Molecular Detection of Diseases

7/28/2019 Molecular Detection of Diseases

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Cystic fibrosis (CF) is the most commonautosomal recessiveinherited disease in Caucasians and affects

approximately 1 in2500 individuals. It occurs with lesser frequenciesin otherpopulations. It is a complex multi-systemdisorder, that mayaffect the following organ systems: Pulmonary

Pancreatic Gastro-intestinal Reproductive

The pathological processes affecting thesesystems arise frommutations in the CFTR gene which encodes thecystic fibrosis

transmembrane conductance regulator, amembrane chloridechannel located in the apical membrane ofsecretory epithelia.

The CFTR protein is a cyclic-AMP dependentchannel:increasing levels of c-AMP inside a secretory

epithelial celltrigger activation of protein kinase A which bindsthephosphorylation site on the (regulatory) R-domain of theCFTR protein thus opening the channel (Collins,1992). TheCFTR chloride channel essentially works as an

electrostatic


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attractant by drawing intracellular andextracellular anionstoward positively charged transmembrane

domains inside thechannel. The CFTR protein has 12transmembrane (TM)domains. Two of these (TM1 and TM6) attract andbindchloride (and/or bicarbonate) ions. As thechloride ions bind

to these sites in the pore, the mutual repulsionacceleratesexpulsion of the ions from the cell (Linsdell,2006). Whennormally functioning CFTR is activated, chlorideions aresecreted out of the cell. However, in addition to

chloride ionsecretion, the epithelial sodium channel (ENaC) isalsoinhibited by CFTR (Konig et al, 2001) and lesssodium isabsorbed into the cell, leaving a greatercombined ionic

gradient to allow water to leave the cell byosmosis providingfluid for epithelial tissue secretions. In cysticfibrosis thesemucus secretions become hyperviscous and it isthis whichaccounts for the principal features of cystic

fibrosis.


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the differing types of mutation into five classes,

Nonsense mutations, frameshift or splicemutations are ClassI.II. CFTR is produced but does not fold correctly,giving rise toimproper maturation (glycosylation) of theprotein. This classincludes the p.Phe508del (F508del) mutationwhich producesa defective protein which is destroyed by theEndoplasmicReticulum (ER)-Associated Degradation (ERAD)pathway(Farinha et al, 2005) thereby reducing the amountof CFTRpresent at the cell surface.

III. These mutations affect chloride channelgating; CFTR isimproperly activated as mutations affect bindingandhydrolysis of ATP or phosphorylation of the R-

domain. e.g.

p.Gly551Asp (G551D), the most commonmissense mutationworldwide.IV. CFTR does not allow proper chloride flux dueto defectiveconduction through the pore, although somemutations cause


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lower chloride channel activity e.g. p.Arg117His(R117H),some may produce a higher current. The class

also includesp.Asp1152His (D1152H) and is frequentlyassociated withmilder phenotypes.V. These mutations affect the regulation of otherion channelssuch as the ENaC sodium channel and the

OutwardlyRectifying Chloride Channel (ORCC).

6. Strategies for Molecular Testing

6.1 Methodology

Although there is no gold standard for routinetesting, initialanalysis of a sample is usually by means of a

commerciallyavailable kit, which will analyse approximately 30sequencevariants, accounting for more than 90% of CFdiseasecausingmutations (depending on local figures); althoughsome laboratories use alternative methods. The

mutationstested should identify at least 80% of mutationsin the UKpopulation e.g. at least p.Phe508del (F508del),p.Gly551Asp, (G551D), p.Gly542X (G542X) andc.489+1G>T (621+1G>T). Reports shouldspecify the


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proportion of mutations identified by the test inthepopulation of origin of the patient; they should

also statethat further testing is available if no mutation oronly onemutation is identified and a clinical diagnosis ofCF ismade.Subsequent analysis will depend on the reason

for referraland might involve whole gene screening ortesting forparticular mutations. Whether commercial kits orin-housemethods are employed, laboratory personnelshould be

proficient in performing the test and interpretingthe rawdata. Furthermore, laboratories should be awareof thelimitations of their chosen method e.g. whichmutations arenot identified, if there is the possibility of false

negative orfalse positive results, and the general robustnessof the test.Methods used in CFTR testing can be divided intotwogroups: those targeted at known mutations (i.e.testing DNAsamples for presence or absence of specificmutation(s), and


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scanning methods (i.e. screening samples for anydeviationfrom the standard sequence). These now include

searchingfor large unknown CFTR rearrangements,including largedeletions, insertions and duplications, by semi-quantitativePCR experiments, i.e. Multiplex Ligation-dependant Probe

Amplification (MLPA) or Quantitative FluorescentMultiplex PCR. Such rearrangements, which canescapedetection using conventional amplificationassays, havebeen shown to occur in up to 2% of alleles in CFpatients

and 1% in CBAVD patients.Even though commercial kits may be CE-markedin vitrodiagnostic devices (IVDD), assay performanceshouldalways be verified by laboratories beforediagnostic use.

