Comprehensive statistical study of 452 BRCA1 missense substitutions with classification of eight recurrent substitutions as neutral.

BACKGROUND: Genetic testing for hereditary cancer syndromes contributes to the medical management of patients who may be at increased risk of one or more cancers. BRCA1 and BRCA2 testing for hereditary breast and ovarian cancer is one such widely used test. However, clinical testing methods with high sensitivity for deleterious mutations in these genes also detect many unclassified variants, primarily missense substitutions. METHODS: We developed an extension of the Grantham difference, called A-GVGD, to score missense substitutions against the range of variation present at their position in a multiple sequence alignment. Combining two methods, co-occurrence of unclassified variants with clearly deleterious mutations and A-GVGD, we analysed most of the missense substitutions observed in BRCA1. RESULTS: A-GVGD was able to resolve known neutral and deleterious missense substitutions into distinct sets. Additionally, eight previously unclassified BRCA1 missense substitutions observed in trans with one or more deleterious mutations, and within the cross-species range of variation observed at their position in the protein, are now classified as neutral. DISCUSSION: The methods combined here can classify as neutral about 50% of missense substitutions that have been observed with two or more clearly deleterious mutations. Furthermore, odds ratios estimated for sets of substitutions grouped by A-GVGD scores are consistent with the hypothesis that most unclassified substitutions that are within the cross-species range of variation at their position in BRCA1 are also neutral. For most of these, clinical reclassification will require integrated application of other methods such as pooled family histories, segregation analysis, or validated functional assay.

Linkage mapping and physical localization of the major histocompatibility complex region of the marsupial Monodelphis domestica.

We used genetic linkage mapping and fluorescence in situ hybridization (FISH) to conduct the first analysis of genic organization and chromosome localization of the major histocompatibility complex (MHC) of a marsupial, the gray, short-tailed opossum Monodelphis domestica. Family based linkage analyses of two M. domestica MHC Class I genes (UA1, UG) and three MHC Class II genes (DAB, DMA, and DMB) revealed that these genes were tightly linked and positioned in the central region of linkage group 3 (LG3). This cluster of MHC genes was physically mapped to the centromeric region of chromosome 2q by FISH using a BAC clone containing the UA1 gene. An interesting finding from the linkage analyses is that sex-specific recombination rates were virtually identical within the MHC region. This stands in stark contrast to the genome-wide situation, wherein males exhibit approximately twice as much recombination as females, and could have evolutionary implications for maintaining equality between males and females in the ability to generate haplotype diversity in this region. These analyses also showed that three non-MHC genes that flank the MHC region on human chromosome 6, myelin oligodendrocyte glycoprotein (MOG), bone morphogenetic protein 6 (BMP6), and prolactin (PRL), are split among two separate linkage groups (chromosomes) in M. domestica. Comparative analysis with eight other vertebrate species suggests strong conservation of the BMP6-PRL synteny among birds and mammals, although the BMP6-PRL-MHC-ME1 synteny is not conserved.

X-linked gene expression in the Virginia opossum: differences between the paternally derived Gpd and Pgk-A loci.

Expression of X-linked glucose-6-phosphate dehydrogenase (G6PD) and phosphoglycerate kinase-A (PGK-A) in the Virginia opossum (Didelphis virginiana) was studied electrophoretically in animals from natural populations and those produced through controlled laboratory crosses. Blood from most of the wild animals exhibited a common single-banded phenotype for both enzymes. Rare variant animals, regardless of sex, exhibited single-banded phenotypes different in mobility from the common mobility class of the respective enzyme. The laboratory crosses confirmed the allelic basis for the common and rare phenotypes. Transmission of PGK-A phenotypes followed the pattern of determinate (nonrandom) inactivation of the paternally derived Pgk-A allele, and transmission of G6PD also was consistent with this pattern. A survey of tissue-specific expression of G6PD phenotypes of heterozygous females revealed, in almost all tissues, three-banded patterns skewed in favor of the allele that was expressed in blood cells. Three-banded patterns were never observed in males or in putatively homozygous females. These patterns suggest simultaneous, but unequal, expression of the maternally and paternally derived Gpd alleles within individual cells (i.e., partial paternal allele expression). The absence of such partial expression was noted in a parallel survey of females heterozygous at the Pgk-A locus. Thus, it appears that Gpd and Pgk-A are X-linked in D. virginiana and subject to preferential paternal allele inactivation, but that dosage compensation may not be complete for all paternally derived X-linked genes. The data establish the similarity between the American and Australian marsupial patterns of X-linked gene regulation and, thus, support the hypothesis that this form of dosage compensation was present in the early marsupial lineage that gave rise to these modern marsupial divisions. In addition, the data provide the first documentation of the differential expression of two X-linked genes in a single marsupial species. Because of its combination of X-linked variation, high fecundity, and short generation time, D. virginiana is a unique model for pursuing questions about marsupial gene regulation that have been difficult to approach through studies of Australian species.

