Protocol:Diagnostic strategy

Diagnostic strategy
The haemoglobinopathies are a diverse group of inherited recessive disorders that include the thalassaemias and sickle cell disease. They were the first genetic diseases to be characterised at the molecular level and consequently have been used as a prototype for the development of new techniques of mutation detection. There are now many different PCR-based techniques that can be used to diagnose the known globin gene mutations, including dot blot analysis, reverse dot blot analysis, the amplification refractory mutation system (ARMS), denaturing gradient gel electrophoresis (DGGE), mutagenically separated polymerase chain reaction, gap- PCR, (MLPA) and restriction endonuclease (RE) analysis [1-4]. Each method has its advantages and disadvantages and the particular one chosen by a laboratory for the diagnosis of point mutations depends not only on the technical expertise available in the diagnostic laboratory but also on the type and variety of the mutations likely to be encountered in the individuals being screened.

DNA diagnosis of the haemoglobinopathies
Table 5.1 summarises the main diagnostic approaches commonly used for the diagnosis of the haemoglobinopathies. A brief summary of the different categories of globin gene mutations and the main diagnostic approaches is presented here, followed by a section containing a detailed protocol for each of the five commonly diagnostic techniques for known mutations: dot blot analysis, reverse dot blot analysis, the amplification refractory mutation system (ARMS), gap-PCR and restriction endonuclease (RE) analysis.

Table 5.1 DNA diagnosis of the haemoglobinopathies

&alpha;-Thalassaemia
Gap-PCR provides a quick diagnostic test for &alpha;+-thalassaemia and &alpha;o-thalassaemia deletion mutations but requires careful application for prenatal diagnosis. Most of the common &alpha;-thalassaemia alleles that result from gene deletions can be diagnosed by gap-PCR. Primer sequences have now been published for the diagnosis of five &alpha;o- thalassaemia deletions and two &alpha;+-thalassaemia deletions [5-9], as listed in Table 5.2. The &alpha;o-thalassaemia deletions diagnosable by PCR are: the --SEA allele, found in Southeast Asian individuals; the --MED and -(&alpha;)20.5 alleles found in Mediterranean individuals; the --FIL allele, found in Fillipino individuals and finally the --THAI allele, found in Thai individuals. The two &alpha;+-thalassaemia deletion mutations are 3.7 kb and the 4.2 kb single &alpha;-gene deletion mutations, designated -&alpha;3.7 and -&alpha;4.2. Amplification of sequences in the &alpha;-globin gene cluster is technically more difficult than that of the &beta;-globin gene cluster, requiring more stringent conditions for success due to the higher GC content of the breakpoint sequences and the considerable sequence homology within the &alpha;-globin gene cluster. Experience in many laboratories has shown some primer pairs to be unreliable, resulting occasionally in unpredictable reaction failure and the problem of allele drop out. The more recently published primers [8,9] seem to be more robust at amplifying than the earlier published sequences, possibly due to the addition of betaine to the reaction mixture, but great care still needs to be taken in the interpretation of the results. They are also designed for a multiplex screening test, although in the Oxford laboratory they are still used in pairs to test for individual mutations due to problems of occasional primer pair failure in the multiplex reaction and thus complete allele drop for one mutation.

The other &alpha;o and &alpha;+-thalassaemia deletion mutations cannot be diagnosed by PCR because their breakpoint sequences have not been determined. These deletion mutations used to be diagnosed by the Southern blotting technique using &zeta;-gene and &alpha;-gene probes, but now this has been replaced by the multiplex ligation-dependent probe amplification ( MLPA) technique. MLPA can be used to detect all the known deletion mutations and also detects novel ones. Southern blotting is perhaps still useful for identifying breakpoints of deletions and is a good method for investigating &alpha;-gene rearrangements (the triple and quadruple &alpha;-gene alleles)[10].

