Tommaso Meo

Mutations and sequence variants in the testis-determining region of the Y chromosome in individuals with a 46,XY female phenotype.

Abstract The testis-determining gene SRY (sex determining region, Y) is located on the short arm of the Y chromosome and consists of a single exon, the central third of which is predicted to encode a conserved motif with DNA binding/bending properties. We describe the screening of 26 patients who presented with 46,XY partial or complete gonadal dysgenesis for mutations in both the SRY open reading frame (ORF) and in 3.8 kb of Y-specific flanking sequences. DNA samples were screened by using the fluorescence-assisted mismatch analysis (FAMA) method. In two patients, de novo mutations causing complete gonadal dysgenesis were detected in the SRY ORF. One was a nonsense mutation 5′ to the HMG box, whereas the other was a missense substitution located at the C terminus of the conserved motif and identical to one previously detected in an unrelated patient. In addition, two Y-specific polymorphisms were found 5′ to the SRY gene, and a sequence variant was identified 3′ to the SRY polyadenylation site. No duplications of the DSS region in 20 of these patients were detected.

Altered C1 Inhibitor Genes in Type I Hereditary Angioedema

Hereditary angioedema is an inherited disease transmitted as an autosomal dominant trait and characterized by deficient activity of C1 inhibitor, a glycoprotein that limits intravascular activation of complement. The unavailability of markers for the C1 inhibitor locus has so far precluded access to the genetic bases of the disorder. Using a recombinant-DNA probe for the C1 inhibitor gene, we identified a cluster of four distinctive DNA restriction sites in a multigeneration family. The strict cosegregation of these markers with a low C1 inhibitor level supports the conclusion that a defective structural gene is responsible for the disease. The occurrence of distinct DNA markers in patients from unrelated kinships indicates that, like forms of the condition displaying dysfunctional C1 inhibitor variants, those characterized by protein deficiency are also genetically heterogeneous. Although multiple restriction-site changes in the C1 inhibitor gene are carried by certain patients, DNA markers for this locus are strikingly infrequent among normal persons. These findings indicate that the gene alterations responsible for hereditary angioedema arise more often from DNA rearrangements than from nucleotide substitutions. Such variations in the structure of the C1 inhibitor gene may provide the basis for early and direct identification of persons at risk.

Molecular cloning of human C1 inhibitor: sequence homologies with α1-antitrypsin and other members of the serpins superfamily

Genetic and acquired diseases in man show that the proteolytic activity of the complement component C1 is crucially regulated by C1 inhibitor (C1-INH), a plasma protein whose suspected relatedness to other serine proteinase inhibitors (serpins) contrasts with its atypically large size and high degree of glycosylation. Indeed we have found that the C1-INH polypeptide precursor synthesized in a cell-free system is a 64-kDa protein, hence it exceeds the length of the precursor forms of typical serpins. Seeking more conclusive sequence information and a probe for the structural locus, we isolated C1-INH cDNA clones from a library representing size-enriched human liver mRNA. Nucleotide sequence analysis of a clone covering the carboxyterminal half of C1-INH conclusively documents the relatedness of this protein with the serpins, and reveals 27% amino acid identity with alpha 1-antitrypsin.

complement genes C1r and C1s feature an intronless serine protease domain closely related to haptoglobin

The exon-intron structure of the human complement C1s gene displays a striking similarity with that of the gene encoding haptoglobin, a peculiar transport protein distantly related to the serine proteases. While the protease regions of the serine zymogens are typically encoded by multiple exons, the protease domains of C1s and of its genetically linked and functionally interacting homolog C1r are encoded as intronless domains, not unlike a region of haptoglobin, which in fact is devoid of proteolytic activity. The close similarity of the C1s gene with haptoglobin includes the precise conservation of exon-intron junctions and it extends to upstream exons encoding the short repeats typical of several complement components, but found also in other functionally unrelated proteins. Additional evidence of the common ancestry of C1r, C1s and haptoglobin is the presence, within the protease domain, of a set of sequence markers that distinguish these three proteins from all known serine proteases. The finding of vertebrate serine protease genes with an uninterrupted protease-encoding exon supports the definition of a novel evolutionary branch of this gene family and rules out the hypothesis that regards this unusual exon as an irrelevant byproduct of the extravagant functional divergence of haptoglobin.