Genetic analysis of liver neuraminidase isozymes in Rattus norvegicus: independent control of NEU-1 and NEU-2 phenotypes.

Two recently identified isozymes of neuraminidase in rat liver were examined for transmission patterns and linkage relationships, and for variation among inbred strains. The isozymes, designated neuraminidase-1 (NEU-1) and neuraminidase-2 (NEU-2), exhibited no electrophoretic mobility variants among the 22 inbred strains examined, but did possess striking interstrain variation in activity phenotypes on electrophoretic gels. The results of a backcross analysis involving the KGH and ACP strains revealed that NEU-1 and NEU-2 phenotypes are independently controlled, each by a single autosomal locus with additively acting alleles. The two loci are unlinked to one another, but the gene controlling NEU-1 is tightly linked to RT1, the rat major histocompatibility complex. This gene is almost certainly identical to Neu-1, a gene identified previously through its effect on "total" activity levels of liver neuraminidase as determined by fluorometric assay of tissue homogenates. NEU-2 and the gene controlling its phenotype were not detected by the fluorometric technique. We designate the genes controlling the NEU-1 and NEU-2 phenotypes as Neu-1 and Neu-2, respectively. Data from this and other studies place Neu-1 between Glo-1 and dw-3. The location of Neu-2 is unknown.

Linkage of essential hypertension to the angiotensinogen locus in Mexican Americans.

Essential hypertension has been linked to a highly polymorphic marker at the angiotensinogen locus, and association with a polymorphism in this locus has been found in some populations. We tested the hypothesis that these same polymorphic markers are linked to essential hypertension in Mexican Americans. The data comprised all the affected relative pairs in 46 extended families chosen at random from a low-income barrio in San Antonio. Specifically, we searched for linkage by testing for excessive marker alleles shared identical by descent (IBD) among hypertensive relative pairs. When women taking oral contraceptives or hormones were excluded, the affected relative pairs shared a significant excess of alleles IBD for the highly heterozygous GT repeat polymorphism (P=.038) and were marginally significant for the M235T variant (P=.079), which has a much lower heterozygosity (0.43 versus 0.85 for the GT repeat). We also assayed plasma levels of angiotensinogen and, using likelihood methods, found no significant association (P=.43) between plasma levels of angiotensinogen and M235T genotypes. These results support the linkage of essential hypertension to the angiotensinogen locus but do not indicate a specific role for the M235T variant.

X-chromosome replication patterns in adult, newborn and prenatal opossums.

Somatic cells from the opossums Monodelphis domestica and Didelphis virginiana were labelled with 5-bromodeoxyuridine (BrdU), treated with colchicine, stained with acridine orange and examined using fluorescence microscopy. BrdU-incorporated metaphase spreads from females of M. domestica at developmental stages from late bilaminar blastocysts to adults showed replication asynchrony of the two (acrocentric) X chromosomes. The long arm of one X chromosome was the latest replicating region in the entire chromosome complement and is presumed to represent transcriptional inactivation and X dosage compensation. The minute short arm of the same X, which contains a nucleolar organizer region, was earlier replicating and synchronous with the short arm of its homologue and is thus assumed to escape inactivation. BrdU-incorporated spreads from cells of fetuses, neonates and adults of D. virginiana also showed a late replicating (submetacentric) X chromosome. The pattern was different from that of M. domestica because of the different morphology and the presence of large blocks of constitutive heterochromatin in both homologues. The timing and pattern of replication of the single X in males of both species resembled the earlier replicating X in females. The array of molecular techniques now available offers the best means for investigating X-chromosome replication and activity states of X-linked genes in the earliest stages of marsupial embryogenesis.

