Mira Malcov

Preimplantation genetic diagnosis for fragile X syndrome using multiplex nested PCR

Fragile X syndrome is caused by a dynamic mutation in the FMR1 gene. Normal individuals have <55 CGG repeats in the 5 untranslated region, premutation carriers have 55-200 repeats and a full mutation has >200 repeats. Female carriers are at risk of having affected offspring. A multiplex nested polymerase chain reaction protocol is described for preimplantation genetic diagnosis (PGD) of fragile X syndrome with simultaneous amplification of the CGG-repeat region, the Sry gene and several flanking polymorphic markers. The amplification efficiency was > or =96% for all loci. The allele dropout rate in heterozygotic females was 9% for the FMR1 CGG-repeat region and 5-10% for the polymorphic markers. Amplification failure for Sry occurred in 5% of single leukocytes isolated from males. PGD was performed in six patients who underwent 15 cycles. Results were confirmed in all cases by amniocentesis or chorionic villous sampling. Five clinical pregnancies were obtained (31% per cycle), four of which resulted in a normal delivery and one miscarried. This technique is associated with high efficiency and accuracy and may be used in carriers of full mutations and unstable high-order premutations.

Multiplex nested PCR for preimplantation genetic diagnosis of Spinal muscular atrophy

Objective: Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder caused in most patients by homozygous deletion of the SMN1 gene. For a carrier couple at a 25% risk of affected offspring, preimplantation genetic diagnosis (PGD) offers an alternative to prenatal diagnosis and termination of affected pregnancies. Our objective was to develop an accurate and reliable single-cell multiplex nested PCR analysis for PGD of SMA. Methods: The method was developed on single blood leukocytes, obtained from healthy controls and an adult SMA type III patient with a known homozygous deletion of SMN1 exon 7 and 8. Multiplex nested PCR on single cells was used to co-amplify exons 7 and 8 of SMN. Additional multiplexing was performed with the ZFX/ZFY gene for sexing. Following successful establishment of the multiplex nested PCR protocol in single leukocytes, the technique was employed for PGD in 4 patients for a total of 7 cycles. In 2 patients, sexing was simultaneously performed using ZFX/ZFY. Results: 220 single leukocytes from a normal individual and 220 from an SMA patient were analyzed. Exon 7 of SMN1 was amplified in 99% of normal single leukocytes and in none of the SMA-affected leukocytes. Exon 7 of SMN2 was amplified in 100% of both normal and SMA-affected leukocytes. Exon 8 of SMN1 was amplified in 98% of normal cells and in none of the SMA-affected leukocytes. Exon 8 of SMN2 was amplified in 96% of both normal and SMA-affected leukocytes. Amplification efficiency was 99% for ZFX/ZFY. There were no false-negative results and no contamination was detected in all wash-drop blanks tested. Seven PGD cycles were performed in 4 SMA-carrier couples with successful molecular analysis of 34 embryos and a total of 15 normal embryos transferred in 7 cycles. One clinical pregnancy has resulted in the delivery of a healthy male. Amniocentesis performed at 17 weeks confirmed the correct diagnosis for both SMA and sexing. Conclusions: These results suggest that our multiplex nested PCR protocol offers an efficient and accurate method for PGD of SMA while enabling the simultaneous analysis of an additional loci.

Preimplantation Genetic Diagnosis of Canavan Disease

Objective: Canavan disease is an autosomal recessive disorder which is relatively common in Ashkenazi Jews. It is characterized by developmental delay, severe hypotonia and early death, and is caused by a deficiency of aspartoacylase which is encoded by the ASPA gene. In Ashkenazi Jews, one major mutation (E285A) and one minor mutation (Y231X) account for the majority of cases. The objective of this study was to develop a preimplantation genetic diagnosis (PGD) protocol for Canavan disease. Methods: Two carrier couples requested PGD for Canavan disease. In 1 couple each was a carrier of a different ASPA mutation (Y231X and E285A). In the other couple both were carriers of the minor mutation (Y231X). A single-cell duplex nested polymerase chain reaction (PCR) protocol was first developed on single leukocytes obtained from the known carrier parents. Following verification in single leukocytes, clinical PGD was offered to both couples. Results: We evaluated 115 single leukocytes from known carriers and found an allele drop out rate of 1.7% for the fragment harboring the Y231X mutation and 0% for the fragment harboring the E285A mutation. One cycle of PGD was performed in each family. In the first, 11 embryos were successfully analyzed and 4 were found to be affected. Two unaffected embryos were transferred, but no pregnancy resulted. In the other family, 4 embryos were analyzed, 1 was affected, 2 were heterozygotes and 1 was homozygous normal. Following transfer of 2 unaffected embryos, a singleton pregnancy resulted, currently ongoing at 18 weeks gestational age. Amniocentesis performed at 16 weeks confirmed the diagnosis. Conclusion: Reliable PGD for Canavan disease is possible using a single-cell nested PCR approach.

Resolving a genetic paradox throughout preimplantation genetic diagnosis for autosomal dominant severe congenital neutropenia.

Severe congenital neutropenia is an inherited disease characterized by low peripheral blood neutrophils, amenable to bone marrow transplantation. Genetic analysis in the family here described detected a ELA2 splice‐site mutation in the affected child and also in his asymptomatic father. The parents requested preimplantation genetic diagnosis (PGD), coupled with HLA matching, to obtain a suitable bone marrow donor for the affected child.