A. Adler

Preimplantation genetic diagnosis in two families at risk for recurrence of Herlitz junctional epidermolysis bullosa

Abstract: The Herlitz type of junctional epidermolysis bullosa (H‐JEB) is a severe inherited bullous disease which leads to the early demise of the affected newborn. Mutations in the genes encoding the 3 polypeptides of the anchoring filament protein laminin 5 underlie this condition. We studied 2 families with affected children who previously died from H‐JEB. Mutation screening using heteroduplex analysis and direct sequencing of the PCR products revealed a previously described hotspot mutation in LAMB3 (R635X), and a novel delayed termination codon in LAMB3 in the first proband. In the second proband, we found a novel initiation codon mutation in LAMB3, and a novel 2 bp deletion in LAMB3. For preimplantation genetic diagnosis (PGD) in these families, we developed nested multiplex PCR assays, amplifying the mutations and informative intragenic polymorphisms in the probands. Single embryonic cells were biopsied from 8‐cell embryos using standard techniques, and subjected to the multiplex PCR assay followed by restriction enzyme digestion. Embryos found not to carry either mutation were transferred to the mothers, and a pregnancy was established in the second family as evidenced by the elevated level of HCG, although the pregnancy did not persist. This study illustrates the feasibility of PGD for an inherited skin disorder for the first time.

Preimplantation Genetic Diagnosis of Human Embryos for Marfan's Syndrome

Purpose:Single-cell nested polymerase chain reaction (PCR) and Dde1 endonuclease digestion were used to detect the presence of a Marfans syndrome mutation in human preimplantation embryos derived from in vitro fertilization (IVF). These procedures were conducted to eliminate the possibility of transmission of the affected allele from the father to his offspring. The mutation on chromosome 15 is transmitted as an autosomal dominant trait, and the chance of having a child affected with the disease is 50%.Methods:A couple presented to the Program for In Vitro Fertilization, Reproductive Surgery and Infertility for preimplantation genetic diagnosis. IVF was performed and embryo biopsy was done on day 3 embryos, Single blastomeres were removed from embryos and subjected to nested PCR analysis and endonuclease digestion to detect a Marfans syndrome mutation located on chromosome 15 inherited from the father.Results:Thirteen oocytes were injected with spermatozoa using intracytoplasmic sperm injection, and nine fertilized normally. Following embryo biopsy and polymerase chain reaction amplification-Dde1 endonuclease digestion, five embryos were detected that were positive for the mutation. The four non-affected embryos were transferred to the uterus, resulting in a healthy and normal ongoing pregnancy.