Integrated cytogenetic BAC map of the genome of the gray, short-tailed opossum, Monodelphis domestica.

The generation of high-quality genome assemblies for numerous species is advancing at a rapid pace. As the number of genome assemblies increases, so does our ability to investigate genome relationships and their contributions to unraveling complex biological, evolutionary, and biomedical processes. A key process in the generation of a genome assembly is to determine and verify the precise physical location and order of the large sequence blocks (scaffolds) that result from the assembly. For organisms of relatively recent common ancestry this process may be achieved largely through comparative sequence alignment. However, as the evolutionary distance between species lengthens, the use of comparative sequence alignment becomes increasingly less reliable. Simultaneous cytogenetic mapping, using multicolor fluorescence in-situ hybridization (FISH) analysis, offers an alternative means to define the cytogenetic location and relative order of DNA sequences, thereby anchoring the genome sequence to the karyotype. In this article we report the molecular cytogenetic locations of 415 bacterial artificial chromosome (BAC) clones that served to anchor sequence scaffolds of the gray, short-tailed opossum (Monodelphis domestica) to its karyotype, which enabled accurate integration of these regions into the genome assembly.

X-linked glucose-6-phosphate dehydrogenase (G6PD) and autosomal 6-phosphogluconate dehydrogenase (6PGD) polymorphisms in baboons.

Electrophoretic polymorphisms of glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) were examined in captive colonies of five subspecies of baboons (Papio hamadryas). Phenotype frequencies and family data verified the X-linked inheritance of the G6PD polymorphism. Insufficient family data were available to confirm autosomal inheritance of the 6PGD polymorphism, but the electrophoretic patterns of variant types (putative heterozygotes) suggested the codominant expression of alleles at an autosomal locus. Implications of the G6PD polymorphism are discussed with regard to its utility as a marker system for research on X-chromosome inactivation during baboon development and for studies of clonal cell proliferation and/or cell selection during the development of atherosclerotic lesions in the baboon model.

X-linked and autosomal inheritance patterns of homologous genes in two species of Tribolium.

The tenebrionid beetles Tribolium castaneum and T. confusum are representative of two distinct species groups within their genus. It has been suggested [Smith, S.G. (1952). J. Morphol. 91:325] that the 8AA + neo-XY karyotype of T. confusum was derived from the ancestral 9AA + XY formula, still present in T. castaneum, via the fusion of one pair of autosomes with the X and Y chromosomes during the early divergence of the confusum and castaneum species groups. In the present paper, electrophoretic variation in malic enzyme and hexokinase-1, detected in laboratory strains in Tribolium, is described. Evidence is presented that the genes encoding variation in both enzymes are autosomal in T. castaneum but are X linked in T. confusum. These species-specific patterns of inheritance of homologous gene loci are consistent with the hypothesized karyotypic history of the genus.

Biochemical characteristics and subcellular localizations of rat liver neuraminidase isozymes: a paradox resolved.

A striking discrepancy in the abilities of two analytical approaches (fluorometric and electrophoretic) to detect the effect of a gene, Neu-2, on rat liver neuraminidase phenotypes led us to examine the biochemical and physical properties of the liver isozymes NEU-1 and NEU-2 that might be responsible for this difference. Cell fractionation via Percoll gradient centrifugation revealed NEU-1 activity almost exclusively in the lysosomal cell fraction, while NEU-2 was strictly cytosolic in distribution. The two isozymes were also found to differ in pH activity curves and optima (optima: 4.6-4.8 and 5.4-5.8 for NEU-1 and NEU-2, respectively) and in solubility characteristics (NEU-2 highly soluble; NEU-1 relatively insoluble but solubilized by freezing/thawing). Both isozymes were found to be freeze-thaw stable in crude, whole-cell extracts, but NEU-1 was destabilized in the enriched (partially purified) lysosomal subcellular fraction. Consideration of these properties relative to those described previously for unidentified cytosolic and membrane bound (lysosomal) rat liver neuraminidases (Tulsiani, D. R. P., and Carubelli, R., J. Biol. Chem. 245:1821, 1970) leads us to believe that NEU-2 also is destabilized by partial purification and that NEU-1 and NEU-2 have very different relative abundances within the cell. The biochemical and physical differences between NEU-1 and NEU-2 can account for the discrepant abilities of the fluorometric and electrophoretic approaches to detect the effects of Neu-2. Ways to increase the sensitivity of the fluorometric approach for quantitative assays of specific NEU-1 and NEU-2 activity are discussed.

