PGD by minisequencing method- detection of single gene disorders in blasotmere biopsy prior to embryo tranfernsfer.

 

 

What is The minisequencing method??      Ans: The minisequencing method applies to a new technique in Blastomere   biopsy & Genotyping single cells in IVF settings  in PGD !!!! From blastocyst single cell DNA amplification have become evident now to detect mutations disorders  by PCR . But now the  age old PCR is not favoured ?? Because  due to its sensitivity, PCR, which also detects  single gene mutations but now not favoured as PCR technology   is highly prone to sources of error . Courtesy : Molecular Human Reproduction July 2003. The minisequencing method:( a diagnostic strategy capable of detecting a wide spectrum of mutations and compound genotypes is more feasible)  an alternative strategy for preimplantation genetic diagnosis of single gene disorders .

Where this technology is 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. Due to its sensitivity, PCR is highly prone to sources of error; thus precautions must be taken in its use for clinical diagnosis.

Since the first PCR‐based PGD cases were performed, several inherent difficulties associated with single cell DNA amplification have become evident. What are the drawbacks of PCR from a single cell for mutation disorders?? Ans: Previous researchers have noted potential A) sample contamination, B) total PCR failure, C) allelic drop‐out (ADO, when one of the alleles fails to amplify to detectable levels), D)and preferential amplification (PA) of one of the alleles. E) Technical challenge as :  PGD  as it is 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. F)  Autosomal dominant disease are often missed . 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 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.

What is the goal of single cell DNA analysis?? Ans:   The goal of centres performing  the single cell DNA analysis is to optimize a strategy that maximizes efficiency, sensitivity, and reliability of the procedure, enabling interpretable and unambiguous results to be obtained as ay abnormal result will discard the embryo transfer and cancelation of cycle .

However , 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 hemophilia 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 DGG 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.

Fluorescent multiplex PCR  was the next modification: 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 .

What is new  in the    minisequencing method      PGD strategy, instead, was 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.

The aim of this study was to evaluate the reliability of minisequencing for its following application in single cell DNA analysis. PCR products from 887 blastomeres from 55 PGD cases of different genetic diseases, such as cystic fibrosis, β‐thalassaemia, sickle cell anaemia, haemophilia A, retinoblastoma, and spinal muscular atrophy (SMA), were analysed simultaneously with both traditional automated sequence analysis, routinely used in our laboratory for single cell mutation detection, and with the minisequencing method.

We have applied a new method of genetic analysis, called ‘minisequencing’, to preimplantation genetic diagnosis (PGD) of monogenic disorders from single cells. This method involves computer‐assisted mutation analysis, which allows exact base identity determination and computer‐assisted visualization of the specific mutation(s), and thus facilitates data interpretation and management. Sequencing of the entire PCR product is unnecessary, yet the same qualitative characteristics of sequence analysis are maintained. The main benefit of the mini-sequencing strategy is the use of a mutation analysis protocol based on a common procedure, irrespective of the mutations involved. To evaluate the reliability of this method for subsequent application to PGD, researchers analysed PCR products from 887 blastomeres including 55 PGD cases of different genetic diseases, such as cystic fibrosis, β‐thalassaemia, sickle cell anaemia, haemophilia A, retinoblastoma, and spinal muscular atrophy. Minisequencing is now  found to be a useful technique in PGD analysis, due to its elevated sensitivity, automation, and easy data interpretation. The method was also efficient, providing interpretable results in 96.5% (856/887) of the Blastomeres tested.