The Clinical Applications of Southern blotting

In many areas, the use of Southern blotting has been superseded by PCR-based techniques, which are quicker, easier, and require less DNA. However, some laboratories still use Southern blotting, particularly for the investigation of 
1. Fragile X and 
2. Myotonic dystrophy. 

Both of which are associated with larger expansion sizes. First, let's have a quick think about how Southern blotting is used to investigate Fragile X syndrome.

Fragile X syndrome

Fragile X is a condition primarily affecting boys, which is characterised by an intellectual disability along with a subtle but recognisable facial appearance, which is characterised by dolichocephaly, describing a long, thin face and large ears. In addition, boys often have hyper-mobile joints. And macroorchidism can be associated.

Fragile X is an example of a triplet repeat expansion disorder. The FMR1 gene located on the X chromosome has a CGG repeat unit in the five prime untranslated region. It is usually present in less than 45 copies. Those with Fragile X syndrome have greater than 200 CGG repeats, known as a full mutation. Those with 59 to 200 repeats are known as premutation allele carriers. Premutation alleles are unstable. And when female premutations carriers pass on the gene to their offspring, there may be repeat expansion within the premutation to full mutation range. This expansion does not occur when the male premutation carriers pass on the gene.The large repeat length in the Fragile X full mutation results in methylation and subsequent inactivation of the FMR1 gene, leading to an affected male phenotype. Females with a full mutation may be more or less affected depending on the degree of X inactivation. Premutation carriers may suffer from a tremor ataxia syndrome in later life. And, in addition, female premutation carriers may suffer from premature ovarian failure.

Let's have a look at a schematic showing some possible results. The first sample is that of a normal female. Here there are two bands because, due to X inactivation, one of her X chromosomes is methylated. And the restriction enzymes used are methylation sensitive.

Skip to 0 minutes and 52 secondFragile X is an example of a triplet repeat expansion disorder. The FMR1 gene located on the X chromosome has a CGG repeat unit in the five prime untranslated region. It is usually present in less than 45 copies. Those with Fragile X syndrome have greater than 200 CGG repeats, known as a full mutation. Those with 59 to 200 repeats are known as premutation allele carriers. Premutation alleles are unstable. And when female premutations carriers pass on the gene to their offspring, there may be repeat expansion within the premutation to full mutation range. This expansion does not occur when the male premutation carriers pass on the geneThe large repeat length in the Fragile X full mutation results in methylation and subsequent inactivation of the FMR1 gene, leading to an affected male phenotype. Females with a full mutation may be more or less affected depending on the degree of X inactivation. Premutation carriers may suffer from a tremor ataxia syndrome in later life. And, in addition, female premutation carriers may suffer from premature ovarian failure. Let's have a look at a schematic showing some possible results. The first sample is that of a normal female. Here there are two bands because, due to X inactivation, one of her X chromosomes is methylated. And the restriction enzymes used are methylation sensiSkip to 2 minutes and 13 seconds

The normal male has only a single band, representing his single active X chromosome. The premutation carrier female has one normal and one premutation allele. And either of these may be subject to X inactivation and methylation, hence giving rise to four bands. The green bands represent the premutation allele. These bands can vary in intensity of their skewing of X inactivation, which can cause difficulties in interpretation. The premutation carrier male has a single band, representing his premutation allele. The full mutation alleles are large and subject to heavy methylation, resulting in poor digestion by the endonuclease and a broad smear of bands. In the full mutation female, this is present in addition to the bands from the normal allele.
However, the previous schematic represents a perfect world. This picture represents what it is more likely to look like. So we have A here, which represents the normal female. And this shows the two bands. B, which is over on the right, shows the premutation carrier female with her four bands. C, in the middle, is the premutation carrier female with skewed inactivation. So you can see the different intensity of the top band, which is the green band from the previous schema. And D shows the full mutation male, which shows that smear at the very top. Let's now think myotonic dystrophy, another triplet repeat disorder. Myotonic dystrophy is a multi-system disorder affecting both the skeletal and smooth muscle.

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The classic feature is of myotonia, where there is sustained muscle contraction. But the condition is also associated with muscle weakness and wasting. Facially, affected individuals can have ptosis, frontal balding, and a myopathic facies, which is often associated with an open, tented mouth. In addition, myotonic dystrophy is associated with cardiac conduction defects, cataracts, and endocrine disturbance including diabetes, thyroid disorders, and hypogonads. However, myotonic dystrophy is highly variable and ranges from mildly to severely affected. The most severe form of the condition is congenital myotonic dystrophy, characterised by severe hypotonia at birth, respiratory insuffiency, and often intellectual disability. Myotonic dystrophy is caused by an expansion of a run of CTG repeats in the DMPK gene.

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Normal DMPK alleles have up to 34 CTG repeats. Repeat lengths greater than this are unstable and may expand during meiosis such that children inheriting the gene from an affected parent are often more severely affected than their parent, a phenomenon known as anticipation. Anticipation most commonly occurs during transmission of the disease from mother to child, although it has also been rarely seen with paternal transmission. Most cases of severe congenital myotonic dystrophy occur when the neonate inherits the expanded allele from their mother. 

Testing for myotonic dystrophy is now mostly done using a triplet-primed PCR technique, which can accurately detect small to medium DMPK gene expansions. It is therefore suitable for most diagnostic and carrier testing.

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However, triplet-primed PCR cannot currently accurately differentiate between medium and large expansions, the latter of which, if detected prenatally, increases the risk of the baby being born with congenital myotonic dystrophy. For this reason, Southern blotting is currently still being used for prenatal myotonic dystrophy testing in many laboratories though it has increasingly been superseded by newer PCR-based techniques.

Source:-https://www.futurelearn.com/courses/molecular-techniques/6/steps/765693