The combined use of all these techniques cannotguaranteedetection of the two disease-causing mutations(in trans i.e. on both parental alleles) in all patients; 1-5%of allelesremain undetermined in CF patients with theclassical form


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and even more in patients with atypicalpresentations.Moreover, the percentage of undetected

mutations increasesfrom Northern-to-Southern European populations.CFTRmutations may be missed by scanningtechniques, especiallywhen homozygous, and even direct sequencingcannot

identify 100% of mutations. Undetected CFTRmutationsmay lie deep within introns or regulatory regionswhich arenot routinely analysed. For example3849+10kbC>T(c.3718-2477C>T) and 1811+1.6kbA>G

(c.1679+1.6kbA>G), the detection of whichrequireparticular methodologies.It should also be noted that locus heterogeneityhas beendocumented in patients with the classical form ofCF,

including a positive sweat test; but this probablyconcernsless than 1% of cases. In addition mutations inthe SCNN1genes, encoding sodium channel (ENaC)subunits, haverecently been found in non-classic CF caseswhere no CFTR


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mutations could be identified by extensivemutationscanning. However, the diagnostic utility of ENaC

testing inroutine practice has not been determined.

Table 1 summarises the approaches to differentkinds ofreferral for CF testing and includes the initial test,subsequent or reflex testing, the possibleoutcomes and the

recommended report style and further actionrequired.6.2 Population frequencies of CF mutations.

There is considerable heterogeneity in CFmutationfrequencies throughout the world. Therefore theproportion

of mutations identified in any one populationusingcommercial kits will vary. It is recommended thatanestimate of this figure be included in individualreports.

Table 2 shows an estimate of CF mutation

frequencies ofindividual populations worldwide [see alsosection 16(i)]7 Fetal Echogenic Bowel

7.1 Background

Fetal echogenic bowel (FEB) is observed in 0.2 -1.8% of2nd trimester pregnancies and appears to have amultifactorial aetiology. Conventionally the bowel


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hyperechogenicity might be graded 1 to 3relative to thesonodensity of the iliac crest, grade 3 being

considered to beas bright as bone. Bowel hyperechogenicity maybeobserved as an isolated finding, in associationwith otherscan anomalies and may be transitory. Fetalhyperechogenicity at grade 2 or above is

associated with arange of perinatal outcomes: normal (65.5%),severemalformation (7.1%), prematurity (6.2%),intrauterinegrowth retardation (4.1%), severe chromosomalabnormality

(3.5%), placental/maternal problem (3.5%), CF(3%), viralinfection (2.9%), in utero fetal death (1.9%)(Simon-Bouyet al, 2003).7.2 Strategy

It is recommended that parental samples are

tested in thefirst instance to determine the likely risk of CFand whetherthe situation is informative for prenatal diagnosis.Prenatalsamples, even if available, are not tested in thefirst instancein order to avoid a carrier test. Analysis involvesa mutation


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screen for a panel of mutations typically usingthe OLA32or CF29 kits. Evidence suggests that cases of CF

ascertained by hyperechogenicity havepancreaticinsufficient (PI) (severe) mutations and mostcommonlyp.Phe508del (F508del). Referral forms shouldideallyspecify the grade of hyperechogenicity, whether

it is anisolated finding, results of any other relevantinvestigations(specifically karyotype and CMV testing),gestational age,ethnic origin, consanguinity and any knownfamily history

of CF.7.3 Risk figuresDue to the subjective nature of the assessmentforhyperechogenicity it is recommended thatlaboratoriesderive their own risk figures by determination of

the overallincidence of CF in their referrals for echogenicbowel anddetermination of the mutation sensitivity for therelevantethnic group. If this is not possible, an estimatemay be usedbased on the recent studies (Scotet et al, 2002;Patel et al,


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2004; Jones et al, 2006) which suggest anincidence of CFin FEB at grade 2 or above of around 2 - 4% in

routinereferrals (Ogino et al2004).7.4 Extended screening

Extended screening is not recommended unlessthe analysiswill significantly increase the mutation detectionrate and

can be completed in an appropriate timescale forthemanagement of the pregnancy.

DNA-based diagnostic techniques forDMD / BMD

Based on the latest results regarding the frequency of DMD-mutations identified causing

Duchenne / Becker muscular dystrophy [see Table,White & den Dunnen 2006,Aartsma-Rus

2006] the most powerful DNA-based techniques currently available to reveal molecular

changes in patients are (to be performed in this order);

1. deletion / duplication screening

NOTE: to reliably predict the consequences of any rearrangmenent (incl. deletions /

duplications) in the DMD gene on the dystrophin reading frame (i.e. in-frame or out-of-frame) it is essential to analyse DMD mRNA.Predictions based on DNA findings

are predcitions only.

o Multiplex Ligation-dependent Probe Amplification (MLPA)

the power ofMLPA-analysis(Schwatz & Duno 2003) is that it screens all 79

exons of the DMD-gene fordeletion and duplicationmutations. MLPA-

analysis can also be performed using agarose gels (Lalic 2005) and arrays

(Zeng 2008).

