Psychiatric disorder in a familial 15;18 translocation and sublocalization of myelin basic protein of 18q22.3.

Two related patients with similar clinical features consisting of a few dysmorphic signs and psychiatric disturbance were reported to have a partial trisomy of chromosomes 15(pter-q13.3) and 18(q23-qter) deriving from a familial translocation t(15;18). One patient is affected by bipolar disorder and the other by schizoaffective disorder. Both cases have a predominantly affective course; nevertheless, a clear diagnosis is difficult in the first patient, who is 15 years of age, and only a longitudinal course will allow us to establish a definite diagnosis. The possibility that these two pathologies belong to a single category is discussed, and the presence of a susceptibility locus on chromosome 18 is hypothesized. Cytogenetic data, FISH, and DNA studies indicate that the myelin basic protein (MPB) gene is not involved in the translocation, and localize it centromeric to the breakpoint on chromosome 18(q22.3). Thus, it is unlikely to be involved in the disease.

Molecular defects in CRM+ factor VII deficiencies: modelling of missense mutations in the catalytic domain of FVII.

The molecular defects causing CRM+ factor VII deficiency were investigated in seven unrelated subjects and several members of their families. Four missense mutations located in the catalytic domain of factor VII were found. The previously reported 304Arg-->Gln substitution was present in the homozygous and heterozygous forms, with different polymorphic haplotypes, thus demonstrating that it is recurrent and frequent in the Italian population. The 310Cys-->Phe substitution was found in the homozygous form and in the compound heterozygous condition with the nonsense mutation 356Trp-->stop. Two missense mutations, 298Met-->Ile and 342Gly-->Arg, were found in the homozygous and in the heterozygous condition respectively. Molecular heterogeneity was further increased by finding of the 353Arg-->Gln polymorphism in the doubly heterozygous condition with the 304 and 342 mutations. Plausible explanations for loss of FVII function were found by inspecting a model of the serine protease domain of factor VIIa. Inefficient activation of the catalytic site is predicted for 298Met-->Ile. 342Gly-->Arg would directly distort the geometry of the 'oxyanion hole' preventing formation of a substrate enzyme intermediate. 310Cys-->Phe is predicted to have an adverse effect on tissue factor interaction. These mutations point to important regions of the factor VII molecule.

Molecular analysis of factor VII deficiency in Italy: a frequent mutation (FVII Lazio) in a repeated intronic region.

Molecular defects and polymorphic haplotypes of coagulation factor VII gene were studied in eight unrelated Italian subjects with factor VII deficiency, seven having the factor VII- variant, one the factor VIIR variant. An intron 7 mutation, which alters the consensus donor splice site sequence, was found in six subjects. The presence of the founder effect is suggested by their common geographical origin (a mountain area in the Lazio region) and by the identical polymorphic haplotype underlying the mutation. A different mutation, also located in the 5' monomer of the repeated intron 7 sequence, was found in the heterozygous condition in a subject from Northern Italy. New polymorphic alleles were detected in the repeated intron 7 region in subjects from Eastern Africa. Two missense mutations in codon 97 (Gly-->Cys, Gly-->Ser), the first found in the compound heterozygous condition with the frequent intron 7 mutation, suggest the presence of a hot spot mutation site in the second epidermal growth factor domain. Two neutral dimorphisms at codon 333Ser and 115His were detected, the last in linkage disequilibrium with the 353Arg/Gln polymorphism, and showing differences in frequency in the FVII deficient and control subjects.

Study of a protein S gene polymorphism at DNA and mRNA level in a family with symptomatic protein S deficiency.

A protein S gene polymorphism, detectable by restriction analysis of amplified exonic sequences, was investigated in a family with members affected by protein S deficiency, deep vein thrombosis and ictus. The clinical laboratory findings as well as RFLP analysis were consistent with the presence of a type WP III protein S deficiency clearly marked by a polymorphic allele, thus enabling us to determine the carrier status in several subjects. The RFLP analysis, extended to platelet mRNA after reverse transcription and amplification, demonstrated that the mRNA produced by the putative defective gene was present in a subject affected by thrombosis.