Developmental progression of Gpd expression from the inactive X chromosome of the Virginia opossum.

Metatherian (marsupial) mammals possess a non-random form of X-chromosome inactivation in which the paternally-derived X is always the one inactivated. To examine the progression of X-linked gene expression during metatherian development, we compared relative levels of the maternally and paternally encoded Gpd gene products in heterozygous female Virginia opossums (Didelphis virginiana) across a major portion of the developmental period. Panels of tissues obtained from fetuses, newborns, and pouch young were examined via polyacrylamide gel electrophoresis of the G6PD protein. As in adults, G6PD phenotypes in these developmental stages were highly skewed in favor of the maternal allele product, but in some tissues there was a marked increase in paternal allele expression with advancing developmental age. However, even by 42 days of post-partum development, expression of the paternal Gpd allele had not attained the adult, tissue-specific activity pattern. Our findings indicate remarkable developmental changes in the activity of the paternal allele in several tissues/organs continuing well into mid pouch-life stages and beyond. Specifically we found that 1) a substantially repressed paternal Gpd gene is present in the cells of female stage 29 fetuses and later developmental stages, 2) the activity state of the paternal Gpd gene is not fixed during early embryonic development in this species, 3) major changes in paternal Gpd expression occur in advanced developmental stages and comprise a maturation of the gene expression pattern during ontogeny, and 4) alterations of paternal Gpd allele activity during development occur in a tissue-specific manner.

X-linked gene expression and X-chromosome inactivation: marsupials, mouse, and man compared.

The existence of paternal X inactivation in Australian and American marsupial species suggests that this feature of X-chromosome dosage compensation is not a recent adaptation, but probably predates the evolutionary separation of the Australian and American marsupial lineages. Although it is theoretically possible that the marsupial system is one of random X inactivation with p greater than 0.99 and q less than 0.01 and dependent on parental source, no instance of random X inactivation (p = q or p not equal to q) has ever been verified in any tissue or cell type of any marsupial species. Therefore, we conclude that the most fundamental difference in X inactivation of marsupials and eutherians is whether the inactive X is the paternal one or is determined at random (with p = q in most but not all cases). The only other unequivocal difference between eutherians and marsupials is that both X chromosomes are active in mice and human oocytes, but not in kangaroo oocytes. Apparently, the inactive X is reactivated at a later meiotic stage or during early embryogenesis in kangaroos. X-chromosome inactivation takes place early in embryogenesis of eutherians and marsupials. Extraembryonic membranes of mice exhibit paternal X inactivation, whereas those of humans seem to exhibit random X inactivation with p greater than q (i.e., preferential paternal X inactivation). In general, extraembryonic membranes of marsupial exhibit paternal X inactivation, but the Gpd locus is active on both X chromosomes in at least some cells of kangaroo yolk sac. It is difficult to draw any general conclusion because of major differences in embryogeny of mice, humans, and marsupials, and uncertainties in interpreting the data from humans. Other differences between marsupials and eutherians in patterns of X-linked gene expression and X-chromosome inactivation seem to be quantitative rather than qualitative. Partial expression of some genes on the inactive X is characteristic of marsupials, with species variation in the behavior of specific loci; some X-linked human genes on the inactive chromosome also are known to exhibit partial activity in vivo and in cultured cells. The X chromosomes of marsupials do not behave as units with respect to transcriptional activity, nor does the human X chromosome. In addition, Barr bodies have recently been detected at interphase in some marsupials, establishing that this manifestation of X chromosome inactivity is not restricted to eutherians.(ABSTRACT TRUNCATED AT 400 WORDS)

Interspecific variation at the Y-linked RPS4Y locus in hominoids: implications for phylogeny.

