diagnosis by DNA tech

8/8/2019 diagnosis by DNA tech

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Pure & Appl. Chem.,Vol. 63, No. 8, pp. 1089-1096, 1991Printed in Great Britain.0 1991 IUPACADONIS 2069222091000965

Diagnosis of genetic diseases by DN A technologyMaurizio Ferrari, Laura Cremonesi, Paola Carrera andPierangelo BoniniIstituto Scientific0 H.S.Raffaele, Lab. Centrale, Milano, Italy

Abstract - The development of recombinant DNA technologyhas allowed the study of the molecular pathology ofinherited diseases in man. Two main molecular approachesare employed: direct study for detection of moleculardefects and indirect detection by linkage analysis.A variety of new technologies and their applications haveprovided a powerful new tool in the diagnosis of inheriteddiseases.

I N T R O D U C T I O NDuring the last twenty years the recombinant DNA technology has developedvery powerful and sensitive techniques, which are listed in Table 1,useful in the study and for the identification of the molecular defectsof human inherited diseases.DNA technology has allowed an enormous increase of basic knowledge oninherited diseases and has had great effect on biodiagnostics; in generalit has increased the knowledge on the human genome. The development of DNAbased diagnostic tests for genetic disorders is still in progress. It willtake time for experimental validation and for standardization of protocols.However their transferibility to the clinical field will depend on otherfactors, like the simplicity of procedures, the speed of execution and thecost of the analysis. Since 1 9 7 3 , 7 5 human genes have been mapped (ref.1). Table 2 shows all the genes mapped in the 1st edition of "HumanGene Mappingt'. Every year, during the Human Gene Mapping conference, theDNA Committee has three primary responsibilities: a) summarizinginformation on cloned and mapped human genes, b) summarizing information onpolymorphisms detected by using molecular techniques, and c) thedevelopment of a nomenclature appropriate for loci identified by anonymousDNA probes and assignment of symbols to those loci. Since 1 9 8 3 , these datahave been collected also on Database to facilitate and to speed up theaccess (ref. 1). There are also others Database with the list of papers oncloned sequences both for genes and for anonimous sequences (ref. 2 ) . Thenumber and chromosome distribution of cloned sequences and polymorphismsare shown in Fig. 1 and Table 3 (10th edn of Human Gene Mapping).The nature of the genetic lesion has now been established at the DNA levelin many diseases: the majority , 5 8 , are due to deletions, 5 2 are caused bypoint mutations and 3 4 by insertions, duplications and gene rearrangements(ref. 3 ) . A variety of chromosomal alterations have also been analysed andwell characterized (ref. 4).The advent of molecular biology techniques has expanded our ability todiagnose inherited diseases. In fact , it is possible to carry out theanalysis at the DNA level and it is not necessary to know the gene product,which gives three advantages: a) the analysis should be done for exampleon limphocytes and not only on the disease target cells, b) it allowscarrier detection and, c) it allows the molecular defect characterization.Two main molecular approaches are possible for genetic studies: a)indirect detection by linkage analysis, b) direct detection of themolecular alterations of genes. 1089


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1090 M. FERRARI et a / .

ProbeABLE 1. Recombinant DNA technologyI I- RestrictionEndonucleases- Cloning- Libraries- Electrophoresis- Blotting- Mapping- PFGE (a)- Chemical Cleavage

10 ktrVectorsOligonucleotidesP C R (b)SequencingProbesDGGE ( c ) I it tA

f7kb 11 3kb ISCP (d)RNAseA mismatchanalysis ?(a) Pulse Field Gel Electrophoresis(b) Polymerase Chain Reaction(c) Denaturing Gradient Gel

(d) Single Strand ConformationalElectrophoresisPolymorphism

m

88

+ +B

A:RwMctlm&teabstmtB :Eterrtrlctioaate p w e n tFig. 2. RFLPs associated withbase changes that alterres-triction endonuclease sites.After the cleavage we willobtain DNA fragments ofdifferent molecular weight.IIIt

Fig. 1. Cumulative annual number of Fig. 3 . Polymorphisms associatedcloned sequences and polymorphisms with variable tandem repeats.

