The labeling efficiency of human telomeres is increased by double-strand PRINS

Telomeres are composed of tandem repeated sequences, TTAGGG, that can be detected either by fluorescence in situ hybridization (FISH), more efficiently by using a peptide nucleic acid (PNA) probe, or by the primed in situ (PRINS) technique. However, the efficiency of human telomere labeling using PRINS is somewhat lower than the efficiency using PNA-FISH. To solve this problem, we developed a double-strand PRINS technique, which uses two primers, (TTAGGG)₇ and (CCCTAA)₇, to label both forward and reverse telomeric DNA strands. A total of 120 lymphocyte metaphases obtained from three normal adults were scored to evaluate the labeling efficiency based upon the telomere signal frequency present in chromatid ends and chromosome arms. As a comparison, 30 metaphases from the same three individuals were evaluated using PNA-FISH. The average labeling efficiency of PRINS was increased to a level very close to that obtained with PNA-FISH. Therefore, we demonstrated that the low labeling efficiency of human telomeres with regular PRINS was likely caused by uneven annealing of primers at the relatively short human telomere sequences, resulting in some telomere sites with very weak or absent labeling. We suggest that the present double-strand labeling protocol is critical to maximize the labeling efficiency of the human telomere sequence when using the PRINS technique.     

Mechanisms governing B cell developmental defects in invariant chain-deficient mice

Invariant chain (Ii)-deficient mice exhibit profound B cell defects that have remained poorly understood, because they could not be simply explained by impaired Ag presentation. We found that Ii deficiency induced cell autonomous defects of two distinct B cell lineages. The life span of mature follicular (FO) B cells was reduced, accounting for their markedly decreased frequency, whereas, in contrast, marginal zone (MZ) B cells accumulated. Other Ii-expressing lineages such as B1 B cells and dendritic cells were unaffected. Surprisingly, the life span of FO B cells was fully corrected in Ii/I-Abeta doubly deficient mice, revealing that Ii-free I-Abeta chains alter FO B cell survival. In contrast, the accumulation of MZ B cells was controlled by a separate mechanism independent of I-Abeta. Interestingly, in Ii-deficient mice lacking FO B cells, the MZ B cells invaded the FO zone, suggesting that intact follicules contribute to the retention of B cells in the MZ. These findings reveal unexpected consequences of Ii deficiency on the development and organization of B cell follicles.     

Kallmann syndrome: mutations in the genes encoding prokineticin-2 and prokineticin receptor-2

Kallmann syndrome combines anosmia, related to defective olfactory bulb morphogenesis, and hypogonadism due to gonadotropin-releasing hormone deficiency. Loss-of-function mutations in KAL1 and FGFR1 underlie the X chromosome-linked form and an autosomal dominant form of the disease, respectively. Mutations in these genes, however, only account for approximately 20% of all Kallmann syndrome cases. In a cohort of 192 patients we took a candidate gene strategy and identified ten and four different point mutations in the genes encoding the G protein-coupled prokineticin receptor-2 (PROKR2) and one of its ligands, prokineticin-2 (PROK2), respectively. The mutations in PROK2 were detected in the heterozygous state, whereas PROKR2 mutations were found in the heterozygous, homozygous, or compound heterozygous state. In addition, one of the patients heterozygous for a PROKR2 mutation was also carrying a missense mutation in KAL1, thus indicating a possible digenic inheritance of the disease in this individual. These findings reveal that insufficient prokineticin-signaling through PROKR2 leads to abnormal development of the olfactory system and reproductive axis in man. They also shed new light on the complex genetic transmission of Kallmann syndrome.     

Expression of the delta-protocadherin gene Pcdh19 in the developing mouse embryo

Protocadherins constitute a large family of transmembrane proteins primarily involved in weak homophilic adhesion in the brain and several other tissues. In a screen for potential regulators of kidney development, we have identified Pcdh19, a poorly characterized member of the delta-protocadherin subfamily. Here, we report the spatio-temporal expression pattern of Pcdh19 during mouse embryonic development. In midgestation embryos, Pcdh19 mRNA was detected in the mesonephros and in the neuroepithelium of the forebrain and midbrain. At later stages, Pcdh19 was expressed in other neural tissues such as the neural retina, nasal epithelium and spinal cord, as well as in the collecting duct and differentiating nephrons of the metanephros, in the glandular stomach, the exocrine pancreas and the hair follicles. Hence, the Pcdh19 gene is developmentally regulated during mouse organogenesis and shows a unique expression profile among protocadherins.     

