Alessandro A. Castellani

Biochemical investigations of different forms of osteogenesis imperfecta. Evaluation of 44 cases.

Forty-four patients with Osteogenesis Imperfecta (O.I.) were divided into groups on the basis of clinical and genetic criteria and the alterations in collagen and glycosaminoglycans (GAG) in the subjects of each group were examined. The largest group of patients as affected with a mild form of O.I. and showed an increased ratio of type III to type I collagen in skin and an increase of the ratio of hydroxylysine diglycoside to monoglycoside in skin collagen. The group of patients affected with a severe nonlethal form of O.I. appeared to be heterogeneous both from a clinical and from a biochemical point of view. A marked increase of the diglycoside to monoglycoside ratio was observed in skin and urine, whereas the ratio of type III to type I collagen in skin was within the normal range or significantly decreased. Some of these patients also showed alterations involving proteoglycans, e.g. in urinary GAGs a decreased galactosamine to glucosamine ratio could be demonstrated. Similar and more marked alterations involving both collagen and GAG metabolism were observed in five children affected with a lethal form of O.I.

Structural characterization of two genetic variants of human serum albumin

In the present paper we report the structural characterization of two genetic mutants of human serum albumin: albumin Vanves, a very rare, electrophoretically fast variant of French origin, and albumin Verona, a slow-migrating variant which is the most frequently observed in Italy and which possesses the same electrophoretic mobility as albumin B. Both variants were isolated from the sera of healthy heterozygous subjects. Analysis of CNBr fragments by isoelectric focusing allowed us to localize the mutation to the COOH-terminal region of the molecule (residues 549-585) in both cases. The modified fragments were then isolated on a preparative scale by HPLC and subjected to tryptic digestion. Sequential analysis of the abnormal tryptic peptide, purified by HPLC, established the mutation responsible for albumin Vanves as 574 Lys----Asn and the molecular defect of albumin Verona as 570 Glu----Lys, both probably due to point mutations in the structural genes. The amino-acid substitutions found in albumins Verona and Vanves are consistent with the electrophoretic mobilities observed for the native proteins at pH 8.6.

The amino acid substitution in albumin Roma: 321 Glu → Lys

Albumin Roma is an electrophoretically slow moving genetic variant of human serum albumin found in 22 unrelated families. The protein was isolated from the serum of a healthy, heterozygous subject. Analysis of CNBr fragments by isoelectric focusing allowed us to localize the mutation to fragment CNBr IV (residues 299–329). This fragment was isolated on a preparative scale by RP‐HPLC and subjected to tryptic digestion. Sequential analysis of two abnormal tryptic peptides, purified by RP‐HPLC, revealed that the variant arises from the substitution of glutamic acid 321 by lysine. This amino acid replacement, probably resulting from a point mutation in the structural gene, causes a change in the net charge of +2 units which is in keeping with the decreased electrophoretic mobility of the native protein.

The molecular defect of albumin Tagliacozzo: 313 Lys+Asn

Albumin Tagliacozzo is a fast‐moving genetic variant of human serum albumin found in 19 unrelated families. The protein was isolated from the serum of a heterozygous healthy subject. Analysis of CNBr fragments by isoelectric focusing allowed us to localize the mutation to CNBr fragment IV (residues 299–329). This fragment was isolated on a preparative scale and subjected to tryptic digestion. Sequential analysis of the abnormal tryptic peptide, purified by RP‐HPLC, revealed the variant was caused by 313 Lys → Asn substitution, probably due to a point mutation in the structural gene. The lack of a lysine residue accounts for the electrophoretic behavior of albumin Tagliacozzo.

Type I procollagen in the severe non-lethal form of osteogenesis imperfecta.

SummaryWe have screened type I procollagen synthesized in vitro by skin fibroblasts from several patients with the severe non-lethal form of osteogenesis imperfecta. Cells from one patient synthesized and secreted both normal and a larger amount of abnormal type I procollagen. The abnormal alpha chains are larger in size due to post-translational overmodifications involving the whole triple helical domain. Abnormal collagen heterotrimers had a melting temperature 2.5°–3°C lower than normal ones or from controls. Chemical analysis of collagen in the medium showed a greater degree of both lysyl hydroxylation and hydroxylysyl glycosylation, the major increase in molecular mass of overmodified alpha chains being due to the higher hydroxylysine-bound hexose content. The probands cells modify proteoglycan metabolism and mineral probands cells modify proteoglycan metabolism and mineral crystals form in the dermis, possibly a response to abnormal collagen-proteoglycan interactions. These findings can be explained by a small defect in the product of one allele for pro-α1(I) chains: three-quarters of the synthesized type I procollagen molecules are composed of trimers containing one or two chains defective near the C-terminus of the triple helix or in the C-propeptide. The data obtained for this patient confirmed that the severity of clinical manifestations in osteogenesis imperfecta strongly depends on the location and nature of the mutations, and that the phenotype could be a consequence of a collagen defect(s) and its influence on collagen-collagen interactions and collagen interactions with other connective tissue components.

Collagen glycosylation in human granulation tissue and scar.

Some biochemical characteristics of collagen extracted from granulation tissue were studied and compared with those of normal skin and scar. By using electrophoretic techniques the type III collagen content was confirmed to be significantly greater in granulation tissue and lower in scar with respect to normal skin. The chromatographic determination of hydroxylysine (Hyl) glycosides in collagen extracted from granulation tissue showed a significant increase in both the degree of Hyl glycosylation and in the di-/monoglycoside ratio, while both parameters turned out to be lower in scar. These data suggest that the degree of Hyl glycosylation and the di-/monoglycoside ratio could represent an index of the degree of collagen fiber maturation.

Severe Nonlethal Osteogenesis Imperfecta: Biochemical Heterogeneity

Our studies on osteogenesis imperfecta (01) started in the middle 1970s as a consequence of the interest of our laboratory in connective tissue macromolecules and their disorders, such as mucopolysaccharidoses, lathyrism, and Larsen’s syndrome. Osteogenesis imperfecta is not a widespread disease in Italy (although quantitative data for its frequency has never been reported). It is mainly for this reason that family physicians and the great majority of clinical doctors are unaware of the disease (very few laboratories recently started their work at both the protein and nucleic-acid level). This situation recently began to improve when the Italian Association of affected families (As.It.O.1.) was founded, thanks to Professor L. Lenzi’s efforts, an orthopedic surgeon who had a great scientific and clinical interest in connective tissue diseases. Previous studies on the metabolism of collagen and proteoglycans showed alterations in several affected patients’-’ concerning differences in the relative amount of type I and I11 collagens in skin, and, more extensively, for the posttranslational modifications (hydroxylation of prolyl and lysyl residues and glycosylation of hydroxyl ysine). A step forward in the understanding of the molecular basis of the disease was made possible with the use of dermal fibroblast cultures that produce a large amount of type I and type 111 collagen, and, more importantly, that express the type I collagen defect in these in vitro conditions. While the majority of the patients whose collagen defects have been elucidated to date were affected with lethal 01, we had the opportunity to deal mainly with subjects affected with the severe nonlethal form of the disease. The knowledge of the pattern of inheritance for many of our patients is often