I N D IR E C T D E T E C T IO N : P O L Y M O R P H I S M S A N D L I N K A G E A N A L Y S I SThe indirect analysis is very useful in the diagnosis of those diseasesin which the affected structural gene is not known. The analysis can bedone by Polymorphisms analysis.DNA Polymorphisms: The human genome (3 billion bp) normally carries onevariation in every 2 0 0 - 3 0 0 nucleotides localized in non-coding regions ofDNA. Two or more allelic variations are known as Polymorphisms if theirfrequency in the general population is not less than 1%. Polymorphismsresulting after digestion of DNA with restriction enzymes are calledRestriction Fragment Length Polymorphisms (RFLPs) (ref. 5). Another kindof polymorphism is due to a Variable Number of Tandem nucleic acid Repeatsequences (VNTRs) (ref.6), useful for example in the diagnosis ofhemophilia A (ref. 7 ) . In Fig. 2 and 3 the RFLP and VNTR mechanisms arerespectively explained.


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Diagnosis of genetic diseases by DNA technology 1091

TABLE 2. Summary of Human Gene Map. New Haven HGM 1 - 1973. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Chromosome Mendelian Markers In Vitro Markers

1 Iqh+, Cae, Fy, AOD PGM1, PGD, PPH,Pep-ClRN5S,AK-2GallPT, Ifl, Hb3 no assignment4 or 5 Hb,ade+B,Es-Act5 If2, Hex B6 Me-1, IPO-B,

7 MPI,PKIII,HexA8 no assignment9 no assignment

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Amyl, Amy2, EL1, Rh UGPP, FH, GuK,

2 Acpl, MNSs IDH-1, MDH-1,

PGM3, HLA

10 GOT-1, HK11 LDH-A,Es-Al,ALAcP2Gly+A, CS12 LDH-B,Pep-BITPI13 RNr14 RNr, NP15 RNr16 aHP APRT17 TK18 Pep-A19 GPI20 ADA21 Ag RNr,IPO-A,AVP22 RNr

x mP,rP,rs,oa,Xg, PGK,aGal,HPRT,ich,cbD,cbP,sp,md, G6PD,TATrmdc,HemA,HemB,Xm,MPS2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .TABLE 3. Chromosome distribution of cloned human DNA (HGM 10). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chrom Genes Anonimous Allmapped cloned poly* DNA segmt cloned DNAtot poly* tot poly*. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1 181 98 33 97 88 278 1212 103 62 26 65 53 168 793 63 40 17 184 46 247 634 71 45 28 157 96 228 1245 66 32 9 103 86 169 956 98 61 28 76 42 174 707 104 41 21 417 155 521 176s 56 30 14 72 36 128 509 58 25 9 44 33 102 4210 55 30 13 87 65 142 7811 133 68 37 404 67 537 10412 86 50 15 37 31 123 4613 27 17 8 61 42 88 5014 50 33 25 33 23 83 4815 48 25 8 71 38 119 4616 55 32 9 255 98 310 10717 83 57 21 217 112 300 13318 22 14 6 28 22 50 2835 15 53 33 128 48

28 3 24 15 52 1821 34 16 7 144 48 178 5575 17192022 55 31 11 108 60 163 71X 17 44 21 478 191 663 212Y 14 8 2 192 9 216 11XY 10 6 6* polymorphism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


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1092 M. FERRARI et a /.

