivative X chromosome been silentcells are deadbecause the autosomal segment is silentWith X chromosome been silentcells survivebecause the derivative X autosome is activeif the X chromosome breakpoint has damaged an gene, the woman will be affected by the diseaseFrom Peter D. Turnpenny, Emerys Ele

33、ments of medical genetics. Wesley H Brooks Yves Renaudineau, 2015, Epigenetics and autoimmune diseases: The X chromosome-nucleolus nexus, Frontiers in Genetics,6:22 Positional cloningFrom Kristel Van Steen, Genetics of DMDIsolation of the Gene for DMDThe dystrophin gene is huge79 exons 2.3 Mb of gen

34、omic DNA14 kb of mRNAmay explain the high mutation rateIt is transcribed in brain and musclemay explain why some boys with DMD show learning difficultiesFrom Genetics of DMDMutations in the Dystrophin GeneDeletionsaccount for two-thirds of all mutationscan be almost any size and locationhotspots are

35、 found in the first 20 exons and exons 45 to 53usually disturb the translational reading framearise almost in maternal meiosisdue to unequal crossing overDuplications account for smaller number of affected males Unequal crossing overUnequal crossing over requires similar sequences for misalignment.W

36、hen two sequences are misaligned, unequal crossing over may create a tandem repeat on one chromosome and a deletion on the other.From (02)02592-1Genetics of DMDMutations in the Dystrophin GeneMutations account for one-third of all mutationsinclude frameshift mutations, stop codons, altered splicing

37、signalsframeshift mutationthe number of nucleotides added or removed is not a multiple of threethe normal sequence of codons is disruptedresult in abnormal protein with an incorrect amino acid sequencemostly lead to premature translational terminationthe little production of proteinarise in paternal

38、 meiosisdue to recurrent rounds of DNA replications in maleFrom Genetics of BMDMutations in the Dystrophin GeneBMD usually do not alter the reading frame the amino-acid sequence of the protein product downstream of the deletion is normalexplaining the relatively mild features in BMDFrom Genetics of

39、DMD The Gene Product DystrophinThe 427-kDa dystrophin is located close to the muscle membrane, where it links intracellular actin with extracellular laminin. Absence of dystrophin leads to muscle cell degeneration. Levels less than 3% of dystrophin are diagnostic. From Ervasti JM, Campbell KP, 1991,

40、 Membrane organization of the dystrophinglycoprotein complex, Cell 66:11211131.Actin is a family of multi-functional proteins that form microfilaments.Laminins are high-molecular weight proteins of the extracellular matrix.Genetics of BMD The Gene Product DystrophinThe 427-kDa dystrophin is located

41、close to the muscle membrane, where it links intracellular actin with extracellular laminin. The dystrophin shows qualitative rather than quantitative abnormalities.From Ervasti JM, Campbell KP, 1991, Membrane organization of the dystrophinglycoprotein complex, Cell 66:11211131.Actin is a family of

42、multi-functional proteins that form microfilaments.Laminins are high-molecular weight proteins of the extracellular matrix.Genetics of other disorders The Gene Product DystrophinThe 427-kDa dystrophin is located close to the muscle membrane, where it links intracellular actin with extracellular lami

43、nin. Glycoprotein complex is binded by C-terminal domain of Dystrophin in the muscle membrane. Abnormalities of this glycoprotein complex cause other rare genetic muscle disorders, including several different types of autosomal recessive limb girdle muscular dystrophy and congenital muscular dystrop

44、hy.From Ervasti JM, Campbell KP,1991, Membrane organization of the dystrophinglycoprotein complex, Cell 66:11211131.Genetics comparison of DMD and BMDDMDBMDInheritanceGeneMutations Gene Product Genetics comparison of DMD and BMDDMDBMDInheritanceX-linked recessive inheritanceX-linked recessive inheri

45、tanceGeneMutations Gene Product Genetics comparison of DMD and BMDDMDBMDInheritanceX-linked recessive inheritanceX-linked recessive inheritanceGenedystrophindystrophinMutations Gene Product Genetics comparison of DMD and BMDDMDBMDInheritanceX-linked recessive inheritanceX-linked recessive inheritanc