Symptomatic type II protein C deficiency caused by a missense mutation (Gly 381-->Ser) in the substrate-binding pocket.

A patient with recurrent deep vein thrombosis and heterozygous type II deficiency, characterized by reduced protein C activity in both amidolytic and clotting functional assays, was investigated by direct sequencing of PCR fragments derived from the coding portion of the protein C gene. AG (8856) to A transition was noted in the patient which was not present in healthy controls. This mutation is predicted to cause the substitution of Ser for Gly 381, an evolutionari'y conserved residue in the substrate binding pocket of serine-proteases (Gly 216, chymotrypsin numbering). A computer model of the structure of the serine-protease domain indicates that the properties of the altered protein C molecule can be explained on the basis of steric hindrance between the substituted serine and the substrate arginine side chains.

In-frame deletion of von Willebrand factor A domains in a dominant type of von Willebrand disease.

von Willebrand disease (vWD), the most common inherited bleeding disorder in humans, is very heterogeneous and has been classified into several subtypes. Missense mutations have been found to be responsible for the dominant type II vWD, characterized by qualitative abnormalities affecting von Willebrand factor (vWF) function. The breakpoints of a heterozygous vWF gene deletion (31 Kb), occurring 'de novo' in a patient with a variant of type II vWD, were localized to introns 25 and 34 and sequenced. An Alu repeat in intron 25 was interrupted between the transcriptional boxes A and B. The new junction present in the abnormal von Willebrand factor mRNA was sequenced after reverse transcription of platelet RNA. The codon 1104 (Cys) is followed in frame by the mutated codon 1926 (Cys to Arg), thus removing the complete A domains, found in a wide variety of genes and characterized by independent assembly 'in vitro'. We propose that the abnormal vWF, which carries intact protein domains responsible for vWF dimer and multimer formation, makes ineffective interactions with the normal molecules in the biosynthetic process, causing the dominant type II phenotype through a novel mechanism.

Characterization of a deleted Y chromosome in a male with Turner stigmata.

A 46,X,+mar karyotype was detected in an 11-year-old male with a clinical picture characterized by obesity, short stature, bilateral cryptorchidism and coarctation of the aorta. The presence of ZFY and SRY genes was demonstrated by PCR amplification, and the origin of the marker chromosome from a deleted Y chromosome was analyzed by in situ hybridization. The proximal limits of a deletion in Yq were defined by the absence of Southern blot hybridization signals upon probing with Yq11 markers. Cytogenetics and molecular methods taken together indicate a deletion in q11.21. In addition, the loss of Yp subtelomeric sequences was suggested by the analysis of Southern blots hybridized with a 29A24 (DXYS14) probe and by the presence of coarctation of the aorta tentatively localized in Yp. The karyotype of the patient was suggested to be: 46,X,del (Y) (p11.3-q11.21).

A polymorphism in the 5' region of coagulation factor VII gene (F7) caused by an inserted decanucleotide.

We describe a polymorphism in the 5' region of the coagulation factor VII (FVII) gene, originating from a decanucleotide (CCTATATCCT) insert present in the less frequent allele. This marker can be detected by restriction analysis of polymerase chain reaction products.

Characterization and mapping of the 5' portion of von Willebrand factor pseudogene.

A genomic fragment containing the 5' boundary of the von Willebrand factor pseudogene was cloned, partially sequenced and used for in situ hybridization experiments on metaphase spreads from a Philadelphia chromosome (Ph1)-positive chronic myelogenous leukemia patient. Data obtained indicate that the von Willebrand factor pseudogenic region is centromeric to the breakpoint cluster region on 22q11.2. This probe could be used for the study of deletions in the DiGeorge syndrome.

Localization of cloned human DNA sequences and analysis of chromosomal alterations by in situ hybridization.