A condition for the diagnostic use of RFLPs is a strong association of thepolymorphism with the disease gene. The probability of recombinationalevents will depend on their distance. The genetic distance is expressed asrecombination fraction and varies from 0 to 0.5 morgan (M). Also for closegenes, recombination might occurr between the RFLPs and the structuralgene; to prevenE false negatives it is advisable to use more than one RFLP.The degree of linkage must be established by extensive studies on families.RFLPs analysis allows to score the non random association between adisease allele and a polymorphic locus (linkage disequilibrium). To beinformative, the family must show heterozigosity for the analysedpolymorphism.RFLPs analysis is usually carried out by Southern blotting technique(ref. 8): peripheral blood limphocytes DNA is extracted and digested byrestriction endonucleases which cut the DNA at specific palindromicsequences. The resulting fragments are then separated by electrophoresis inan agarose gel, denatured and blotted onto nylon membranes. DNA fragmentsare then hybridized with isotopically labeled complementary probes.In Fig.4 a prenatal diagnosis based on RFLP analysis is shown.

kbf m aci ac2 CVS - .0C4/Taq I - 6.0

-5.4- ,

c -3.7i rPrr21OH/Taq Iiu -3.2

Fig. 4 . Prenatal diagnosis in a family with 21-hydroxilasedeficiency.The probe/enzyme combination C4B550/TaqIevidentiates two polymorphic bands (6.0 and 5.4 kb)on the C4B gene, close to 21OHase-B gene (min 0.1%recombination) and a 7.0 kb constant band on theC4A gene. The probe/enzyme combination 21OH/TaqIrecognizes the 210Hase-B active gene (3.7 kb) andthe 210Hase-A pseudogene (3.2 kb).In this family the RFLP analyzed is fullyinformative since both parents are heterozygotesfor the polymorphism. The proband (CVS),inheritingboth normal alleles from parents, results to benormal.F.: father, M.: mother, AC1, AC2: affected children,CVS: chorionic villus sample.

Table 4 lists some of the most frequent inherited diseases for which thiskind of analysis is carried out. Actually RFLPs analysis is used for thediagnosis of I 4 diseases both autosomic and X-linked (ref. 3 ) .

DIRECT DETECTIONThe direct detection is possible when the affected gene is known and itsstructure is well defined. This approach is very sensitive and quick, inparticular for those diseases in which the molecular defect shows littlevariability. In fact, the higher the heterogeneity of mutations, the morecomplex the design of diagnostic approach. The direct approach isfeasible in the diagnostic field only after the definition of thedifferent kinds of mutations in the various populations. Thus, when thecomplexity of genetic alterations is vast or when research is still atpreliminary stages, the direct detection is used in combination with RFLPsanalysis.


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Diagnosis of genetic diseases by DNA technology 1093

TABLE 4. Diagnosis of human inherited diseasesb DNA technology.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Direct Indirect- - - - - - --------Autosomal DominantAdult Polycystic Kidney - +Huntington Chorea +osteogenesis Imperfecta ( t IV) - +Myotonic Dystrophy +Tuberous Sclerosis +Autosomal Recessive

---

Thalassaemia beta and alfaPhenylketonuriaAlpha-1-antitrypsin def.21-hydroxylase dc-ic.Cystic FibrosisAdenosine deaminase def.Wilson diseaseRetinoblastomaWilmsX-linkedDMD DMB + +Haemophilia A and B + +Ornithine transcarbamylase def. + +Granulomatous disease chronic + -Retinitis pigmentosa - +Fragile X syndrome (Xq27-3) - +_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ - - - - _ _ - - - - - - - - - - - - - - - - - - - - - .