46、eGenedystrophindystrophinMutations Usually disturb the translational reading frameDo not alter the reading frame Gene Product Genetics comparison of DMD and BMDDMDBMDInheritanceX-linked recessive inheritanceX-linked recessive inheritanceGenedystrophindystrophinMutations Usually disturb the translati

47、onal reading frameDo not alter the reading frame Gene Product Levels less than 3% of dystrophin are diagnostic. The dystrophin shows qualitative rather than quantitative abnormalities.Clinical Applications of DMDCarrier DetectionDNA testinghas e increasingly usedPedigree information combined with se

48、rum creatine kinase (CK) assayonly useful sometimesLinkage studies may still be useful, when no DNA is available from an affected male, but perhaps available from normal males in the same familyCare has to be taken, because of the high bination rate of 12% across the DMD gene.Serum creatine kinase (

49、CK) assay CK levels are commonly used to judge the severity of muscle damage. CK levels are increased in about two-thirds of all carriers.From Tippett PA, Dennis NR,Machin D, et al1982, Creatine kinase activity in the detection of carriers of Duchenne muscular dystrophy: comparison of twomethods, Cl

50、in Chim Acta, 121:345359Clinical Applications of DMDProspects for Treatment of DMDAt present, there is no cure for DMD or BMD.Physiotherapy is beneficial maintaining mobility preventing muscle spasm and joint contracturesGene therapy offers the only realistic hope of a cure transgenic direct injecti

51、on of myoblast implantation transfection a dystrophin mini genemice with dystrophin-negative muscular dystrophyshow spontaneous muscle repair a way of switching on an alternative compensatory protein, such as utrophinFrom Carmen Bertoni 2014,Emerging gene editing strategies for Duchenne muscular dys

52、trophy targeting stem cells,Front. Physiol.,5:148-165HemophiliaHemophiliaHemophilia AHemophilia BHemophilia CFrom HemophiliaHemophilia Awas recognized by the Jewish authorities 2000 years ago Queen Victoria was a carrier transmitted the disorder to most of the royal families of Europe.is the most co

53、mmon severe inherited coagulation disorderCoagulation (clotting) is the process by which blood changes from a liquid to a gelhas an incidence of 1 : 5000men are more affected than womencaused by a deficiency of factor VIIIPedigree showing the segregation of hemophiliaQueen Victoria was a carrier hav

54、ing an affected son.She transmitted the disorder through two of her daughters to most of the royal families of Europe.From Peter D. Turnpenny, Emerys Elements of medical genetics. Coagulation cascadeFactor VIII activates prothrombin to thrombin.Thrombin converts fibrinogen to fibrin.fibrin forms the

55、 structure of clotted blood. From HemophiliaHemophilia Baffects about 1 : 40,000 malesis caused by deficiency of factor IXFactor IX is a upstream factor of factor VIII.From Clinical Features of HemophiliaSimilar in both forms of hemophilia Vary frommild bleeding following spontaneous bleeding into m

56、uscles and jointsAffected family members generally show the same degree of severity.The degree of severity shows a close correlation with the reduction in factor VIII or IX activity. Levels below 1% are usually associated with a severe bleeding tendency from birth.Spontaneous bleeding into muscles a

57、nd jointscauses severe pain and swellingFrom Peter D. Turnpenny, Emerys Elements of medical genetics. Genetics of HemophiliaBoth forms of hemophilia show X-linked recessive inheritance.The loci lie close together near the distal end of Xq.From Genetics of Hemophilia AThe factor VIII comprises 26 exo

58、ns and spans 186 kb with a 9-kb mRNA transcript. Deletions account for 5% of all cases usually cause complete absence of factor VIII expressionarise mainly in the femaledue to unequal crossing over In addition, hundreds of frameshift, nonsense, and missense mutations have been described.usually orig

59、inate in male due to recurrent rounds of DNA replicationsFrom /wiki/Factor_VIIIGenetics of Hemophilia ABesides, insertions and a flip inversion, which represented a new form of mutation.The inversion disrupts the factor VIII gene, resulting in very low factor VIII activity. Inversions account for 50

60、% of all severe cases with 1% factor VIII activity.The flip inversions show 10-fold higher mutation rate compared with male and female.The inversion disrupts the factor VIII geneThey are caused by bination between a small gene called A, which are located within intron 22 of the factor VIII gene and