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Cystic fibrosis (CF) is the most common autosomal recessive inherited disease in Caucasians and affects approximately 1 in 2500

individuals. It occurs with lesser frequencies in other populations. It is a complex multi-system disorder, that may affect the following organ systems: Pulmonary Pancreatic Gastro-intestinal Reproductive The pathological processes affecting these systems arise from mutations in the CFTR gene which encodes the cystic fibrosis transmembrane conductance regulator, a membrane chloride channel located in the apical membrane of secretory epithelia. The CFTR protein is a cyclic-AMP dependent channel: increasing levels of c-AMP inside a secretory epithelial cell trigger activation of protein kinase A which binds the phosphorylation site on the (regulatory) Rdomain of the CFTR protein thus opening the channel (Collins, 1992). The CFTR chloride channel essentially works as an electrostatic

attractant by drawing intracellular and extracellular anions toward positively charged transmembrane domains inside the channel. The CFTR protein has 12 transmembrane (TM) domains. Two of these (TM1 and TM6) attract and bind chloride (and/or bicarbonate) ions. As the chloride ions bind to these sites in the pore, the mutual repulsion accelerates expulsion of the ions from the cell (Linsdell, 2006). When normally functioning CFTR is activated, chloride ions are secreted out of the cell. However, in addition to chloride ion secretion, the epithelial sodium channel (ENaC) is also inhibited by CFTR (Konig et al, 2001) and less sodium is absorbed into the cell, leaving a greater combined ionic gradient to allow water to leave the cell by osmosis providing fluid for epithelial tissue secretions. In cystic fibrosis these mucus secretions become hyperviscous and it is this which accounts for the principal features of cystic fibrosis.

the differing types of mutation into five classes, Nonsense mutations, frameshift or splice mutations are Class I. II. CFTR is produced but does not fold correctly, giving rise to improper maturation (glycosylation) of the protein. This class includes the p.Phe508del (F508del) mutation which produces a defective protein which is destroyed by the Endoplasmic Reticulum (ER)-Associated Degradation (ERAD) pathway (Farinha et al, 2005) thereby reducing the amount of CFTR present at the cell surface. III. These mutations affect chloride channel gating; CFTR is improperly activated as mutations affect binding and hydrolysis of ATP or phosphorylation of the Rdomain. e.g. p.Gly551Asp (G551D), the most common missense mutation worldwide. IV. CFTR does not allow proper chloride flux due to defective conduction through the pore, although some mutations cause

lower chloride channel activity e.g. p.Arg117His (R117H), some may produce a higher current. The class also includes p.Asp1152His (D1152H) and is frequently associated with milder phenotypes. V. These mutations affect the regulation of other ion channels such as the ENaC sodium channel and the Outwardly Rectifying Chloride Channel (ORCC). 6. Strategies for Molecular Testing 6.1 Methodology Although there is no gold standard for routine testing, initial analysis of a sample is usually by means of a commercially available kit, which will analyse approximately 30 sequence variants, accounting for more than 90% of CF diseasecausing mutations (depending on local figures); although some laboratories use alternative methods. The mutations tested should identify at least 80% of mutations in the UK population e.g. at least p.Phe508del (F508del), p.Gly551Asp, (G551D), p.Gly542X (G542X) and c.489+1G>T (621+1G>T). Reports should specify the

proportion of mutations identified by the test in the population of origin of the patient; they should also state that further testing is available if no mutation or only one mutation is identified and a clinical diagnosis of CF is made. Subsequent analysis will depend on the reason for referral and might involve whole gene screening or testing for particular mutations. Whether commercial kits or in-house methods are employed, laboratory personnel should be proficient in performing the test and interpreting the raw data. Furthermore, laboratories should be aware of the limitations of their chosen method e.g. which mutations are not identified, if there is the possibility of false negative or false positive results, and the general robustness of the test. Methods used in CFTR testing can be divided into two groups: those targeted at known mutations (i.e. testing DNA samples for presence or absence of specific mutation(s), and

scanning methods (i.e. screening samples for any deviation from the standard sequence). These now include searching for large unknown CFTR rearrangements, including large deletions, insertions and duplications, by semiquantitative PCR experiments, i.e. Multiplex Ligationdependant Probe Amplification (MLPA) or Quantitative Fluorescent Multiplex PCR. Such rearrangements, which can escape detection using conventional amplification assays, have been shown to occur in up to 2% of alleles in CF patients and 1% in CBAVD patients. Even though commercial kits may be CE-marked in vitro diagnostic devices (IVDD), assay performance should always be verified by laboratories before diagnostic use. The combined use of all these techniques cannot guarantee detection of the two disease-causing mutations (in trans i.e. on both parental alleles) in all patients; 1-5% of alleles remain undetermined in CF patients with the classical form

and even more in patients with atypical presentations. Moreover, the percentage of undetected mutations increases from Northern-to-Southern European populations. CFTR mutations may be missed by scanning techniques, especially when homozygous, and even direct sequencing cannot identify 100% of mutations. Undetected CFTR mutations may lie deep within introns or regulatory regions which are not routinely analysed. For example 3849+10kbC>T (c.3718-2477C>T) and 1811+1.6kbA>G (c.1679+1.6kbA>G), the detection of which require particular methodologies. It should also be noted that locus heterogeneity has been documented in patients with the classical form of CF, including a positive sweat test; but this probably concerns less than 1% of cases. In addition mutations in the SCNN1 genes, encoding sodium channel (ENaC) subunits, have recently been found in non-classic CF cases where no CFTR