:ABLE 5.Diagnosis of human inheriteddiseases by PCR (1988-89) (ref.10)Adenosine deaminase deficiencyApoB deficiencyBecker and Duchenne Muscular DystrophyZystic FibrosisFamilial hypercholesterolemiaBlucose-6-phosphate dehydrogenaseBauchers diseaseHaemophilia AHaemophilia BHereditary persistence of fetalHypoxanthine phosphoribosyltransferaseInsulin resistance type ALesch-Nyhan syndromeLeukocyte adhesion deficiencyNon insulin dependent diabetes mellitusOsteogenesis imperfectaOrnithine transcarbamylase deficiencyPhenylketonuriaRetinoblastomaSickle cell anaemiaTay-Sachs diseaseThalassaemia betaThalassaemia deltavon Willebrand disease type IIA

haemoglobindeficiency

Polymerase Chain Reaction.The reaction allows the specific amplification ofa particular DNA segment flanked by twooligonucleotide primers. It involves repeatedcycles of heat denaturation (a) of DNA, annealingof primers (b) to their complementary sequencesand extension (c) of the annealed primers byTaq DNA Polymerase. At the end of the reaction wewill obtain an exponential enrichment of thespecific target fragment.

0

C Shorl ProductLongProduct


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The rapid expansion of DNA technology and the development of new moresensitive techniques has remarkably increased the usefulness andpotentiality of direct approach in prenatal diagnosis and carrierscreening.The most appropriate diagnostic methods will be chosen for each kind ofdisease even combining different techniques.As we reported, at the DNA level there should be many different kinds ofalterations: extensive deletions, insertions and rearrangements, can bescored on Southern blots as a band shift. On the other hand, if we considersmaller deletions/insertions or mutations, such as point mutations, theanalysis by Southern blotting is not very sensitive because it is feasibleonly when the mutation alters a restriction enzyme site. In the last fewyears the introduction of the Polymerase Chain Reaction (PCR) technique(ref. 9) has advantaged the possibility to carry out genetic diseasediagnoses that were impossible using conventional methods.The PCR is a powerful method that allows to amplify over a millionfoldspecific DNA target single copy sequences. The reaction principles areshown ia Fig. 5 . The PCR has made possible to diagnose many inheritablediseases as is shown in Table 5 .When a polymorphism does not alter any restriction sequence or when wemust screen for point mutations, the employment of Allele SpecificOligonucleotides (ASO) is useful. This tecnique, not very sensitive onsingle copy genes (ref. ll), is more efficient if utilized on amplified DNAspotted on dot blots (ref. 12). It allows to discriminate between differentalleles by the use of synthetic oligonucleotides that must be hybridizedunder conditions that permit stability of only perfectly matched duplexes.The application of this technology is particularly feasible for thescreening of populations and for carrier detection (example: beta-thalassemia , Cystic Fibrosis, alfa-1-Antitrypsin, Phenylketonuria). Theuse of PCR combined with AS0 is also realible by non isotopic means ofdetection (ref. 13) with a potential widespread use in diagnosticlaboratories.PCR is also employed for detection of large deletions as for example onthe dystrophin gene. By two sets of simultaneous amplifications of codingregions at the human dystrophin gene it is possible to detect about 98%deletions in Duchenne and Becker Muscular Dystrophy patients (ref. 14, 1 5 ) .This strategy is very appropriate in these patients because of the largepercentage of cases (about 60%) due to the presence of heterogeneousintragenic deletions.In those cases in which the mutation is unknown, RFLP analysis coupled toPCR can be used. By cutting specific amplified DNA with a restrictionenzyme it is possible to directly analyse the RFLP on a agarose gel,without the use of radioisotopes. This strategy takes less than one day incomparison with longer Southern blotting and hybridization method. InFigure 6 is shown an amplification for detection of KM-19 polymorpfism forprenatal diagnosis in a family with Cystic Fibrosis.

N E W T E C H N O L O G I E SWe will briefly consider the most recent and significant technologies andwe will outline the more advantageous aspects. In general these newtechnologies have the advantage of enhancing the resolution and thediagnostic potentiality and to increase the basic knowledge on researchand characterization of new mutations and polymorphisms.DN A sequencingThe two most used DNA sequencing methods are the chemical Maxam-Gilbertmethod (ref.16) and Sanger's enzymatic chain-terminating method (ref. 17).Such methods are subjected to cloning, mantainance and use of systemsdependent on vectors and living host cells. More recently, the possibilityto directly sequence PCR products (ref. 181, both from genomics or DNA-RNAtemplates, has greatly simplified sequencing because it does not needlibraries construction, screening and subcloning. This method alsofacilitates automation for large-scale sequencing (ref. 19).