&alpha;+-Thalassaemia is also caused by point mutations in one of the two &alpha;-globin genes. These non deletion alleles can be detected by PCR using a technique of selective amplification of each &alpha;-globin gene followed by a general method of mutation analysis such as DGGE [11] or preferably, by DNA sequence analysis [12]. Several of the non deletion mutations alter a restriction enzyme site and may be diagnosed by selective amplification and restriction endonuclease analysis in a similar manner to that reported for the mutation which gives rise to the unstable &alpha;-globin chain variant Hb Constant Spring [13].

&beta;-Thalassaemia
The &beta;-thalassaemia disorders are a very heterogeneous group of defects with more than 170 different mutations characterised to date [14]. The majority of the defects are single nucleotide substitutions, insertions or deletions. Only 13 large gene deletions have been identified and eight of these can be diagnosed by gap-PCR, as listed in Table 5.2. Different methods are required for the detection of other mutations although the basic principles are the same. That is to say that mutation is region specific and each at-risk population has a few common mutations together with a larger variable number of rare ones. Thus for any given ethnic region a PCR method designed to detect the common specific mutations simultaneously is employed initially. Such an approach will identify the mutation in more than 80% of cases for most ethnic groups. Further screening of the known rare mutations will identify the defect in another 10-15% of cases if necessary. Mutations remaining unidentified at this stage are characterised by DNA sequencing.

The first PCR based method to gain widespread use was the hybridisation of allele- specific oligonucleotide probes (ASOs) to amplified DNA bound to nylon membrane by dot-blotting [15]. Although still in use, the method is limited by the need for separate hybridisation steps to test for multiple mutations. This was overcome by the development of the reverse dot-blotting technique, in which amplified DNA is hybridised to a panel of mutation specific probes fixed to a nylon strip. This technique is compatible with the optimum strategy for screening &beta;-thalassaemia mutations, using a panel of the commonly found mutations for the first screening and a panel of rare ones for the second screen [16].

The amplification refractory mutation system (ARMS) [10] fulfils the main requirements of a PCR technology, i.e. speed, cost, convenience and the ability to test for multiple mutations simultaneously. No labelling of primers or amplified DNA is required. The simplest approach is to screen for mutations with simultaneous PCR assays although the multiplexing of ARMS primers in a single PCR assay is possible [17].

Denaturing gradient gel electrophoresis (DGGE) [18] is the most widely used indirect method to characterise &beta;-thalassaemia mutations. This detects at least 90% of &beta;- thalassaemia mutations by a shifted band pattern to normal and provides an alternative approach to ASO probes or ARMS in countries where a very large spectrum of &beta;-thalassaemia mutations occur [19].

&delta;&beta;-Thalassaemia and HPFH
&delta;&beta;-Thalassaemia and the hereditary persistence of fetal hemoglobin (HPFH) disorders result from large gene deletions affecting both the &beta;- and &delta;-globin genes. Restriction enzyme mapping has enabled the characterisation of more than fifty different deletions starting at different points between G&gamma;-gene and the &delta;-gene and extending up to 100kb down stream of the &beta;-globin gene. In two cases, the Macedonian/Turkish (&delta;&beta;)o-thalassaemia gene and the Indian (A&gamma;&delta;&beta;)o- thalassaemia gene, the mutation is a complex rearrangement consisting of an inverted DNA sequence flanked by two deletions.

A small number of (&delta;&beta;)-thalassaemia and HPFH deletions have had their breakpoint sequences characterised and can be diagnosed simply by gap-PCR [20]. Gap-PCR can also be used for the diagnosis of the fusion haemoglobins Hb Lepore, created by a deletion of the DNA sequence between the &delta;- and &beta;-globin genes, and Hb Kenya, created by a deletion of the DNA sequence between the &gamma;- and &beta;-globin genes. Hb Lepore is associated with a severe &beta;-thalassemia phenotype., whereas Hb Kenya has a HPFH phenotype. However all the (&delta;&beta;)-thalassaemias and HPFH deletion mutations can now be diagnosed using the MLPA technique.