mutations could be identified by extensive mutation scanning. However, the diagnostic utility of ENaC testing in routine practice has not been determined. Table 1 summarises the approaches to different kinds of referral for CF testing and includes the initial test, subsequent or reflex testing, the possible outcomes and the recommended report style and further action required. 6.2 Population frequencies of CF mutations. There is considerable heterogeneity in CF mutation frequencies throughout the world. Therefore the proportion of mutations identified in any one population using commercial kits will vary. It is recommended that an estimate of this figure be included in individual reports. Table 2 shows an estimate of CF mutation frequencies of individual populations worldwide [see also section 16(i)] 7 Fetal Echogenic Bowel 7.1 Background Fetal echogenic bowel (FEB) is observed in 0.2 1.8% of 2nd trimester pregnancies and appears to have a multifactorial aetiology. Conventionally the bowel

hyperechogenicity might be graded 1 to 3 relative to the sonodensity of the iliac crest, grade 3 being considered to be as bright as bone. Bowel hyperechogenicity may be observed as an isolated finding, in association with other scan anomalies and may be transitory. Fetal hyperechogenicity at grade 2 or above is associated with a range of perinatal outcomes: normal (65.5%), severe malformation (7.1%), prematurity (6.2%), intrauterine growth retardation (4.1%), severe chromosomal abnormality (3.5%), placental/maternal problem (3.5%), CF (3%), viral infection (2.9%), in utero fetal death (1.9%) (Simon-Bouy et al, 2003). 7.2 Strategy It is recommended that parental samples are tested in the first instance to determine the likely risk of CF and whether the situation is informative for prenatal diagnosis. Prenatal samples, even if available, are not tested in the first instance in order to avoid a carrier test. Analysis involves a mutation

screen for a panel of mutations typically using the OLA32 or CF29 kits. Evidence suggests that cases of CF ascertained by hyperechogenicity have pancreatic insufficient (PI) (severe) mutations and most commonly p.Phe508del (F508del). Referral forms should ideally specify the grade of hyperechogenicity, whether it is an isolated finding, results of any other relevant investigations (specifically karyotype and CMV testing), gestational age, ethnic origin, consanguinity and any known family history of CF. 7.3 Risk figures Due to the subjective nature of the assessment for hyperechogenicity it is recommended that laboratories derive their own risk figures by determination of the overall incidence of CF in their referrals for echogenic bowel and determination of the mutation sensitivity for the relevant ethnic group. If this is not possible, an estimate may be used based on the recent studies (Scotet et al, 2002; Patel et al,

2004; Jones et al, 2006) which suggest an incidence of CF in FEB at grade 2 or above of around 2 - 4% in routine referrals (Ogino et al 2004). 7.4 Extended screening Extended screening is not recommended unless the analysis will significantly increase the mutation detection rate and can be completed in an appropriate timescale for the management of the pregnancy.

DNA-based diagnostic techniques for DMD / 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 outof-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 of MLPA-analysis (Schwatz & Duno 2003) is that it screens all 79 exons of the DMD-gene for deletion and duplication mutations. MLPAanalysis 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 a lot to the diagnosis, while the cost of the assay is significantly higher.

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 borders of the rearrangements detected, as well as to pick up duplications. Defining the deletion / duplication borders is important to discriminate 'open reading frame' from 'reading frame disrupting' changes. 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 in carriers 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 after MyoD-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, see Primers for DMD RNA RT-PCR)

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. 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. 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) . 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

electrophoresis of all (>79) exon fragments each using several electrophoretic conditions).

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 specialised equipment.

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, haplotype analysis (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 diseasecausing 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). These techniques, 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 within or surrounding any particular gene. However, because more than 95% of

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 EcoI MstII CvnI MspI TaqI CAG^CTG G^AATTC CC^TNAGG CC^TNAGG C^CGG 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 in Figure 1, individuals lacking the second Pvu 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 cuts between the G-C. The length of the restriction fragments generated by

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) by a 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 enzyme Xba 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.

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 the orientation 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 a probe 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 time required 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 sequencespecific 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

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 sources of 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 restriction site 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 more mendelian disorders will become feasible in the not-too-distant future.

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 adrenal hyperplasia 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

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 acid substitution 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 enzymes Mst II, CvnI, and Bsu 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 with Bsu 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 PCRamplified 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.

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 in every 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 these limitations, 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 in some families. One method for detecting the delta F508 mutation utilizes PCR, dot blotting the amplified product, and hybridization to sequencespecific oligonucleotide (SSO) probes (Fig. 6).16 This 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

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 the deletion 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 the KM. 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 the KM.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. The KM.19 alleles can be used to track the inheritance of the CFTR in

families. B. Diagnosis of cystic fibrosis using the KM.19 RFLP in PCRamplified DNA in a family with three affected children. After PCR, DNA was digested with restriction enzyme Pst I and electrophoresed. DNA is labeled with ethidium bromide and visualized under ultraviolet light. Chromosomes that lack the Pst I cutting site (-) appear as a single 950 bp band. Chromosomes that have the cutting site (+) appear as 650 and 300 bp 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 CF gene. Thus, the recombination frequency between the KM. 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 (<1%). On the other hand, if the fetus was genotype, +,+, the probability that it will have CF is >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 of progesterone and 17-hydroxyprogesterone to 11deoxycorticosterone 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 to testosterone 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

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 a frequency 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 HLAB and HLA-DR.21, 22, 23 Gene probes for the 21-hydroxylase gene have been developed and molecular genetic studies of this region have demonstrated that 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 21hydroxylase 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 21hydroxylase 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 or absence 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.