Molecular karyotyping of patients with MCA/MR: the blurred boundary between normal and pathogenic variation

Molecular karyotyping has revealed that microdeletions/duplications in the human genome are a major cause of multiple congenital anomalies associated with mental retardation (MCA/MR). The identification of a de novo chromosomal imbalance in a patient with MCA/MR is usually considered causal for the phenotype while a chromosomal imbalance inherited from a phenotypically normal parent is considered as a benign variation and not related to the disorder. Around 40% of imbalances in patients with MCA/MR in this series is inherited from a healthy parent and the majority of these appear to be (extremely) rare variants. As some of these contain known disease-causing genes and have also been found to be de novo in MCA/MR patients, this challenges the general view that such familial variants are innocent and of no major phenotypic consequence. Rather, we argue, that human genomes can be tolerant of genomic copy number variations depending on the genetic and environmental background and that different mechanisms play a role in determining whether these chromosomal imbalances manifest themselves.     

Family specific rates of protein evolution

MOTIVATION: Amino acid changing mutations in proteins are contstrained by purifying selection and accumulate at different rates. We estimate evolutionary rates on multiple alignments of eukaryotic protein families in a maximum likelihood framework and spot sets of slow and fast evolving proteins. RESULTS: We find that the evolution of indispensable proteins is constrained by selection and that protein secretion is coupled to an increased evolutionary rate.     

Development of a microarray assay that measures hybridization stoichiometry in moles

Microarray data is most useful when it can be compared with other genetic detection technologies. In this report, we designed a microarray assay format that transforms raw data into a defined scientific unit (i.e., moles) by measuring the amount of array feature present and the cDNA sequence hybridized. This study profiles a mouse reference universal RNA sample on a microarray consisting of PCR products. In measuring array features, a labeled DNA sequence was designed that hybridizes to a conserved sequence that is present in every array feature. To measure the amount of cDNA sample hybridized, the RNA sample was processed to ensure consistent dye to DNA ratio for every labeled target cDNA molecule, using labeled branched dendrimers rather than by incorporation. A dye printing assay was then performed in order to correlate molecules of cyanine dye to signal intensity. We demonstrate that by using this microarray assay design, raw data can be transformed into defined scientific units, which will facilitate interpretation of other experiments, such as data deposited at the Gene Expression Omnibus and ArrayExpress.     

Germ cell transplantation in an azoospermic Klinefelter bull

Germ cell transplantation is a technique that transfers donor testicular cells into recipient testes. A population of germ cells can colonize the recipient testis, initiate spermatogenesis, and produce sperm capable of fertilization. In the present study, a nonmosaic Klinefelter bull was used as a germ cell recipient. The donor cell suspension was introduced into the rete testis using ultrasound-guided puncture. A pulsatile administration of GnRH was performed to stimulate spermatogenesis. The molecular approach to detect donor cells was done by a quantitative polymerase chain reaction with allele discrimination based on a genetic mutation between donor and recipient. Therefore, a known genetic mutation, associated with coat-color phenotype, was used to calculate the ratio of donor to recipient cells in the biopsy specimens and ejaculates for 10 mo. After slaughtering, meiotic preparations were performed. The injected germ cells did not undergo spermatogenesis. Six months after germ cell transplantation, the donor cells were rejected, which indicates that the donor cells could not incorporate in the testis. The hormone stimulation showed that the testosterone-producing Leydig cells were functionally intact. Despite subfertility therapy, neither the recipient nor the donor cells underwent spermatogenesis. Therefore, nonmosaic Klinefelter bulls are not suitable as germ cell recipients. Future germ cell recipients in cattle could be mosaic Klinefelters, interspecies hybrids, bulls with Sertoli cell-only syndrome, or bulls with disrupted germ cell migration caused by RNA interference.     

Free human mitochondrial GrpE is a symmetric dimer in solution

The co-chaperone GrpE is essential for the activities of the Hsp70 system, which assists protein folding. GrpE is present in several organisms, and characterization of homologous GrpEs is important for developing structure-function relationships. Cloning, producing, and conformational studies of the recombinant human mitochondrial GrpE are reported here. Circular dichroism measurements demonstrate that the purified protein is folded. Thermal unfolding of human GrpE measured both by circular dichroism and differential scanning calorimetry differs from that of prokaryotic GrpE. Analytical ultracentrifugation data indicate that human GrpE is a dimer, and the sedimentation coefficient agrees with an elongated shape model. Small angle x-ray scattering analysis shows that the protein possesses an elongated shape in solution and demonstrates that its envelope, determined by an ab initio method, is similar to the high resolution envelope of Escherichia coli GrpE bound to DnaK obtained from single crystal x-ray diffraction. However, in these conditions, the E. coli GrpE dimer is asymmetric because the monomer that binds DnaK adopts an open conformation. It is of considerable importance for structural GrpE research to answer the question of whether the GrpE dimer is only asymmetric while bound to DnaK or also as a free dimer in solution. The low resolution structure of human GrpE presented here suggests that GrpE is a symmetric dimer when not bound to DnaK. This information is important for understanding the conformational changes GrpE undergoes on binding to DnaK.     

Functional characterization of three novel tissue-specific anion exchangers SLC26A7, -A8, and -A9

A second distinct family of anion exchangers, SLC26, in addition to the classical SLC4 (or anion exchanger) family, has recently been delineated. Particular interest in this gene family is stimulated by the fact that the SLC26A2, SLC26A3, and SLC26A4 genes have been recognized as the disease genes mutated in diastrophic dysplasia, congenital chloride diarrhea, and Pendred syndrome, respectively. We report the expansion of the SLC26 gene family by characterizing three novel tissue-specific members, named SLC26A7, SLC26A8, and SLC26A9, on chromosomes 8, 6, and 1, respectively. The SLC26A7-A9 proteins are structurally very similar at the amino acid level to the previous family members and show tissue-specific expression in kidney, testis, and lung, respectively. More detailed characterization by immunohistochemistry and/or in situ hybridization localized SLC26A7 to distal segments of nephrons, SLC26A8 to developing spermatocytes, and SLC26A9 to the lumenal side of the bronchiolar and alveolar epithelium of lung. Expression of SLC26A7-A9 proteins in Xenopus oocytes demonstrated chloride, sulfate, and oxalate transport activity, suggesting that they encode functional anion exchangers. The functional characterization of the novel tissue-specific members may provide new insights to anion transport physiology in different parts of body.