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Diagnosis of genetic diseases by DNA technology

4 F V allele PbP U m b P

n i la

&&I

1095

A ) Amplification scheme,A 1 and B1: primersflanking the Pst Ipolymorphic site.

B) Agarose gel analysisof amplified fragmentsafter Pst I digestion.

F M C F V F: father, heterozygotes,alleles 1/2M: mother, homozygotes,alleles 2/2

bP AC: affected child: homozygo-- 50 tes, alleles 2/2- 50- 00 V: proband: heterozygotes,alleles 1/2

Fig- 5. RFLPs analysis on PCR amplified D N A .Chemical cleavageThis procedure allows detection, definition and localization of singlebase pair mutations. Labeled heteroduplex DN A molecules containingmismatched base pairs, are incubated with osmium tetroxide or hydroxylamineand then treated with piperidine to cleave the DN A at the modifiedmismatched base. DN A cleavage is detected by denaturing gel elecrophoresis(ref. 20).Denaturing gradient gel electrophoresis (DGGE)DGGE is very useful for the initial screening and detection of newpolymorphisms or mutations, before precisely defining the nature ofnucleotidic variation. This tecnique allows the resolution of DN A moleculesdiffering by single base changes. Using a polyacrilamide gel containing alinear gradient of DN A denaturants (urea/formamide) (ref.21) it is possibleto resolve mutant from normal homoduplexes because of the different meltingproperties of the DNA molecules. These different melting behaviors can beincreased by adding a GC-rich clamp ending a primer (ref. 22, 23).Single strand conformation polymorphisms (SSCP)This technique, described by Orita et a1 in 1989, (ref. 24, 25) is amethod to detect base changes on amplified or cloned DN A sequences.Specific DN A regions labeled and denatured are analysed in non-denaturingpolyacrilamide gels. The electrophoretic mobility of single-strandedmolecules will depend on their size and also on their sequence allowingdetection of allelk variants or mutations.RNAseA mismatch analysisThis strategy is based on the capability of the enzyme to digest only nothybridized RNA molecules. Thus it is possible to identify the site of alesion in a gene by hybridizing a normal R NA molecule to the RNA or DN A tobe analyzed. The mismatch region is identified by gel elecrophoresis afterthe R N A s e A cleavage (ref. 26).Pulse field gel electrophoresis (PFGE)PFGE is a technique for resolving megabase restriction fragment lengthvariations. By alternating the electric field, D N A s are able to reorientand differentially move through the gel (ref. 27). Conventionalelecrophoresis does not resolve DN A fragments larger than 5 0 kb.


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This technique has many applications: a) detection of polymorphisms (ref.2 8 ) , b) analysis of large rearrangments due to recombinational events asunequal crossovers (ref. 2 9 1 , c) molecular mapping and construction oflong-range genomic restriction maps (ref. 3 0 ) .C O N C L U S I O N S

DNA technology has led possible the diagnosis of many genetic diseases. Itwill play an increasing role in research and diagnosis of a growing numberof inherited diseases, that will become amenable. The increase of knowledgeand the technical development will be exploited and quickly transferred tothe clinical laboratories. However, the widespread diffusion and thetransferibility of these technologies will need to develop protocols and totake care of possible ethical problems.

1.

2 .3 .4 .5 .6 ,7 .

8 .9 .1 0 .11.1 21 3 .1 4 .1 5 .1 6 .1 7 .1 8 .1 9 .2 0 .2 1 .2 2 .2 3 .2 4 .2 5 .2 6 I2 7 .2 82 9 .30

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