SNP-Single gene disorder detection

 

What is new in PGD?? What is Mutation‐directed PGD protocols? What is  minisequencing for detection of SNP  by application in single cell DNA analysis Blastomere biopsy & Genotyping from single cells in IVF settings  in PGD-a procedure that is not uncommon in ART practice more so Recurrent Implantation Failure ! From single cell of blastocyst   “ DNA amplification” have become evident now to detect mutations disorders  by PCR  technology . But now the age old PCR is not favoured –Why ??

 Because  due to its high  sensitivity, PCR,  also detects  many single gene mutations but now not favoured as PCR technology.PCR is therefore  highly prone to sources of error . Courtesy : Molecular Human Reproduction July 2003. The minisequencing method: an alternative strategy for preimplantation genetic diagnosis of single gene disorders .

Where this technology was most relevant??

Preimplantation genetic diagnosis (PGD) is presently a valid alternative for couples at high risk of pregnancy with genetic anomalies. PGD enables unaffected embryos generated by IVF to be identified and transferred and it therefore permits couples to avoid termination of affected pregnancies.

Protocols for genotyping single cells for monogenic disorders are based on the PCR , which represents the only method sensitive enough to detect single gene mutations. AS mentioned , due to  its sensitivity .PCR is highly prone to sources of error; thus precautions must be taken in its use for clinical diagnosis.

  What exactly was wrong with PCR? Since the first PCR‐based PGD cases were performed, several inherent difficulties associated with single cell DNA amplification have become evident. Drawbacks of PCR from a single cell for mutation disorders?? Ans: Previous researchers have noted potential sample contamination, total PCR failure, allelic drop‐out (ADO, when one of the alleles fails to amplify to detectable levels), and preferential amplification (PA) of one of the alleles.

PGD , therefore remained and continued to be a technical challenge, as only one or two blastomeres are available for analysis, which must be performed within 1 day. A major limitation of PGD practice comes from the need to develop single cell DNA analysis protocols.

They should be sensitive enough to provide the greatest amplification efficiency, thus allowing the maximum number of embryos to be diagnosed. This is very important when PGD is performed for an autosomal dominant disease, in which 50% of the embryos could theoretically be affected.

 PGD protocols should also meet high standards of accuracy, have a low ADO rate (allelic drop‐out ADO) ,and contamination controls, ensuring transfer of only unaffected embryos. Therefore a PGD protocol must be put through an extensive preclinical trial before it can be applied to clinical cases.

The goal of centres performing single cell DNA analysis is thus to optimize a strategy that maximizes efficiency, sensitivity, and reliability of the procedure, enabling interpretable and unambiguous results to be obtained.

Techniques involving non‐automated gel analysis are successfully used for mutation screening in the majority of PGD cases to detect the presence or absence of restriction sites  electrophoretic mobility shift, as in single strand conformation polymorphism (SSCP) or in denaturing gradient gel electrophoresis (DGGE) ,

Computer‐assisted highly sensitive mutation detection is also performed, for the above techniques, by means of fluorescent PCR;  and for allele specific amplification (ARMS: amplification refractory mutation system.

For diseases involving a heterogeneous spectrum of mutations identified, such as cystic fibrosis, β‐thalassaemia or haemophilia A, the development of a mutation‐based PGD strategy is not practical because it requires time and resources for standardization of PCR protocols unique for the specific mutations of interest.

 

For these kinds of monogenic diseases, the use of a diagnostic strategy capable of detecting a wide spectrum of mutations and compound genotypes is more feasible. Genotyping methods based on DGGE  or SSCP have been used to facilitate mutation detection for the above anomalies, and have also addressed many of the inherent potential problems associated with PCR‐based genotyping of single cells.

An alternative procedure to mutation‐directed PGD protocols was proposed to overcome these problems: fluorescent multiplex PCR indirect diagnosis performed by the use of polymorphic markers, allowing identification of the pathogenic haplotype instead of the mutation .

PGD strategy, can also be  based on the use of a single mutation analysis protocol that could be fluorescence‐based (i.e. highly sensitive), computer‐assisted (i.e. facilitating data interpretation and management), and involving the use of a common procedure for each mutation to be analysed.

Automated fluorescence‐based DNA sequencing combines the above characteristics, allowing the identification and computer‐assisted visualization of a specific mutation. Moreover, it enables the simultaneous analysis of more than one mutation in a single PCR fragment. However, while representing a valid genetic analysis technique, guaranteeing good interpretative reliability, its application to PGD analysis is unwieldy, time consuming, and requires good quality amplification products for analysis. Furthermore it requires experience for data interpretation.

In order to overcome some of these limitations, especially in the case of larger blastomere numbers, the application of a new mutation analysis method, based on a primer extension technique , primarily devised to detect single nucleotide polymorphisms (SNP), was investigated. This method, more generally known as minisequencing  permits identification of the specific mutations without sequencing the entire PCR product, yet it still maintains the same qualitative characteristics of sequence analysis.