Marijo Kent-First

High-resolution human genome structure by single-molecule analysis

Variation in genome structure is an important source of human genetic polymorphism: It affects a large proportion of the genome and has a variety of phenotypic consequences relevant to health and disease. In spite of this, human genome structure variation is incompletely characterized due to a lack of approaches for discovering a broad range of structural variants in a global, comprehensive fashion. We addressed this gap with Optical Mapping, a high-throughput, high-resolution single-molecule system for studying genome structure. We used Optical Mapping to create genome-wide restriction maps of a complete hydatidiform mole and three lymphoblast-derived cell lines, and we validated the approach by demonstrating a strong concordance with existing methods. We also describe thousands of new variants with sizes ranging from kb to Mb.

Y chromosome assessment and its implications for the development of ICSI children

The aetiology of compromised spermatogenesis is often genetic in nature. There are only a few reports of father/son cohorts that have been evaluated to assess heritability of mutations associated with male factor infertility and the psychological well-being of the children. In the present study, multiple tissues were sampled from consenting male patients and their sons derived from intracytoplasmic sperm injection (ICSI) and underwent chromosomal and genetic analyses. Paediatric and psychological examinations were also conducted. In 87 men and 47 boys, 22 sequence tagged sites (STS) were analysed by multiplex PCR and deletion breakpoints were defined with additional loci. In one patient, the breakpoints map to the highly unstable palindrome-rich region within AZFb and proximal AZFc was investigated. A total of 86 blood, 26 semen, and 73 cheek cells samples were collected from adults, and 36 blood samples and 44 cheek cell specimens were obtained from the boys. Though all of the fathers had normal karyotypes, the incidence of chromosomal abnormalities in the somatic cells of male progeny was 8.3% (3/36). The incidence of germ line aneuploidy in these men was 0.5-2.8%. A CF mutation (Delta508) was detected in one of 87 men (1.2%) and microdeletions in Yq AZF were detected in 3.4% of 87 adults and in 2.1% of their sons (n = 47). In conclusion, screening for Y chromosome microdeletions provides crucial information in the counselling of couples seeking infertility treatment. Moreover, DNA extraction and Y deletion assessments of cheek cells provide a non-invasive approach. Inheritance of Yq deletions appears not to affect the psychological and physical development of children derived from ICSI.

Use of mononucleotide repeat markers for detection of microsatellite instability in mouse tumors

Tumors lacking DNA mismatch repair activity (MMR) from patients with Hereditary Nonpolyposis Colorectal Cancer (HNPCC) or those with sporadic colorectal cancer can be identified by the presence of high levels of instability in repetitive sequences known as microsatellites (MSI). The assessment of MSI phenotype in human tumors helps to establish a clinical diagnosis and is accomplished with a reference panel of five mononucleotide repeats. By contrast, detection of MSI in mouse tumors has proven to be problematic and lack of a uniform set of markers for classification of MSI has impeded comparison of results between studies. We tested for MSI in intestinal tumors from MMR‐deficient mice with four mononucleotide repeats with polyA24‐37 tracts and three new markers with extended polyA59‐67 tracts. All seven markers were sensitive to MSI in MMR‐deficient tumors, but those with extended mononucleotide tracts displayed larger deletions, which were easily distinguishable from the germline alleles. With a panel of the five most sensitive and specific mononucleotide repeats, a high level of MSI was detected in 100% of MMR‐deficient tumors, but not in tumors with MMR activity. This novel panel is an improvement over existing combinations of mono‐ and dinucleotide repeat markers and should facilitate MSI screening and standardize results from different studies.

Length of Androgen Receptor—CAG Repeats in Fertile and Infertile Egyptian Men

Androgens play key roles in spermatogenesis, and they exert their effect via the androgen receptor (AR). The AR gene has a repetitive DNA sequence in exon 1 that encodes a polyglutamine tract. Instability in the glutamine (CAG) repeat unit length is polymorphic across ethnic groups. Previous studies of the relationship between the repeat unit length and male infertility have been contradictory. To establish the range of wild-type alleles in Egyptian men, we determined the range of repeat lengths in a population of normally fertile, ethnically selected Egyptian men. We also investigated the association between trinucleotide repeat length within the AR gene and male factor infertility in a population of ethnically selected Egyptian infertile men, who were compared with fertile, ethnic group-matched and age-matched controls. The study included 129 clinically selected infertile Egyptian men who were scheduled for intracytoplasmic sperm injection and 52 ethnically matched fertile controls. The experimental population was grouped according to sperm counts ranging from nonobstructive azoospermia to normozoospermia. The CAG repeat N-terminal domain region of the AR gene was amplified in peripheral blood DNA, and allele size was determined by fragment analysis. Allele size and single-nucleotide polymorphism and mutation rates were determined by sequencing individual amplified alleles. The mean CAG repeat length in the azoospermia group was 18.55 +/- 2.0; in the severe oligozoospermia group it was 18.21 +/- 3.42; in the oligozoospermia group it was 18.27 +/- 2.93; and in the infertile with normal sperm count group it was 17.72 +/- 2.0. In the control group, the mean CAG repeat length was 18.18 +/- 3.63. No significant correlation was found between CAG repeat length and the risk of male factor infertility in an ethnically defined population of Egyptian men. However, a significant and positive correlation between CAG repeat length and serum testosterone concentration was demonstrated. This suggests the involvement of epigenetic regulation linked to this region.

The critical and expanding role of genetics in assisted reproduction.

With the progress of the human gene mapping initiative, it is expected that the entire genome will be mapped within two years. A significant use for these data will centre on testing for genetic disease. Professionals associated with assisted reproduction are presented with a very special subset of the population, namely, couples suffering from infertility. Infertility may occur in the male, the female or both partners and may be heritable. Infertility, subfertility or recurrent spontaneous miscarriage is associated with chromosomal or genetic anomalies, suggesting that basic developmental genetics should be a part of the education of the physician or clinical embryologist. A review of the most common infertility‐associated chromosomal and genetic diseases for which genetic testing has become routine in infertile parents and in the products of assisted reproduction through preimplantation genetic diagnosis (PGD) and prenatal testing follows. Less common genetic diseases that have compromising effects on reproduction and which are likely to be encountered by providers of assisted reproduction are also considered. Copyright

Isolation and derivation of mouse embryonic germinal cells.

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos. Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF). Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days. The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state. Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

A Historical Perspective of the Cloning of Cattle

This chapter focuses on the history of cloning of cattle, the comparative methodology, successes, and failures, possible causes of failures with a particular emphasis on imprint maintenance and epigenetics, and applications of cloning of cattle.