Hb Variants
More than 700 hemoglobin variants have been described to date, most of which were identified by protein analysis and have never been characterised at the DNA level. Positive identification at the DNA level is achieved by selective globin gene amplification and DNA sequence analysis. However the clinically important variants, Hb S, Hb C, Hb E, Hb D-Punjab and Hb O-Arab, can be diagnosed by simpler DNA analysis techniques. All these variants can be diagnosed by ASO hybridisation, the ARMS technique or, except Hb C, by restriction endonuclease digestion of the PCR product [10]. The sickle cell gene mutation abolishes a Dde I recognition site at codon 6 and diagnosis by Dde I digestion of amplified product remains the simplest method of DNA analysis for sickle cell disease. Similarly, the mutations giving rise to Hb D- Punjab and Hb O-Arab abolish an EcoR I site at codon 121. However the Hb C mutation at codon 6 does abolish the Dde I site is diagnosed by other methods. Hb E interacts with &beta;-thalassaemia trait to produce a clinical disorder of varying severity ranging from thalassaemia intermedia to transfusion dependent thalassaemia major. The Hb E mutation can be diagnosed by ASO hybridisation, ARMS or restriction endonuclease analysis as the mutation abolishes a Mnl I site in the &beta;-globin gene sequence.

Commonly used diagnostic methods
The principal methods of analysis in diagnostic use worldwide are:
 * 1) ASO-probe methods:
 * The principle of dot blot (DB)and reverse dot blot (RDB) methods is that a single-strand DNA molecule of defined sequence (the "oligoprobe") can hybridize to a second DNA molecule that contains a complementary sequence (the "target") with the stability dependent on the extent of base pairing that it can occurs. In both methods specific amplified fragments of DNA are need to be hybridized with ASO probes that are fixed on surface of a membrane (RDB) or by radioactive-labelled probe to DNA samples fixed to the membrane in the form of dots (DB). DB needs different hybridization and wash temperatures, and screens many samples for one mutation per membrane. RDB is a non- radioactive method, which requires the same temperature for hybridisation and washing. RDB can screen one sample for many mutations per membrane.
 * 1) Amplification refractory mutation system (ARMS):
 * It is a PCR-based system that discriminates between normal and mutant alleles by selecting primers that have nucleotide at their 3' end corresponding to either normal or mutant sequence. PCR amplified a DNA fragment only if there is perfect identity with the genomic sequence to which the primer is annealing. This is a rapid, simple and non radioactive method. It can use for screening multiple mutations in one patient but it needs good experience to understand false negatives.
 * 1) Restriction enzyme analysis:
 * It is a rapid, simple and non radioactive method used if a mutation may create or abolish the site present in the normal DNA sequence. The PCR fragment is cut with a restriction enzyme that will cut or not cut when the particular point mutation is present. The limitations of this method that the majority of globin gene mutations do not create or destroy a restriction enzyme site, and some enzymes for those that do are expensive.
 * 1) DNA sequencing:
 * DNA sequencing is sometimes used for the diagnosis of known mutations when there is no quick, simple method for the diagnosis of a mutation, eg a very rare or novel mutation, and is also used as a second method for confirmation of prenatal diagnosis results.
 * 1) Gap-PCR:
 * This is a rapid, simple and no radioactive method that allows the identification DNA deletions or gene rearrangements. This method was used for to detect α-thalassaemia deletions, δβ-thalassaemia deletions, Hb Lepore rearrangements, etc. The limitation of this method is that the deletions endpoints must be known
 * 1) MLPA:
 * Multiplex ligation-dependent probe amplification (MLPA) is a new rapid technique that that allows the identification DNA deletions and is an alternative or complimentary method to gap–PCR. Unlike gap-PCR, the advantage of MLPA is that it detects all known deletion mutations in the &alpha;- and &beta;-globin gene clusters. It also detects novel deletion mutations and thus is an extremely useful technique for the molecular diagnosis of large thalassaemia deletion mutations (56). However it cannot identify a deletion mutation precisely, as the primer locations are far apart and two different deletion mutations with endpoints between the same two primers sets will give the same result pattern. Thus a similar MLPA profile to one for a known mutation gives a presumed diagnosis as the result is consistent with that for the known mutation.

Evaluation of Methods
The advantages and disadvantages of each method of analysis for known mutations are summarised in the table below.