The in situ hybridization technique was used for the localization on human chromosomes of single-copy and repeated sequences and, in addition, for the characterization of altered human chromosomes. Two anonymous clones, single or low-copy, obtained from a human X chromosome library were localized on the distal part of the long arm and in the paracentromeric region of X chromosome, respectively. A genomic fragment of the single-copy thyroglobulin (TG) gene was used to confirm the localization on the distal part of the long arm of chromosome 8. The localization and distribution on human chromosomes of the glyceraldehyde-3-phosphate dehydrogenase (GAPD) multigene family obtained by in situ hybridization and by somatic cell hybrids were compared. A phosphoglycerate kinase (PGK) c-DNA clone, which detects genic and pseudogenic sequences on the X chromosome, was used for the characterization of three small ring markers present in unrelated female patients.

Detection of two missense mutations and characterization of a repeat polymorphism in the factor VII gene (F7).

The 3' portion of the coagulation factor VII gene, containing the activation and serine protease domains, was investigated in four subjects with factor VII deficiency by temperature gradient gel electrophoresis and sequencing of polymerase chain reaction (PCR) products. Molecules displaying an altered melting behaviour were detected in three subjects, and direct sequencing showed two mutations. A G-to-T transversion causing a missense mutation, Cys-310 to Phe, suppresses a disulphide bond conserved in the catalytic domain of all serine proteases. This mutation, which in the homozygous form causes a severe reduction in protease activity (4%), was found in two patients from different Italian regions. A G-to-A transition, which gives rise to a missense mutation, Arg-304 to Gln, and is associated with the factor VII padua variant, was found in the heterozygous form in a subject also affected by von Willebrand disease. Two polymorphic alleles, which differ in one repeat monomer element, were precisely mapped in a region spanning the exon-intron 7 border of the factor VII gene and studied in families with factor VII or X deficiency.

Rapid detection of a protein C gene mutation present in the asymptomatic and not in the thrombosis-prone lineage.

The presence of mutations in the serine protease domain of protein C was investigated by temperature gradient gel electrophoresis of PCR products in five patients with protein C deficiency and thrombosis. Molecules with an altered melting behaviour were detected in one subject with a history of venous and arterial thrombosis. Direct sequencing showed that a G deletion, present in the heterozygous state, caused a reading frame shift at Trp 300 and subsequently a premature termination at the codon 335. The resulting suppression of the protein C catalytic function explains the reduction of protease activity to half. In addition the mutation caused a reduction of the antigen level in plasma. Temperature gradient gel electrophoresis enabled the rapid detection of the gene alteration in the family of the propositus. Several members of the paternal lineage had had severe thrombotic episodes. Unexpectedly the mutation was found to be inherited from the clinically asymptomatic maternal lineage, thus suggesting that an additional unknown defect from the paternal lineage is present in the thrombosis-prone propositus.

Chromosomal localization of human p85 alpha, a subunit of phosphatidylinositol 3-kinase, and its homologue p85 beta.

The human phosphatidylinositol (PI) 3-kinase p85 alpha subunit gene and its homologue p85 beta were assigned to human chromosomes by analysis of their segregation in a panel of somatic cell hybrids using human-specific polymerase chain reaction primers. The p85 alpha locus was only present in hybrids retaining the human chromosome 5q. The presence of the p85 beta locus coincided with the presence of chromosome 19. The precise chromosomal sublocalization of these two genes was then determined by in situ hybridization. We confirmed the localization of the p85 alpha gene at 5q12-q13, as recently described (Cannizzaro, L.A., Skolnik, E.Y., Margolis, B., Croce, C.M., Schlesinger, J. & Huebner, K. (1991). Cancer Res., 51, 3818-3820) and positioned the p85 beta locus at 19q13.2-q13.4.

Characterization of the pseudogenic and genic homologous regions of von Willebrand factor.