Recently, arrayCGH approaches have been published, using oligonucleotide

tiling arrays spanning the DMD-gene (Hegde 2008,del Gaudio 2008,Saillour

2008). Compared to MLPA these arrays precisely determine the deletion /

duplication borders in the introns. Thusfar this information seems not to add alot to the diagnosis, while the cost of the assay is significantly higher.
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o multiplex PCR (Beggs & Chamberlain kits)

multiplex PCR screens only 18 of the 79 exons of the DMD-gene and it will

not detect duplications present in 5-7% of the patients (den Dunnen 1989,

White & den Dunnen 2006). Furthermore, additional analysis, e.g. Southern

blotting, will be required to determine the exact bordersof the rearrangements

detected, as well as to pick up duplications. Defining the deletion / duplicationborders is important to discriminate 'open reading frame' from 'reading frame

disrupting' changes.

o other methods

many other quantitative methods have been used but none of them have found

wide-spread application. qPCR (quantitative-PCR - e.g. Ashton 2008) seems

simple but is technically demanding, especially when performed in mutliplex

mode.Multiplex-Amplifiable Probe Hybridisation - (MAPH - White 2002) is a

simple and effective alternative for MLPA-screening, but it requires more

input DNA and it is more laborious. FISH, CA-repeat marker analysis and

exon-specific qPCR are valuable tools to confirm known rearrangments incarriers but they are not effective to screen patients directly.

2. point mutation screening

we consider RNA-based point mutation screening as the most powerful technique to

screen for deleterious, non-exon-deletion / duplication changes in the DMD-gene. By

amplifying the entire DMD coding region from an RNA template, all deleterious

truncating mutations will be resolved, including those affecting RNA-splicing. The

Protein Truncation Test (PTT), an RNA-based screening mehtod, has been proven to

be very effective. However, PTT is not the simplest method to implement and an

RNA sample, preferably from a muscle biopsy, is not always available. PTT on

lymphocyte RNA is possible, but more difficult to perform (Tuffery-Giraud 2004).An alternative is to use RNA obtained afterMyoD-induced in vitro muscle

differentiation. The cDNA fragments obtained after RT-PCR can also be used for

sequencing to determine the mutations present (Hamed 2006, seePrimers for DMD

RNA RT-PCR)

o high-resolution Melting Curve Analysis (hrMCA)

for DNA-based point mutation screening we currently prefer hrMCA (Al

Momani,submitted- details available on request). hrMCA is simple, cheap

and very sensitive (>98%). Applied as a pre-sequencing tool, resolving those

fragments that contain variants, it is very cost-effective.

o Denaturing Gradient Gel-Electrophoresis (DGGE)

DGGE (Hofstra 2004), having a close to 100% sensitivity, is once

implemented a very effective technique. However, DGGE is laborious, it uses

several PCR and electrophoresis conditions and it difficult to automate.

o direct sequencing

dirct sequencing (or SCAIP -single condition amplification/internal primer,

Flanigan 2003) is a straightforward and effective method but it is rather costly

(>79 separate exon fragments to analyse) .

o Single-Strand Conformation Analyis (SSCA)

SSCA / DOVAM (detection of virtually all mutations, Mendell 2001 / Buzin

2005) is simple,cheap and effective but rather laborious (e.g. demanding
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electrophoresis of all (>79) exon fragments each using several electrophoretic

conditions).

o Denaturing High Performance iquid Chromotography (DHPLC)

characterisitcs for DHPLC (Bennett 2001) are similar to those for SSCA.

However, DHPLC is easier to automate but requires specific specialisedequipment.

Compared to DGGE we consider SSCA and DHPLC as good but more laborious

alternatives. Direct sequencing is very powerful, but also more costly.

With few exceptions, mostly only the protein coding regions of the DMD gene are

analysed. Studies analysing other regions (promoters, 5'UTR and 3'UTR) have so far

not revealed many changes (e.g.Tubiello 1995,Flanigan 2003).

3. haplotyping

when no change can be detected using the above mentioned techniques, haplotypeanalysis (i.e. identifying the risk chromosome) is the only available technique to

perform a DNA-based analysis. In rare cases, a cytogenetic analysis may reveal

translocations or large inversions.