Within- and between-species variation in restriction endonuclease recognition sites was examined at the Y-linked RPS4Y locus of six hominoid species: human (Homo sapiens), gorilla (Gorilla gorilla), chimpanzee (Pan troglodytes), bonobo (Pan paniscus), orangutan (Pongo pygmaeus), and gibbon (Hylobates lar). RPS4Y is an expressed gene that maps to the non-recombining region of the Y chromosome. An approximately 1,490 base pair fragment of the RPS4Y gene, including all of intron 3, was amplified by PCR from DNA extracted from each of the six species. Forty-seven restriction sites were identified on the six-species composite map derived from double-digest restriction analyses of the amplified fragment. As expected, maximum parsimony analysis indicated that chimpanzee and bonobo are the two most closely related living hominoids. The same analysis suggested that the closest living relative of Homo is Gorilla, not Pan, although support for this relationship was relatively weak. These results disagree with recently published phylogenies based on analyses of mtDNA sequences (Horai et al. [1995] Proc. Natl. Acad. Sci. U.S.A. 88:7401-7404) and the Y-linked ZFY locus (Dorit et al. [1995] Science 268:1183-1185). A combined data set derived from three distinct Y-linked loci-RPS4Y, SRY, and ZFY-was also analyzed. The maximum parsimony topology for the combined data provided only weak support for a shared common ancestor for Homo and Pan subsequent to divergence from the Gorilla lineage. Taken together, the data from the Y chromosome do not provide unequivocal support for any single, dichotomously branching species tree linking Homo, Pan, and Gorilla.

X-linked gene expression in metatherian fibroblasts: evidence from the Gpd and Pgk-A loci of the Virginia opossum and the red-necked wallaby.

Fibroblasts cultured from ear pinna biopsies of Virginia opossums (Didelphis virginiana) and red-necked wallabies (Macropus rufogriseus) were examined electrophoretically to determine the relative expression levels of the maternally and paternally derived alleles at X-linked, enzyme-coding loci. Only the maternally derived allele was expressed at the Pgk-A locus in fibroblasts of heterozygous D. virginiana (M. rufogriseus not examined), but fibroblasts of both species exhibited evidence of paternal allele expression at the Gpd locus. Furthermore, the heterozygous G6PD phenotypes in both species were skewed in favor of the maternal gene product, as expected if the paternal allele is only partially (incompletely) expressed. For M. rufogriseus this result is contrary to a previous finding which suggested equal expression of both Gpd alleles in cultured fibroblasts of this species. The present results suggest that X-linked genes in metatherian fibroblasts are subject to the same kind of determinate, paternal allele inactivation, incomplete at some loci, described previously for X-linked genes in adult tissues and that the pattern of paternal X-linked gene expression in these cells is independent of the patterns in the tissues from which the fibroblasts are derived.

Analysis of neuraminidase isozyme phenotypes in mammalian tissues: an electrophoretic approach.

A simple cellulose acetate electrophoretic method for visualizing mammalian neuraminidase isozymes has been developed. Application of the method with rat and mouse liver extracts reveals the presence of two distinct isozymes in each species. Each isozyme exhibits tremendous variation in activity between inbred strains. The two isozymes vary independently of one another suggesting that their activities are controlled by different genes. The neuraminidase phenotypes detected in these inbred strains via electrophoresis are consistent with published accounts of neuraminidase phenotypes determined fluorometrically in whole liver homogenates, but also indicate the presence of a second isozyme not perceived by this other procedure.

Electrophoretic analysis of liver neuraminidase-1 variation in mice and additional evidence concerning the location of NEU-1.

