MTHFR C677T genotype frequency in patients of Middle Eastern descent as determined by real-time PCR and melting curve analysis.

The 5,10-methylenetetrahydrofolate reductase gene (MTHFR) 677C>T polymorphism produces an elevation in plasma homocysteine concentrations when present in the homozygous state. Increased homocysteine levels have been associated with a greater risk for vascular diseases, including cardiovascular disease and ischemic stroke. In this study, we genotyped 42 nucleic acid samples for the C677T allele from our database of Middle Eastern patients as routine validation of the MTHFR 677C>T assay. Our study is the first to evaluate MTHFR C677T genotype frequency in a population of Middle Eastern patients residing in the United States. Among the patients, 47.6% were wild type, 40.5% were heterozygous, and 11.9% were homozygous for the C677T variant. Although C677T genotype frequency in our patient population is slightly higher than that reported by Golbahar et al. (2005), statistical analysis showed no statistically significant difference beyond chance in genotype profiles (chi(2) = 1.54, df = 2, p = 0.1675). However, our findings implicate the need for a larger sample size to explore the need to implement standard clinical screening of MTHFR 677C>T. We also highlight the robust, reliable, and reproducible assay afforded by the use of anchor and sensor hybridization probes within the LightCycler platform to perform amplification and melting curve analysis protocols. Melting curve profiles that are produced display distinct and robust T(m) peaks based on the degree of anchor and sensor hybridization to amplicons produced from template DNA that is either wild-type, heterozygous, or a homozygous variant at the MTHFR 677C>T locus. A 10 degrees C gap between T(m) peaks allows for rapid and accurate qualitative identification of genotype.

Darmin is a novel secreted protein expressed during endoderm development in Xenopus.

Endoderm development is an area of intense interest in developmental biology, but progress has been hampered by the lack of specific markers for differentiated endodermal cells. In an unbiased secretion cloning screen of Xenopus gastrula embryos we isolated a novel gene, designated Darmin. Darmin encodes a secreted protein of 56 kDa containing a peptidase M20 domain characteristic of the glutamate carboxypeptidase group of zinc metalloproteases. We also identified homologous Darmin genes in other eukaryotes and in prokaryotes suggesting that Darmin is the founding member of a family of evolutionarily conserved proteins. Xenopus Darmin showed zygotic expression in the early endoderm and later became restricted to the midgut. By secretion cloning of Xenopus cleavage-stage embryos we isolated another novel protein, designated Darmin-related (Darmin-r) due to its sequence similarity with Darmin. Darmin-r was maternally expressed and showed at later stages expression in the lens and pronephric glomus. The endoderm-specific expression of Darmin makes this gene a useful marker for the study of endoderm development.

Isthmin is a novel secreted protein expressed as part of the Fgf-8 synexpression group in the Xenopus midbrain-hindbrain organizer.

Patterning of the central nervous system is regulated by a signaling center located at the midbrain-hindbrain boundary (MHB), or isthmus organizer. Fibroblast growth factors secreted from the MHB are required and sufficient to direct the ordered growth and regionalization of the midbrain and anterior hindbrain. In an unbiased secretion cloning screen of Xenopus gastrula embryos we identified a novel gene, which we designated as Isthmin (xIsm) due to its prominent expression at the MHB. xIsm encodes a secreted protein of 449 amino acids containing one copy of the thrombospondin type 1 repeat (TSR). We also found orthologous Isthmin genes in human (hIsm) and mouse (mIsm), as well as a gene encoding an Isthmin-like human unknown protein (hIsm-l). The conservation of a unique carboxy-terminal region between hIsm and hIsm-l suggests that Isthmin is the founding member of a new family of secreted proteins. xIsm was strongly expressed maternally in the Xenopus egg and showed zygotic expression in the ventral blastopore lip, notochord, and MHB. Additional expression domains were detected in neural crest, ear vesicle, and developing blood islands. Interestingly, xIsm was co-expressed with Fibroblast growth factor-8 (xFgf-8) at multiple sites including the MHB, indicating that these two genes are part of a synexpression group which also includes sprouty and sef homologs.