molecular detection of diseases
TRANSCRIPT
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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.