Complex recombination with deletion in the F8 and duplication in the TMLHE mediated by int22h copies during early embryogenesis.

Haemophilia A (HA) is a common X-linked recessive bleeding disorder and almost one half of patients with severe HA are caused by intron 22 inversion (Inv22) in the F8. Inv22 is considered to be almost exclusively of meiotic origin in germ cells during spermatogenesis and only one mosaic Inv22 female carrier with the mutation possibly occurring during mitosis of the embryo has been reported so far. Previously we have identified a novel complex recombination mediated by int22h copies in a sporadic severe HA pedigree and herein we have localised the sequences flanking the breakpoint region using genome walking technique, AccuCopy technique, gene chip and real-time PCR. The disease causing genetic variant registered an 18.1 kb deletion including part of int22h-1 through the intron 23 of F8 and a 113.3 kb duplication of part of int22h-2 through the intron 1 of TMLHE inserted in the religated region of the F8. Two intrinsically linked mechanisms of recombination-dependent DNA replication: microhomology-mediated break-induced replication (MMBIR) followed by break-induced replication (BIR) might be responsible for the incident of the complex recombination during early embryogenesis of the proband's mother.

Prenatally Diagnosed Hemophilia in a Newborn: a Case Report.

Hemophilia is the most common inherited coagulation disorder, and approximately one-half of patients are diagnosed as newborns. For prenatal diagnosis of hemophilia A, genetic tests are performed using chorionic villi (biopsy PCR chorionic villi sampling [CVS]) at 10 weeks' of gestation. The result in this fetus demonstrated an inversion within intron 1 in part for hemophilia A. This male infant, who was his parents' first offspring, was born after an uneventful pregnancy. An uncle suffered from hemophilia A. This report describes a newborn who was prenatally diagnosed with hemophilia A. The timely diagnosis of hemophilia in a newborn enabled the provision of adequate therapy, which led to a favorable outcome.

Fetal muscle gene therapy/gene delivery in large animals.

Gene delivery to the fetal muscles is a potential strategy for the early treatment of muscular dystrophies. In utero muscle gene therapy can also be used to treat other genetic disorders such as hemophilia, where the missing clotting proteins may be secreted from the treated muscle. In the past few years, studies in small animal models have raised the hopes that a phenotypic cure can be obtained after fetal application of gene therapy. Studies of efficacy and safety in large animals are, however, essential before clinical application can be considered in the human fetus. For this reason, the development of clinically applicable strategies for the delivery of gene therapy to the fetal muscles is of prime importance. In this chapter, we describe the protocols for in utero ultrasound-guided gene delivery to the ovine fetal muscle in early gestation. In particular, procedures to inject skeletal muscle groups such as the thigh and thoracic musculature and targeting the diaphragm in the fetus are described in detail.

Preimplantation genetic diagnosis of haemophilia.

Preimplantation genetic diagnosis (PGD) aims to increase the number of options available to couples who could have a child affected with haemophilia and reduce the anxiety these couples often associate with reproduction. The female partner must undergo an in vitro fertilization cycle, and the eggs or embryos are then biopsied. Embryos which are unaffected by haemophilia can then be transferred to the uterus. The clear advantage of this technique is that the woman knows from the very beginning that the pregnancy is unaffected by haemophilia, and she can avoid conventional invasive prenatal diagnosis and the difficult decision on whether or not to terminate an affected pregnancy. Several strategies for this single cell genetic diagnosis have been described. These include embryonic sexing using polymerase chain reaction, embryonic sexing using fluorescent in situ hybridization, specific diagnosis using restriction enzymes, sequencing and haplotype analysis. Over the years, PGD has attracted much ethical commentary, both supportive and critical, regarding the fundamental principles of embryo selection and destruction. New scientific advances and their potential applications are considered.

Laser-assisted derivation of human embryonic stem cell lines from IVF embryos after preimplantation genetic diagnosis.

BACKGROUND: Human embryonic stem cells (hESCs) suitable for future transplantation therapy should preferably be developed in an animal-free system. Our objective was to develop a laser-based system for the isolation of the inner cell mass (ICM) that can develop into hESC lines, thereby circumventing immunosurgery that utilizes animal products. METHODS: Hatching was assisted by micromanipulation techniques through a laser-drilled orifice in the zona pellucida of 13 abnormal preimplantation genetic diagnosed blastocysts. ICMs were dissected from the trophectoderm by a laser beam and plated on feeders to derive hESC lines. RESULTS: eight ICMs were isolated from nine hatched blastocysts and gave rise to three hESC lines affected by myotonic dystrophy type 1, hemophilia A and a carrier of cystic fibrosis 405 + 1G > A mutation. Five blastocysts that collapsed during assisted hatching or ICM dissection were plated whole, giving rise to an additional line affected by fragile X. All cell lines expressed markers of pluripotent stem cells and differentiated in vitro and in vivo into the three germ layers. CONCLUSIONS: These hESC lines can serve as an important model of the genetic disorders that they carry. Laser-assisted isolation of the ICMs may be applied for the derivation of new hESC lines in a xeno-free system for future clinical applications.

Inversion mutation as a major cause of severe hemophilia A in Italian patients.

We investigated the presence of a recombinant event between the F8A gene located in intron 22 of the factor VIII gene and the two additional copies of F8A lying 500 Kb upstream of FVIII in severe hemophilic patients. The genomic DNA of 146 unrelated Italian patients with severe hemophilia A (HA) was hybridized with an F8A gene probe to detect the abnormal band patterns. A recombinant event was found in 71/146 patients, confirming the high incidence of this mutation in the Italian hemophilic population also. We conclude that the high frequency of the mutation in HA subjects allows us to make a direct and safe diagnosis in about 50% of our families without resorting to RFLP analysis.

Prenatal diagnosis of hemophilia involving grandpaternal mosaicism.

Our Factor VIII and RFLP analyses identified previously unreported grandpaternal hemophilia A mosaicism in a male who transmitted the disease allele to 2 of 4 daughters and 2 of 4 grandsons. An uncommon flanking polymorphic DXS52 allele cosegregated with this grandpaternal mutant allele. This and other reports of mosaic hemophilia A carriers indicate that parental mosaicism can explain unusual segregation of low Factor VIII activities and DNA polymorphisms in about 1% of hemophilia A pedigrees.