Assignment of the complement serine protease genes C1r and C1s to chromosome 12 region 12p13.

SummaryC1r and C1s are distinct, but structurally and functionally similar, serine protease zymogens responsible for the enzymatic activity of the first component of complement (C1). Recent comparisons indicate a significant degree of sequence similarity between C1r and C1s and support the hypothesis that they are related by gene duplication. Complementary DNA probes for human C1r and C1s do not cross-hybridize even at mild stringency conditions and are therefore genespecific. Using a panel of 25 human-rodent cell hybrids, we have independently assigned the C1r and the C1s genes to chromosome 12. In situ hybridization analyses were consistent with these assignments, showing in addition that both C1r and C1s are located on the short arm of the chromosome in the region p13. These data suggest that the homologous C1r and C1s genes have remained closely linked after duplication of a common ancestor. The C1r and C1s loci also provide useful polymorphic DNA markers for the short arm of chromosome 12.

Fluorescence-assisted mismatch analysis (FAMA) for exhaustive screening of the alpha-galactosidase A gene and detection of carriers in Fabry disease.

Abstract We used the fluorescence-assisted mismatch analysis (FAMA) method to screen rapidly the α-galactosidase A gene in patients with Fabry disease in order to identify unknown mutations and help define genotype-phenotype correlations in this X-linked lysosomal storage disorder. Chemical cleavage at mismatches on heteroduplex DNA end-labeled with strand-specific fluorescent dyes, reliably detects sequence changes in DNA fragments of up to 1.5 kb and locates them precisely. Exhaustive scanning of the α-galactosidase gene was accomplished on four polymerase chain reaction-generated amplicons, covering all seven exons, the exon-intron boundaries, and 700 bp of 5′-flanking sequence. Mutations were identified in each of the 15 patients studied from nine unrelated kindreds. Among the seven previously undescribed sequence changes, three are obviously pathogenic because they lead to premature protein termination. The other four, a splice-site mutation and three missense mutations, were the only changes found upon complete scanning of the gene and its promoter. In addition, FAMA also detects female heterozygous carriers more dependably than direct sequencing, and thus provides a valuable diagnostic test. In Fabry disease, this molecular criterion is especially important for genetic counseling since heterozygotes can be asymptomatic and their enzymatic values within the normal range.

TCR Vβ genes in man and mouse and the factors that shape the linkage pattern of immune receptor genes

An effective immune system must rely on an adequate degree of diversification in the structures involved in antigeq binding. The hypothetical optimum of variability may be expected to vary with the particular history, the habitat and the constitution of the organisms whose fitness the immune system is so eminently apt to secure. Neve~heless, it is reasonable to suppose that the more frugal the genetic and/or somatic repertoire of immune recognition molecules, the higher the chances that the whole system may drift as a vulnerable set of genes. For the organisms exposed to a changing antigenic land- scape, the rapid deployment of a suitable set of recog- nition molecules is a forbidding task, inasmuch as gen- etic adaptation can be of little use or even deleterious in a changing world. As first pointed out and persuasively advocated by Susumu Ohno (see Ref. 1 for a recent restatement), the genetic solution to the problem of having to anticipate an inscrutable future is rather extravagant. It amounts to generating, in an antigen- independent fashion and beyond actual needs, a vast stock of antigen-combining sites encoded by genes, whose individual evolutionary fate is largely unaffected by encounters with antigens. In this way, the superfamily of immune receptors has a unique privilege in genetics because their properties may implicate natural selection whether it be to maximize or to reduce variation. For instance, diversity and redun- dancy of the variable gene segments are properties directly beneficial to the organism and hence subject to positive selection. On the other hand, it is logical to expect that V genes that have proven their competence in past epidemics should enjny a survival advantage over untested nc~ve~ties. Furthermore, as is true for any pro- tein, the V regtons as well as other structural elements must be prese~ed for reasons of architectural integrity, i.e., conventional constraints on folding of the polypeptide o- on other sites relevant to the assembly, transport and functions of the complete molecule. Thus, ~t is a recurring temptation to view instances of