DNA-based analysis relies on the fact that there are virtually unlimited

numbers of nucleotide-sequence differences in the DNA of different

individuals. These differences can be detected by restriction fragment

length polymorphisms (RFLPs), or RFLP analysis. Although most sequence

differences have no pathologic significance, they can serve as markers for

mutant genes that cause disease. RFLPs allow diagnosis of affected

individuals in families with a known genetic disease, even if the exact

defect in the gene (i.e., mutation) is unknown. In cases where the disease-

causing mutation is known, RFLP analysis may be used (e.g., sickle cell

anemia), or, alternatively, a variety of methods may be employed for direct

mutation detection (e.g., cystic fibrosis).

DNA-based analyses of genetic disease utilizes the following technologies:

restriction endonuclease (restriction enzyme) digestions, immobilization of

DNA by Southern or dot blotting, separation of DNA fragments by

electrophoresis, visualization by hybridization to cloned DNA probes, and

amplification of DNA using the polymerase chain reaction (PCR). Thesetechniques, applied in a variety of ways, provide powerful tools for the

diagnosis of genetic disorders.

Restriction Fragment Length Polymorphisms

Restriction endonucleases are bacterial enzymes that recognize and cut

double-stranded DNA at specific nucleotide sequences. More than 400

restriction enzymes have been identified. (Examples of restriction enzymes

are listed in Table 1) The sequences of DNA recognized by specific

restriction enzymes are called restriction sites. Restriction sites are

randomly distributed throughout the genome sites and may be found withinor surrounding any particular gene. However, because more than 95% of
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human DNA does not code for gene products, most restriction sites are, by

chance, located within the noncoding portion of genes.

TABLE 1. Examples of Restriction Enzymes and Nucleotide Recognition

Sites

Enzyme Recognition Sequence

PvuII CAG^CTG

EcoI G^AATTC

MstII CC^TNAGG

CvnI CC^TNAGG

MspI C^CGG

TaqI T^CGA

^ = cutting site; N = any nucleotide

The DNA between two restriction sites is called a restriction fragment, and

the size of a restriction fragment is determined by the distance between two

restriction sites. Thus, when DNA is digested with a restriction enzyme it is

cut into many fragments of varying lengths. Because nucleotide sequences

vary from person to person, individuals will vary with respect to the

number of restriction sites and the size of restriction-fragment lengths (i.e.,restriction-fragment lengths are polymorphic). For example, in the

theoretical case depicted inFigure 1, individuals lacking the secondPvu II

restriction site will have one restriction fragment 5000 base pairs (or 5

kilobase pairs [kbp]) long, whereas individuals with the second restriction

site will have two fragments of 3 kb and 2 kb in length. Differences

between individuals with respect to the lengths of fragments are referred to

as RFLPs. A polymorphism refers to the occurrence in a population of two

or more forms of a gene, the least common having a frequency of at least

1%. Classic examples of human genetic polymorphisms are the ABO blood

groups, serum transferrin, or the red-cell enzyme G6PD. Unlike RFLPs,

however, the number of antigen, serum, or red-cell polymorphisms are

limited in the human genome to less than 50 loci. RFLPs, on the other hand,

provide geneticists with a virtually unlimited source of genetic markers

through which diseases can be traced in families.

Fig. 1. Simplified scheme of the

way in which restriction

endonucleases cut specific DNA

sequences to generate RFLPs. DNA

nucleotide sequence is shown as

single stranded.Pvu II recognizes the sequence CAGCTG and cutsbetween the G-C. The length of the restriction fragments generated by
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Pvu II is determined by the distance between the two sites. In this case, 2

kb and 3 kb fragments are generated. In individuals lacking the middle

Pvu II site, a 5-kb fragment only would result from digestion with this

enzyme.

Detection of RFLPs by Electrophoresis, Southern Blotting, and

Hybridization

When genomic DNA is digested with a particular restriction enzyme,

fragments of many sizes result. The various size fragments can be

physically separated on the basis of size by agarose gel electrophoresis

(larger restriction fragments migrate through the gel more slowly than

smaller restriction fragments). After electrophoresis, digested DNA appears

under ultraviolet light as a smear (Fig. 2). To immobilize and preserve the

DNA in the gel, the fragments are denatured (i.e., made single-stranded)

and transferred to a membrane (such as nitrocellulose or charged nylon) bya method called Southern blotting4 that maintains the spatial orientation

of the restriction fragments. Thus, the band patterns on the membrane are

identical to those in the gel. To locate a particular gene within the many

fragments on the blot, the membrane is soaked in a solution containing a

radioactively (or enzymatically) labeled, single-stranded probe (i.e., DNA

that is complementary to the gene of interest or to sequences that are

located very close to {i.e., linked to} the gene of interest). The probe will

hybridize (i.e., pair) to DNA that is complementary to it because both are

single stranded. The radioactive Southern blot is then exposed to

photographic film; fragments to which the probe hybridized are visualized

as dark bands. These steps are illustrated in Figure 3.