The homologous pseudogenic and genic regions of von Willebrand factor (vWF) were studied in DNA from a patient with homozygous deletion of vWF genes and compared with a normal control. This analysis indicates informative restriction patterns for the investigation of restriction fragment length polymorphisms (RFLPs) and gene lesions, and for molecular cloning. A useful new genic XbaI RFLP was found and characterized. A large BgIII fragment of the pseudogenic region was cloned and mapped, and single sequences (9 kb) were used as probes. Corresponding genic and pseudogenic fragments, which contain exons 23-28, and specific restriction patterns were identified, including a new polymorphic TaqI site that was mapped in the gene. A cloned fragment contains the 5' boundary of the pseudogene and recognizes an additional and unknown homologous sequence in the genome. The chromosomal localization of the vWF pseudogene and of the breakpoint cluster region (BCR) gene were compared by 'in situ' hybridization: overlapping patterns were detected. The cloning, characterization and mapping of the pseudogenic region improves the analysis of this portion of chromosome 22 affected by several somatic and constitutional alterations, and also of the corresponding genic region on chromosome 12.

Characterization of polymorphic markers in the von Willebrand factor gene and pseudogene.

Three TaqI restriction fragment length polymorphisms (RFLP) detected by the central portion of von Willebrand factor cDNA, which recognizes the true gene and in addition pseudogenic sequences, were characterized and mapped. Small cDNA fragments which hybridized with DNA from families with von Willebrand disease were used. Two of the RFLP, recognized by 1.7 and 0.45 kb cDNA fragments, are not in linkage either with von Willebrand disease or with RFLP located in the von Willebrand factor (vWF) gene, which indicates their pseudogenic location. These markers located in 22q11, near to the bcr gene, provide new tools for the study of several somatic and constitutional alterations affecting this chromosomal region. The third RFLP is recognized by a cDNA fragment corresponding to the N-terminal portion of mature vWF and is localized in the true gene. Since significant linkage disequilibrium with other informative RFLP is not present, this marker contributes to the definition of family haplotypes associated with von Willebrand disease.

A de novo and heterozygous gene deletion causing a variant of von Willebrand disease.

An abnormal von Willebrand factor (vWF) gene restriction pattern has been found in a patient with von Willebrand disease. Because this gene alteration is not present in his parents or in 50 normal and 25 affected subjects, and the restriction fragment length polymorphism haplotypes are inherited normally in the patient's family, we suggest that a de novo mutation is present. Bands with reduced intensity and additional fragments, observed in several restriction digests, hybridize with noncontiguous copy DNA (cDNA) portions, thus indicating the presence of a heterozygous gene deletion. The deletion removes a genomic region containing at least codons 1147 through 1854 and corresponding to the D3-A3 homologous protein domains. The extent of the vWF pseudogene on chromosome 22 is roughly similar to that of the deleted area. However, the pseudogenic nature of the deletion is excluded by the mapping of bands with reduced intensity in the patient to the true vWF gene. The vWF antigen levels are one fourth of normal and ristocetin cofactor activity is severely impaired. The reduction of high molecular weight multimers in plasma and platelets and the altered triplet morphology are compatible with the presence of a dominant variant of type II von Willebrand disease.

Direct detection of a missense mutation causing severe hemophilia A by PCR amplification and fluorescence scanning.

The amplification of Factor VIII gene-specific sequences, obtained by polymerase chain reaction, was used for hemophilia A carrier detection. Exon 24 sequences were employed in the carrier status determination of a missense mutation causing severe hemophilia A in two unrelated patients. After agarose gel electrophoresis, the digested DNA was subjected to quantitative determination of fluorescence. This technique significantly improves the digest analysis.

Sublocalization of von Willebrand factor pseudogene to 22q11.22-q11.23 by in situ hybridization in a 46,X,t(X;22)(pter;q11.21) translocation.

The von Willebrand factor pseudogene, previously mapped to chromosome 22, was sublocalized by in situ hybridization using as probe a von Willebrand factor cDNA fragment completely contained in the pseudogenic region. Chromosome spreads were from a patient carrying a unique balanced de novo translocation 46,X,t(X;22)(pter;q11.21). Silver grain analysis indicated that the human von Willebrand factor pseudogene is located on 22q,11,22-q11,23, a region relevant for several somatic and constitutional chromosomal alterations.