Detection of mitochondrial single nucleotide polymorphisms using a primer elongation reaction on oligonucleotide microarrays

We have developed a novel allele-specific primer elongation protocol using a DNA polymerase on oligonucleotide chips. Oligonucleotide primers carrying polymorphic sites at their free 3'end were covalently bound to glass slides. The generation of single-stranded targets of genomic DNA containing single nuclotide polymorphisms (SNPs) to be typed was achieved by an asymmetric PCR reaction or exonuclease treatment of phosphothioate (PTO)-modified PCR products. In the presence of DNA polymerase and all four dNTPs, with Cy3-dUTP replacing dTTP, allele-specific extension of the immobilized primers took place along a stretch of target DNA sequence. The yield of elongated products was increased by repeated reaction cycles. We performed multiplexed assays with many small DNA targets, or used single targets of up to 4.4 kb mitochondrial DNA (mtDNA) sequence to detect multiple SNPs in one reaction. The latter approach greatly simplifies preamplification of SNP-containing regions, thereby providing a framework for typing hundreds of mtDNA polymorphisms.     

Limb mammary syndrome: a new genetic disorder with mammary hypoplasia, ectrodactyly, and other Hand/Foot anomalies maps to human chromosome 3q27.

We report on a large Dutch family with a syndrome characterized by severe hand and/or foot anomalies, and hypoplasia/aplasia of the mammary gland and nipple. Less frequent findings include lacrimal-duct atresia, nail dysplasia, hypohydrosis, hypodontia, and cleft palate with or without bifid uvula. This combination of symptoms has not been reported previously, although there is overlap with the ulnar mammary syndrome (UMS) and with ectrodactyly, ectodermal dysplasia, and clefting syndrome. Allelism with UMS and other related syndromes was excluded by linkage studies with markers from the relevant chromosomal regions. A genomewide screening with polymorphic markers allowed the localization of the genetic defect to the subtelomeric region of chromosome 3q. Haplotype analysis reduced the critical region to a 3-cM interval of chromosome 3q27. This chromosomal segment has not been implicated previously in disorders with defective development of limbs and/or mammary tissue. Therefore, we propose to call this apparently new disorder "limb mammary syndrome" (LMS). The SOX2 gene at 3q27 might be considered an excellent candidate gene for LMS because the corresponding protein stimulates expression of FGF4, an important signaling molecule during limb outgrowth and development. However, no mutations were found in the SOX2 open reading frame, thus excluding its involvement in LMS.     

Gene expression profile of mouse bone marrow stromal cells determined by cDNA microarray analysis

Bone marrow stromal cells (BMSC) have gained increased attention because of their multipotency and adult stem cell character. They have been shown to differentiate into other cell types of the mesenchymal lineage and also into non-mesenchymal cells. The exact identity of the original cells, which are isolated from bone marrow by their selective adherence to plastic, remains unknown to date. We have established and characterized mouse BMSC cultures and analyzed three independent samples by cDNA microarrays. The expression profile was compared with two previous expression studies of human BMSC and revealed a high degree of concordance between different techniques and species. To gain clues about the positional context and biology of the isolated cells within the bone marrow stroma, we searched our data for genes that encode proteins of the extracellular matrix, cell adhesion proteins, cytoskeletal proteins and cytokines/cytokine receptors. This analysis revealed a close association of BMSC with vascular cells and indicated that BMSC resemble pericytes.     

Molecular cloning of Xp11 breakpoints in two unrelated mentally retarded females with X;autosome translocations

Mental retardation is a very common and extremely heterogeneous disorder that affects about 3% of the human population. Its molecular basis is largely unknown, but many loci have been mapped to the X chromosome. We report on two mentally retarded females with X;autosome translocations and breakpoints in Xp11, viz., t(X;17)(p11;p13) and t(X;20)(p11;q13). (Fiber-) FISH analysis assigned the breakpoints to different subbands, Xp11.4 and Xp11.23, separated by approximately 8 Mb. High-resolution mapping of the X- chromosome breakpoints using Southern blot hybridization resulted in the isolation of breakpoint-spanning genomic subclones of 3 kb and 0. 5 kb. The Xp11.4 breakpoint is contained within a single copy sequence, whereas the Xp11.23 breakpoint sequence resembles an L1 repetitive element. Several expressed sequences map close to the breakpoints, but none was found to be inactivated. Therefore, mechanisms other than disruption of X-chromosome genes likely cause the phenotypes.     

New perspectives for the elucidation of genetic disorders

For almost 15 years, genome research has focused on the search for major risk factors in common diseases, with disappointing results. Only recently, whole-genome association studies have begun to deliver because of the introduction of high-density single-nucleotide-polymorphism arrays and massive enlargement of cohort sizes, but most of the risk factors detected account for only a small proportion of the total genetic risk, and their diagnostic value is negligible. There is reason to believe that the complexity of many "multifactorial" disorders is primarily due to genetic heterogeneity, with defects of different genes causing the same disease. Moreover, de novo copy-number variation has been identified as a major cause of mental retardation and other complex disorders, suggesting that new mutations are an important, previously overlooked factor in the etiology of complex diseases. These observations support the notion that research into the previously neglected monogenic disorders should become a priority of genome research. Because of the introduction of novel high-throughput, low-cost sequencing methods, sequencing and genotyping will soon converge, with far-reaching implications for the elucidation of genetic disease and health care.     

On the incidence of unilateral and bilateral colour blindness in heterozygous females

Summary In 303 mothers of colour-blind sons, both eyes were tested with pseudoisochromatic plates and with the anomaloscope. Two hundred thirty healthy normal and 56 colour-blind males served as controls. In good agreement with the expected proportion of homozygotes in our sample, 17 colour-blind mothers were detected. Eight others had difficulty reading pseudoisochromatic plates and were conspicuous at the anomaloscope. In these, both eyes were affected to a very similar, moderate degree. Monocular disturbances of colour vision were not observed in the entire series. Our data suggest that (1) in most (if not all) of the carriers with colour vision impairment, there is no complete lack of normal retina cones, and (2) the proportion of defective retina cones is remarkably similar in both eyes of individual heterozygotes.The latter observation may indicate that at the time of X-differentiation there is a common primordial cell pool for both retinas.     

Distribution of G6PD phenotypes in red blood cells of Southern African Negroids: Evidence for somatic selection

Summary In a recent population study, we observed a striking deficit of G6PD heterozygotes among Southern African Negroid females. This finding was interpreted tentatively as evidence for a small number of hematopoetic stem cells in man. In a follow-up study we examined peripheral blood and cord blood in 547 mothers and in their newborn offspring. In mothers and sons, the frequencies of the G6PD alleles are apparently quite different. When the allele frequencies determined in sons are used for calculation of the expected phenotype frequencies in mothers and daughters, there is a large deficit of maternal G6PD AB phenotypes, and an equivalent surplus of G6PD homozygotes. However, no relevant heterozygote deficit is observed in newborn daughters. This discrepancy may be explained by the assumption that in peripheral blood of heterozygotes carrying the Gd^A- allele, G6PD-deficient cells progressively become eliminated during development from birth to adulthood. In other words, the large heterozygote deficit observed in adult females may be due to somatic selection rather than to a small pool of hematopoetic cells at the time of X differentiation.H-.H.R. is supported by the Deutsche Forschungsgemeinschaft