Fig. 2. DNA digested with restriction

enzymeXba I after electrophoresis in an

agarose gel. After electrophoresis, DNA

was stained with ethidium bromide and

visualized under an ultraviolet light. Each

lane represents DNA from a different

individual. The largest bands are on the top

and the smallest are on the bottom. Bands in

the lane on the right are lambda-size markers corresponding to ( top to

bottom) 23,130, 9,416, 6,557, 4,361, 2,322, and 2,027 base pairs.
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Fig. 3. Visualizing RFLPs. DNA is cleaved with

restriction enzyme, and the digested DNA is separated

by size using agarose gel electrophoresis. The DNA is

then transferred to a membrane, such as nitrocellulose,

by a technique called Southern blotting. The orientation

of the bands on the membrane is identical to theorientation of the bands in the gel. The membrane is

hybridized to a radioactively labeled probe. DNA

fragments that hybridize to the probe are visualized after

autoradiography.

Dot (or Slot) Blots

DNA can also be stabilized by dotting undigested DNA directly onto a

membrane under vacuum. These dot, or slot, blots may be hybridized to aprobe as described above. After autoradiography hybridization signals are

viewed as a dark dot. Dot blots are most often used with PCR-amplified

DNA and sequence-specific oligonucleotide (SSO) probes.5,6

Polymerase Chain Reaction (PCR)

PCR is an in-vitro method for making multiple copies of specific DNA

sequences.5 This powerful technique is capable of synthesizing over one

million copies of specific DNA sequences in just a few hours. PCR allows

diagnoses to be made on very small amounts of DNA (i.e., eliminating the

need to culture cells to obtain larger amounts of DNA) and reduces the timerequired to make a diagnosis from a few weeks to a few days. Diagnoses

can be made on amplified DNA either by direct visualization of the

amplified product under ultraviolet light, or after hybridization to sequence-

specific oligonucleotide probes. An oligonucleotide probe is a sequence of

nucleotides (usually 25 base pairs) that is synthesized in the laboratory.

The usefulness of these short sequences is that, under particular conditions,

the probe will not hybridize unless the nucleotides in the DNA being tested

exactly matches the nucleotide sequence in the probe. Thus, an

oligonucleotide probe will differentiate between sequences that differ by a

single base pair (e.g., sickle vs normal -globin gene). A major limitation of

PCR is that the DNA sequence on both sides of the mutation (i.e., the

flanking sequences) must be known. However, as this limitation is

overcome with more information about disease genes, analyses utilizing

PCR are becoming the method of choice.

DNA-BASED PRENATAL DIAGNOSIS

There are two general approaches to prenatal diagnosis through DNA

analysis. The first, called the direct method, is the preferred method for

prenatal diagnosis, but requires that the disease-causing mutation is known

and detectable in a particular family. The second, called the indirect

method, is based on linkage analysis and is more generally applicable, but


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less accurate than the direct method of diagnosis.

Direct Method Diagnosis

With this approach, DNA from the at-risk fetus is directly tested for the

presence or absence of the abnormal gene. There are few potential sourcesof error with this method, provided the clinical diagnosis of the disease is

correct. If more than one mutation results in a similar clinical phenotype

(such as in families with cystic fibrosis and Duchenne muscular dystrophy),

however, the direct test can be employed only if the specific mutation in

that family is known. The applicability of the direct method is thus limited

to genetic diseases in which the precise molecular defect is known.

Although this is considered the ultimate goal for diagnosis of genetic

disorders, at this time few genetic diseases can be so diagnosed.

Indirect Method of Diagnosis

This approach requires identifying RFLPs that are linked (lie within

approximately 1000 kbp) to the disease gene. (RFLPs located this close to

the gene will usually segregate with the gene, rather than undergo

recombination at meiosis.) As discussed previously, RFLPs are randomly

distributed throughout the genome. Thus, it is possible to identify RFLPs

that demonstrate linkage to a disease in family studies, even if the abnormal

gene itself has not been characterized. The presence of the RFLP can then

be used to predict the presence of the abnormal gene in members of a

family with affected individuals.

Despite the potential power of this approach, there are several limitations

and sources of error. One requisite is that multiple family members, usually

including at least one living affected relative, must be available. Also,

costly and time-consuming studies must be performed in each at-risk family

to determine whether the method will be applicable (i.e., informative) in the

particular case. A third limitation is the possibility of genetic recombination

in one of the parents' gametes between the disease gene and the linked

marker. Because of this possibility, the accuracy of diagnosis using linked

RFLPs is always less than 100%. The probability of recombination,

however, is proportional to the chromosomal distance between the mutant

gene and the RFLP. Thus, the smaller the distance between the restrictionsite and the disease gene, the more accurate the test. Whenever possible,

several different linked RFLPs that map to either side of the defective gene

should be used so that, if recombination occurs, it will be detected. The last

potential source of error is false paternity. Obviously, if the biological

father is not correctly identified in family studies, erroneous diagnoses may

result.