Detection of mutations by fluorescence-assisted mismatch analysis (FAMA).

Fluorescence‐assisted mismatch analysis (FAMA) methodology uses bifluorescent double‐stranded DNA substrates to maximize the reliability and sensitivity of mutation‐scanning procedures that rely on cleavage of mismatches using chemical. This unit presents a nested PCR procedure to fluorescently label each DNA strand, followed by chemical cleavage to detect the point mutations. Fluorescent end labeling with strand‐specific fluorophores, electrophoretic separation of cleavage products in an automated Perkin‐Elmer ABI 373 or 377 DNA sequencer and analysis using the Perkin‐Elmer GENESCAN software expands sensitivity by highlighting weak signals through superimposition of strand‐specific fluorescence electropherograms for different samples.

Mice Triallelic for the Ig Heavy Chain Locus: Implications for VHDJH Recombination

VHDJH recombination has been extensively studied in mice carrying an Ig heavy chain rearranged transgene. In most models, inhibition of endogenous Ig rearrangement occurs, consistently with the feedback model of IgH recombination. Nonetheless, an incomplete IgH allelic exclusion is a recurrent observation in these animals. Furthermore, transgene expression in ontogeny is likely to start before somatic recombination, thus limiting the use of Ig-transgenic mice to access the dynamics of VHDJH recombination. As an alternative approach, we challenged the regulation of somatic recombination with the introduction of an extra IgH locus in germline configuration. This was achieved by reconstitution of RAG2−/− mice with fetal liver cells trisomic for chromosome 12 (Ts12). We found that all three alleles can recombine and that the ratio of Ig allotype-expressing B cells follows the allotypic ratio in trisomic cells. Although these cells are able to rearrange the three alleles, the levels of Ig phenotypic allelic exclusion are not altered when compared with euploid cells. Likewise, we find that most VDJ rearrangements of the silenced allele are unable to encode a functional μ-chain, indicating that the majority of these cells are also genetically excluded. These results provide additional support for the feedback model of allelic exclusion.

Fluorescence-assisted mismatch analysis (FAMA) for exhaustive screening of the α-galactosidase A gene and detection of carriers in Fabry disease

We used the fluorescence-assisted mismatch analysis (FAMA) method to screen rapidly the α-galactosidase A gene in patients with Fabry disease in order to identify unknown mutations and help define genotype-phenotype correlations in this X-linked lysosomal storage disorder. Chemical cleavage at mismatches on heteroduplex DNA end-labeled with strand-specific fluorescent dyes, reliably detects sequence changes in DNA fragments of up to 1.5 kb and locates them precisely. Exhaustive scanning of the α-galactosidase gene was accomplished on four polymerase chain reaction-generated amplicons, covering all seven exons, the exon-intron boundaries, and 700 bp of 5′-flanking sequence. Mutations were identified in each of the 15 patients studied from nine unrelated kindreds. Among the seven previously undescribed sequence changes, three are obviously pathogenic because they lead to premature protein termination. The other four, a splice-site mutation and three missense mutations, were the only changes found upon complete scanning of the gene and its promoter. In addition, FAMA also detects female heterozygous carriers more dependably than direct sequencing, and thus provides a valuable diagnostic test. In Fabry disease, this molecular criterion is especially important for genetic counseling since heterozygotes can be asymptomatic and their enzymatic values within the normal range.