A quantitative trait locus for body fat on chromosome 1q43 in French Canadians: linkage and association studies

Objective: To explore a quantitative trait locus (QTL) on human chromosome 1q affecting BMI, adiposity, and fat‐free mass phenotypes in the Quebec Family Study cohort. Research Methods and Procedures: Non‐parametric sibpair and variance component linkage analyses and family‐based association studies were performed with a dense set of chromosome 1q43 microsatellites and single‐nucleotide polymorphism markers in 885 adult individuals. Results: Linkage was observed between marker D1S184 and BMI (p = 0.0004) and with body fat mass or percentage body fat (p ≤ 0.0003), but no linkage was detected with fat‐free mass. Furthermore, significant linkages (p < 0.0001) were achieved with subsamples of sibpairs at both ends of phenotype distributions. Association studies with quantitative transmission disequilibrium tests refined the linkage to a region overlapping the regulator of G‐protein signaling 7 (RGS7) gene and extending to immediate upstream gene loci. Discussion: The present study indicates that the QTL on chromosome 1q43 specifically affects total adiposity and provides a genetic mapping framework for the dissection of this adiposity locus.     

Amnion and ocular surface problems

The amniotic membrane, the most internal placental membrane, has various properties useful in ophthalmology. Collected on delivery by elective Caesarean section, the amnion is prepared under sterile conditions, and, usually, cryopreserved until its use as a biological bandage or as a substrate for epithelial growth in the management of various ocular surface conditions. Specifically, the amnion is used to : (1) limit formation of adhesive bands between eyelids and eyeball (symblepharon) or the progression of a fibrovascular outgrowth towards the cornea (pterygium) or to (2) facilitate the healing of corneal ulcers, bullous keratopathy, and corneal stem cell deficiency. In this last condition, either hereditary or acquired after a thermal or a chemical burn, corneal stem cells, located at a transitional zone between the cornea and conjunctiva, are lost. These cells are essential for renewal of corneal epithelium in normal and in diseased states. The loss of these cells leaves the corneal surface free for invasion by conjunctival epithelium. Not only, does conjunctival epithelium support the development of vascularisation on the normally avascular cornea, but some conjunctival cells differentiate into mucus secreting goblet cells. Such a change in phenotype leads to loss of corneal transparency and visual disability. The removal of this fibro-vascular outgrowth in combination with transplantation of both amniotic membrane and corneal stem cells are used to treat this condition. The amnion stimulates the proliferation of less differentiated cells which have the potential to reconstruct the cornea. This potential is at the origin of the hypothesis that the amnion may provide an alternative niche for limbal stem cells of the corneal epithelium. It abounds in cytokines and has antalgic, anti-bacterial, anti-inflammatory and anti-immunogenic properties, in addition to allowing, like fetal skin does, wound healing with minimal scar formation. These desirable properties are responsible for the increasing use of amniotic membrane in ophthalmology. The complete understanding of the mechanisms of action of amniotic membrane for ocular surface diseases has yet to be understood. Once revealed by research, they may provide new pharmacological avenues to treat ocular surface diseases.     

Length, orientation, and plant host influence the mutation frequency in microsatellites.

Microsatellites are simple, tandem DNA repeats that represent unstable regions of the genome. They undergo frequent changes in tract length by base additions or deletions due to DNA polymerase slippage during replication. To characterize factors affecting the frequency of spontaneous mutations occurring in microsatellites in plants, a reporter system was used in Arabidopsis thaliana and tomato (Lycopersicon esculentum). The beta-glucuronidase (GUS) reporter system was used to measure the mutation frequency in various microsatellites (G(7), G(10), G(13), G(16), and C(16)) in somatic tissues. Our results indicate that this frequency increases with the number of repeats: a G(16) tract was almost 80-fold more mutable than a G(7) tract. Furthermore, the frequency of mutations depends on repeat orientation, as G(16) was 3-fold more mutable than C(16). The mutation rate was also found to differ markedly in Arabidopsis and tomato for an identical microsatellite. Indeed, Arabidopsis showed a 5-fold higher mutation frequency than tomato with the same G(7) reporter construct. Finally, mutation in a G(16) tract was frequent enough that mutations transmitted germinally to the next generation could be detected at a relatively high frequency.     

Pleiotropic QTL on chromosome 12q23-q24 influences triglyceride and high-density lipoprotein cholesterol levels: the HERITAGE family study

To determine whether a common quantitative trait locus (QTL) influences the variation of fasting triglyceride (TG) and high-density lipoprotein cholesterol (HDL-C) levels, we used a bivariate multipoint linkage analysis with 654 polymorphic markers in 99 white and 101 black families. The phenotypes were investigated under two conditions: at baseline and after a 20-week exercise training intervention. A maximum genome-wide bivariate LOD score of 3.0 (p = 0.00010) was found on chromosome 12q23-q24, located within the IGF1 gene (insulin-like growth factor 1, at 107 cM) for TG and HDL-C at baseline in whites. This bivariate linkage peak is considerably higher than the univariate linkage results at the same chromosome location for either trait (for TG, LOD = 2.07, p = 0.00108; for HDL-C, LOD = 2.04, p = 0.00101). The genetic correlations between baseline TG and HDL-C levels were -0.14 for the residual and -0.33 for the QTL components. Moreover, association analysis showed that TG, HDL-C, and IGF1 are significantly associated (p = 0.04). In conclusion, these results suggest that a QTL on chromosome 12q23-q24 influences the variation of plasma TG and HDL-C levels. Further investigation should confirm whether IGF1 or another nearby gene is responsible for the concomitant variation in TG and HDL-C levels.