Despite the above caveats, rapid progress in this area already has made

possible the prenatal diagnosis of many important genetic diseases, using a

combination of the laboratory techniques described above (Table 2). Given

the rapidity of progress in this area, the prenatal diagnosis of many moremendelian disorders will become feasible in the not-too-distant future.


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TABLE 2. Examples of Mendelian Disorders That Can Be Prenatally

Diagnosed Using DNA-Based Diagnosis

Direct Method of Diagnosis

Sickle-cell anemia

-thalassemia

-thalassemia*

-1, antitrypsin deficiency

Duchenne muscular dystrophy*

Cystic fibrosis*

Congenital adrenal hyperplasia*

Fragile X syndrome (X-linked mental retardation)

Indirect Method of Diagnosis (Linkage Studies)

Diseases for which the gene has been cloned, but all mutations are not

detectable

-thalassemia

Duchenne muscular dystrophy

Cystic fibrosis

Congenital adrenal hyperplasia

Diseases for which the gene has not been cloned

Huntington's disease

Adult-onset polycystic kidney disease (dominant form)

Myotonic dystrophy

Spinal muscular atrophy

* Available in families in whom exact mutation is known

Examples of how these techniques are used for both the direct and indirect

diagnosis of sickle cell anemia, cystic fibrosis, and congenital adrenalhyperplasia due to 21-hydroxylase deficiency are described below. It should

be noted that a variety of techniques may be used to diagnose these

disorders; the following methods were chosen for illustrative purposes only.

EXAMPLES OF DNA-BASED DIAGNOSIS


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Sickle Cell Anemia

Sickle cell anemia is an autosomal recessive disorder affecting one out of

400 African-American children in the United States. The defect results

from a single nucleotide substitution (A to T) in the sixth codon (GAG to

GTG) of the -globin gene. This mutation leads to the amino acidsubstitution of valine for glutamine at position 6 of the -globin

polypeptide. Previously, diagnosis of sickle cell anemia was based on the

detection of the abnormal type of hemoglobin in fetal blood. However, fetal

blood sampling has become almost obsolete, as DNA-based diagnosis has

developed.

The mutation causing sickle cell anemia coincidentally resides within the

recognition sites of restriction enzymesMstII, CvnI, andBsu II. Individuals

with the sickle cell mutation lack the restriction site present in individuals

with normal -globin genes. One procedure for diagnosing fetuses affected

with sickle cell anemia using RFLP analysis is illustrated in Figure 4.

Recently, more rapid diagnosis of sickle cell has been possible using PCR.6

With this method, a 725 bp region that includes the sickle cell mutation is

amplified. The amplified DNA is digested withBsu II, and then the

digested product is separated by electrophoresis. The gel is stained with

ethidium bromide, and the banding patterns can be directly visualized under

ultraviolet light (Fig. 5). This procedure is much faster and more efficient

because it eliminates the need to perform Southern blotting, hybridization

with labeled probe, and autoradiography. This straightforward, direct

method of diagnosis can be applied to any disease in which the mutation

causing the disease removes or creates a restriction site.

Fig. 4. Use of a radioactively labeled -globin

probe to diagnose sickle cell anemia. The mutation

causing the disease coincides with a Cvn I site.

Chromosomes with the sickle mutation lack the site

that chromosomes with normal -globin genes

have. After digestion with Cvn I and hybridization

to a -globin probe, DNA from individuals with

sickle-cell anemia yields a 1.3 kb fragment, DNA

from individuals with two normal -globin genes yields a 1.1 kb

fragment, and carriers have 1.1- and 1.3-kb fragments.Fig. 5. Diagnosis of sickle cell anemia in PCR-

amplified DNA. Amplified DNA containing codon 6

of the -globin gene is digested with Cvn and

electrophoresed.6 DNA is labeled with ethidium

bromide and visualized under ultraviolet light. The

sickle mutation eliminates the recognition sequence of

this enzyme. Therefore, the HbS allele is visualized as

a 340-bp band and the HbA allele as 200- and 140-bp

bands. A constant band of 100 base pair is present in all individuals.


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Cystic Fibrosis

Cystic fibrosis (CF) is the most common autosomal recessive genetic

disorder in the northern European population. Approximately one in 25

Caucasians are carriers (i.e., heterozygotes) for the gene, and one child inevery 2500 births is affected with CF. Affected individuals rarely live past

their thirties and suffer from debilitating pulmonary and digestive disorders

throughout their lives. By demonstrating linkage between CF and RFLPs on

chromosome 7, the CF gene was mapped to this chromosome in 1985,7,8,9,10

but only recently was the CF gene itself identified and sequenced.11,12,13 A

three-base pair deletion, resulting in the loss of a phenylalanine residue at

position 508 (called delta F508), was found in 75% of CF chromosomes

studied by Kerem and associates. The remainder of CF chromosomes each

carry one of many (more than 100) less common mutations.