Neuraminidase-1 (NEU-1) is one of two neuraminidase isozymes which can be detected electrophoretically in mouse liver extracts. The inheritance of variation in NEU-1 and the linkage relationships of the gene controlling this variation were studied through a backcross analysis involving the SM/J and MA/MyJ inbred strains, and by examination of NEU-1 phenotypes in three congenic strains: B10.SM, B10.SM(22R) and B10.RVB. The data indicate that NEU-1 is controlled by Neu-1, a gene previously identified by its effect on total liver neuraminidase activity in whole tissue homogenates. Analysis of the congenic strains revealed identical low activity (SM/J-type: Neu-1a/Neu-1a) NEU-1 phenotypes in all three strains. This indicates that Neu-1 lies in the segment of the SM/J-derived H-2 region that is common to all three strains: H-2E alpha to H-2D. In addition, we examined the relationship between NEU-1 and phenotypic variation in liver acid phosphatase (AP; for which a new typing method is described) and linkage order among several other enzyme-coding genes linked to H-2. In all animals that could be scored confidently for AP, the NEU-1 and AP phenotypes were concordant, adding support to the hypothesis that both phenotypes are controlled by Neu-1. Recombination rates among six H-2-linked marker loci were unexpectedly low, but were sufficient to verify the position of Upg-1 as the telomeric flanking marker relative to Glo-1, H-2 (C4), Neu-1 (Apl), Ce-2 and Pgk-2.

Molecular studies of marsupial X chromosomes reveal limited sequence homology of mammalian X-linked genes.

To explore the extent to which the X chromosome has been conserved during mammalian evolution, we compared six loci that are X-linked in the human genome with the corresponding genes of the North American marsupial, the Virginia opossum (Didelphis virginiana). Our analysis shows that in the opossum genome there are sequences highly homologous to those of human cDNAs for housekeeping genes, glucose-6-phosphoribosyltransferase (HPRT), phosphoglycerate kinase A (PGK1), and alpha-galactosidase A (GLA). However, ornithine transcarbamylase and blood clotting Factor IX--tissue-specific genes that are X-linked in eutherians mammals--have no highly conserved homologs in the marsupial genome. By cloning opossum G6PD and HPRT, we found that these genes are X-linked in the opossum and that homologous sequences are limited to coding regions. As all genomic fragments hybridizing with the human GLA probe show dosage effects, it is likely that the opossum counterpart is X-linked. Finally, the pattern of hybridization suggests that the autosomal pseudogenes of HPRT and PGK1 in the opossum have remained highly homologous to the human X-linked genes.

A new DNA marker, U15557, is linked to protease inhibitor and adenylate kinase-1 in the laboratory opossum, Monodelphis domestica.

A 323-bp DNA fragment (U15557) was isolated, cloned, and sequenced after polymerase chain reaction (PCR) amplification from Monodelphis domestica genomic DNA. A HindIII restriction fragment length polymorphism was identified in this species using the U15557 PCR, fragment as a hybridization probe. DNA samples exhibited either a 6.4 kb band, a 7.2 kb band, or both bands simultaneously. Behaviour of these two variants in family studies was consistent with codominant autosomal inheritance. Linkage between this marker and the loci encoding protease inhibitor (PI) and adenylate kinase 1 (AK1) was found in M. domestica.

Genome-wide linkage analysis of blood pressure in Mexican Americans.

The genetic mechanisms that control variation in blood pressure level are largely unknown. One of the first steps in understanding those mechanisms is the localization of the genes that have a significant effect on blood pressure. We performed genome scans of systolic (SBP) and diastolic blood pressure (DBP) on a population-based sample of families in the San Antonio Family Heart Study. A likelihood-based Mendelian model incorporating genotype-specific effects of sex, age, age(2), BMI, and blood pressure (SBP or DBP, as appropriate) as covariates was used to perform two-point lodscore (Z) linkage on 399 polymorphic markers. Results showed that the genotype-specific covariate effects were highly significant for both SBP and DBP. Linkage results showed that a quantitative trait locus (QTL) influencing DBP was significantly linked to D2S1790 (Z = 3.92, theta = 0.00) and showed suggestive linkage to D8S373 (Z = 1.92, theta = 0.00). A QTL influencing SBP showed suggestive linkage to D21S1440 (Z = 2.82, theta = 0.00) and D18S844 (Z = 2.09, theta = 0.11). Without the genotype-specific effects in the model, the linkage to D2S1790 was not even suggestive (Z = 1.33, theta = 0.09); thus genotype-specific modeling was crucial in detecting this linkage. A comparison with linkage studies based in other populations showed that the significant linkage to D2S1790 has been replicated at the same marker in the Quebec Family Study. The replicated significant linkage at D2S1790 may begin to establish the locations of the genes that significantly affect blood pressure across several human ethnic groups.