The direct detection of the delta F508 mutation does not require studying a

living affected relative with CF and could be used to screen the general

population for CF carrier status.13 Because only approximately 75% of CF

carriers have the delta F508 mutation, however, 25% of carriers would go

undetected if we screened for this mutation only. As a result, 25% of true

carriers (or approximately 1% of individuals screened) will have a negative

test, but will in truth be CF carriers. To further complicate matters, the

frequency of the delta F508 mutation is lower in ethnic and racial groups

other than northern European, non-Ashkenazi Caucasian populations. In

these groups (such as African Americans, Ashkenazi Jews, southern and

eastern Europeans), the frequency varies from 30% to 60%. Due to theselimitations, screening for CF mutations is recommended currently only for

relatives of individuals with CF and for spouses of known CF carriers.

Screening for CF-carrier status in the general population is not

recommended until additional mutations can be tested for, thereby reducing

the false-positive rate and increasing the sensitivity of the screen.14,15 It is

anticipated that these problems will be overcome in the near future, and

screening for CF carriers will become available at genetic centers.

Direct testing for delta F508 or other known mutations is useful in many

families with a CF child. Because not all CF carriers have mutations that

can be detected, however, linkage analysis using RFLPs is still required insome families. One method for detecting the delta F508 mutation utilizes

PCR, dot blotting the amplified product, and hybridization to sequence-

specific oligonucleotide (SSO) probes (Fig. 6).16This method quickly

determines whether an individual carries zero, one, or two copies of this

mutation.

Fig. 6. Direct detection of the delta

F508 mutation in DNA from nine

children with CF. DNA is amplified

using PCR and placed onto a

membrane in duplicate as dot
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blots. Each blot is then hybridized to one of two SSOs. The nucleotides

in the first SSO (oligo-N) are complementary to the DNA in the

nondeleted gene ( i. e ., the normal sequence); the sequence of the second

SSO (oligodelta F508) is complementary to the sequence with the

deletion. DNA from individuals homozygous for the deletion hybridizes

only to the second SSO, DNA from individuals heterozygous for thedeletion hybridizes to both oligos, and DNA from individuals

homozygous for the normal sequence hybridizes only to the first SSO. In

the figure, five children are homozygous for the mutation, and three are

heterozygous for the mutation. These three children presumably have a

nondelta F508 mutation on their other chromosome. One child lacks the

delta F508 mutation and presumably has nondelta F508 mutations on

both chromosomes.

In CF families in which only one or neither parent carries a mutation that

can be detected, prenatal diagnosis relies on family-based linkage studies.Figure 7A illustrates the relationship between the CF gene and a closely

linked RFLP,KM. 19, in a family with three affected and two unaffected

children. The pedigree of the family and RFLP banding patterns after

electrophoresis of PCR-amplified DNA that was digested with the

restriction enzyme,PstI, is shown in Figure 7B. The parents (I.1 and I.2)

are presumed to be heterozygous carriers of the CF gene because they have

three affected children. The affected children are assumed to be

homozygous for the CF gene. The unaffected children can be either

heterozygous carriers of the CF gene or can inherit normal genes from both

parents and be homozygous normal. After DNA analysis, it is determined

that both parents are heterozygous for theKM. 19 polymorphism (i.e.,heterozygous for the presence of the restriction site; genotype +,-). The

three affected children (II.1, II.3, II.5) are homozygous for the presence of

the restriction site (genotype, +,+). Thus, we can deduce that each affected

child inherited the chromosome containing the + allele from both parents,

and the CF gene must be on these parental chromosomes. Both parental

chromosomes with the - allele must therefore carry the normal gene. We

can further deduce that the brother with genotype, -,- (II.2), inherited the

normal gene from each parent and is not a carrier for CF. The sister with

genotype +,- (II.4), inherited the chromosome with the normal gene from

one parent and the chromosome with the CF gene from the other parent and

is presumed to be a CF carrier.17

Fig. 7. A. The relationship between the CFTR gene

and a closely linked RFLP,KM.19, on chromosome

7. In this example, one chromosome has the + allele

at theKM.19 locus (presence of a restriction site),

and one chromosome has the - allele at this locus

(absence of a restriction site). The + allele at the

KM.19 locus is on the same chromosome as the

abnormal CFTR gene (designated by a solid triangle)

and the - allele is on the same chromosome as the normal CFTR gene.

TheKM.19 alleles can be used to track the inheritance of the CFTR in


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families. B. Diagnosis of cystic fibrosis using theKM.19 RFLP in PCR-

amplified DNA in a family with three affected children. After PCR, DNA

was digested with restriction enzymePstI and electrophoresed. DNA is

labeled with ethidium bromide and visualized under ultraviolet light.

Chromosomes that lack thePstI cutting site (-) appear as a single 950 bp

band. Chromosomes that have the cutting site (+) appear as 650 and 300bp bands. The smallest 300 bp band cannot always be seen in the

heterozygote.

Prenatal diagnosis of CF in subsequent pregnancies in this family is also

possible. Fetal DNA derived from chorionic villi or from amniotic fluid

cells can be analyzed using the methods described above. As previously

discussed, the major potential source of error in linkage studies is the

probability of recombination between the restriction site and the abnormal

gene in the parents' gametes. Thus, results from indirect DNA testing are

given as a probability. For example,KM. 19 is within 100 kbp of the CFgene. Thus, the recombination frequency between theKM. 19 gene and the

CF gene, determined from family studies, is small (less than 1%). If

prenatal testing of a subsequent pregnancy in the couple in Figure 7

revealed the +,- genotype, the couple should be counseled that the

probability that the fetus will have CF is equal to the probability that

recombination occurred between the CF gene and the RFLP in the

chromosome with the - allele (99%. If the

probability based on recombination calculations proves too uncertain, or for

families in whom DNA diagnosis is not informative, measurement of

amniotic fluid microvillar intestinal enzymes (i.e., alkaline phosphatase, -glutamyl transpeptidase, leucine amniopeptidase) may be useful, albeit not

100% sensitive or specific.18

Congenital Adrenal Hyperplasia (CAH)

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder of

cortisol biosynthesis, which is caused in 95% of cases by a deficiency in the

enzyme steroid 21-hydroxylase.19 This enzyme is required for the

conversion ofprogesterone and 17-hydroxyprogesterone to 11-

deoxycorticosterone and 11-deoxycorticosterol, which are intermediate

products in mineralocorticoid and glucocorticoid biosynthesis, respectively.Due to a lack of feedback in CAH suppression of this pathway, there is a

compensatory increase in ACTH, leading to adrenal hyperplasia and

excessive secretion of precursor steroids. The increased secretion of adrenal

androgens (such as DHEA and DHEAS) leads to increased conversion of

these products totestosterone and dihydrotestosterone.

CAH occurs in a number of forms, ranging from mild to severe. The milder

forms often present during childhood. More attenuated forms present at

puberty (or even after puberty), with menstrual irregularities and infertility.

The severe form is characterized by early virilization in utero, leading to

marked masculinization of the external genitalia. Affected females may, in
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fact, be mistaken for males at birth, with bilateral cryptorchidism and

hypospadias. The diagnosis may not be as obvious in males because the

external genitalia are normal. Unrecognized salt wasting in neonates with

CAH is often fatal because the inadequacy of glucocorticoids can lead to

vascular collapse, shock, and death. The severe form of CAH occurs with afrequency of one in five to 10,000 births, and the milder (or attenuated)

forms occur at a frequency of approximately one in 1000 births.20

The virilization process in utero begins early in pregnancy because the

genital ridge forms at 9 to 10 weeks of gestation. Fortunately, the

virilization process of female fetuses in utero can be inhibited by treatment

of the mother during pregnancy with dexamethasone, thereby eliminating

the need for extensive corrective surgery after birth. Because steroid

treatment of the mother is not totally benign, however, treatment should be

discontinued if the fetus is determined to be either homozygous or

heterozygous for the normal 21-hydroxylase genes, or if the fetus is male.

Thus, early and correct diagnosis of this genetic disease is of great

importance. However, prenatal detection of CAH using biochemical tests

for increased concentrations of amniotic fluid 17-hydroxyprogesterone are

often inconclusive and are usually only feasible after 13 to 14 weeks'

gestation.

The gene encoding the enzyme 21-hydroxylase has been mapped to the

short arm of chromosome 6, positioned between the genes encoding HLA-

B and HLA-DR.21,22,23 Gene probes for the 21-hydroxylase gene have been

developed and molecular genetic studies of this region have demonstratedthat two copies of the 21-hydroxylase gene (called A and B) are present in

this region.23,24 These two genes can be differentiated by hybridizing 21-

hydroxylase gene probes to TaqI digested DNA: The A gene is detected on

a 3.2 kb fragment, whereas the B gene resides on a 3.7 kb fragment.

Detailed sequence analysis of the two genes has revealed that they are

>90% homologous. Three deleterious mutations in the A gene render it

nonfunctional, whereas the B gene is a functional gene, encoding the

enzyme.25,26 Analysis of southern blots of genomic DNA from patients with

salt wasting CAH has revealed that, in about 25% of patients, the 21-

hydroxylase B gene is either deleted or converted to the A gene.27 In these

families, a direct diagnosis can be made by determining the presence orabsence of the 3.7 kb fragment. In most cases, however, patients with CAH

have the 3.7 kb TaqI fragment (B gene). In these families, the prenatal

diagnosis of CAH depends on the indirect method, even though the

biochemical defects for this disorder are known. RFLPs detected by probes

for the HLA-B or HLA-DR loci have proven particularly useful for

linkage studies in CAH families.

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Molecular Detection of Diseases