Clavulanic acid sensitization seems more involved in cutaneous than systemic reactions in amoxicilline-clavulanate drug reactions.

Summary: BLs allergy is considered a major health issue, as BLs are the most frequently involved in drug allergic reactions. Amoxicillin (AX) is the most frequently involved drug in sensitization among all BLs. AX is commercialized alone or combined to clavulanic acid (CLA) in order to increase the antibiotic spectrum. The growing prescriptions of AX-CLA formulations contributed to increase the role of CLA as an allergy inducer. At present, little is known about the clinical characteristics of hypersensitivity reactions to clavulanate. The aim of this study was to assess the difference in the prevalence of cutaneous vs systemic reactions in patients who had a documented history of allergic reactions to amoxicillin-clavulanate and tested positive for clavulanate or penicillin/amoxicillin. Our study suggests that patients who presented only muco-cutaneous reactions were more often sensitized to CLA rather than AX.

Acetylsalicylic acid (ASA) desensitization in an allergic pregnant woman post-vascular scaffolds implantation.

Summary: The use of acetylsalicylic acid (ASA) desensitization for patients with coronary artery disease (CAD) is growing, but no data are available on desensitization protocol in patients with ASA sensitivity and CAD during pregnancy. This case report shows that ASA desensitization protocol during pregnancy could be safe and effective in a tertiary centre with a multidisciplinary team.

RNA-binding proteins RBM20 and PTBP1 regulate the alternative splicing of FHOD3.

Regulation of alternative splicing events is an essential step required for the expression of functional cytoskeleton and sarcomere proteins in cardiomyocytes. About 3% of idiopathic dilated cardiomyopathy cases present mutations in the RNA binding protein RBM20, a tissue specific regulator of alternative splicing. Transcripts expressed preferentially in skeletal and cardiac muscle, including TTN, CAMK2D, LDB3, LMO7, PDLIM3, RTN4, and RYR2, are RBM20-dependent splice variants. In the present study, we investigated the RBM20 involvement in post-transcriptional regulation of splicing variants expressed by Formin homology 2 domain containing 3 (FHOD3) gene. FHOD3 is a sarcomeric protein highly expressed in the cardiac tissue and required for the assembly of the contractile apparatus. Recently, FHOD3 mutations have been found associated with heart diseases. We identified novel FHOD3 splicing variants differentially expressed in human tissues and provided evidences that FHOD3 transcripts are specific RBM20 and PTBP1 targets. Furthermore, we demonstrated that the expression of RBM20 and PTBP1 promoted the alternative shift, from inclusion to exclusion, of selected FHOD3 exons. These results indicate that RBM20 and PTBP1 play a role in the actin filament functional organization mediated by FHOD3 isoforms and suggest their possible involvement in heart diseases.

A novel mutation in ABCD1 unveils different clinical phenotypes in a family with adrenoleukodystrophy.

X-linked adrenoleukodystrophy (X-ALD) is the most common peroxisomal disorder. The disease is the consequence of mutations in the ABCD1 gene that encodes the peroxisomal membrane protein ALDP which is involved in the transmembrane transport of very long-chain fatty acids. We describe a family with six members carrying a novel heterozygous mutation IVS4+2T>A (c.1393+2T>A) of the ABCD1 gene, highlighting the wide range of phenotypic manifestations of ALD and the importance of genetic screening before any pregnancy in asymptomatic women whose carrier status is unknown.

Morfometria de frutos e sementes e germinação de carobinha (Jacaranda decurrens subsp. symmetrifoliolata Farias & Proença), após o armazenamento / Fruit and seed morphometry and germination of "carobinha" (Jacaranda decurrens subsp. symmetrifoliolata) seeds after storage

O trabalho teve como objetivos avaliar a morfometria dos frutos e sementes de carobinha (Jacaranda decurrens subsp. symmetrifoliolata) e o efeito de temperaturas na germinação de sementes armazenadas em ambiente sem controle de temperatura e umidade relativa. O estudo foi realizado no Laboratório de Sementes da UFGD, Dourados, MS. Em amostra de 120 frutos e de 550 sementes foi determinado: o comprimento, a largura, a espessura, o número de sementes por fruto, e a massa dos frutos. A germinação foi estudada após três períodos de armazenamento das sementes (120, 480 e 720 dias) a temperatura ambiente e sob quatro temperaturas de germinação: três constantes (18, 25 e 30ºC) com luz contínua e sob condição de tempera alternada, 20-30ºC, com fotoperíodo de 12 horas. O delineamento experimental foi inteiramente ao acaso, em arranjo fatorial 3 x 4, com quatro repetições. As variações nas medidas de frutos e sementes representam indício da alta variabilidade genética populacional. As sementes armazenadas por 120 dias e submetidas à germinação na temperatura de 25ºC resultaram em 69,4% de germinação e índice de velocidade de germinação - IVG, de 0,983. Sob temperaturas de germinação de 30ºC, constante, e de 20-30ºC, alternada, obtiveram-se 63,9% e 63,8% de germinação e 0,486 e 0,413 de IVG, respectivamente. Sob temperatura de 18ºC a germinação foi lenta e menor, 27,7% e IVG de 0,119. As sementes armazenadas por 480 e 720 dias não germinaram. Com base no comportamento fisiológico das sementes é possível classificar Jacaranda decurrens subsp. symmetrifoliolata como espécie intermediária (entre ortodoxa e recaucitrante).

This study aimed to evaluate the morphometry of fruits and seeds of Jacaranda decurrens subsp. symmetrifoliolata ("carobinha") and the effect of temperature on the germination of seeds that were stored in environment without controlled temperature and relative humidity. The study was carried out at Seed Laboratory of Federal University of Grande Dourados, Mato Grosso do Sul, Brazil. For a sample of 120 fruits and 550 seeds, the following variables were determined: length, width and thickness, besides the number of seeds per fruit and fruit biomass. Germination was studied after three seed storage periods (120, 480 and 720 days) at environment temperature and four germination temperatures: three constant temperatures (18, 25 and 30ºC) with continuous light, and one alternating temperature, 20-30ºC, with 12-hour photoperiod. Experimental design was completely randomized, in 3 x 4 factorial schemes, with four replicates. The variations in the measures of fruits and seeds evidence a high genetic variability in the population. The seeds stored for 120 days and subjected to germination test at 25ºC resulted in 69.4% germination and germination velocity index - GVI of 0.983. At 30ºC, constant temperature, and 20-30ºC, alternating temperature, germination was 63.9% and 63.8% while GVI was 0.486 and 0.413, respectively. At 18ºC, germination was slow and lower, 27.7%, and GVI was 0.119. Seeds stored for 480 and 720 days did not germinate. Based on the physiological behavior of seeds, it is possible to classify Jacaranda decurrens subsp. symmetrifoliolata as an intermediate species (between orthodox and recalcitrant).

Desenvolvimento e produção da carobinha (Jacaranda decurrens Cham. subsp. symmetrifoliolata Farias & Proença) cultivada sob dois arranjos de plantas, com ou sem cobertura de cama-de-frango no solo / Development and yield of carobinha (Jacaranda decurrens Cham. subsp. symmetrifoliolata Farias & Proença) cultivated under two plant arrangements, with or without chicken manure covering on the soil

O objetivo deste trabalho foi avaliar o desenvolvimento e a produção da carobinha (Jacaranda decurrens subsp. symmetrifoliolata) cultivada ex situ sob dois arranjos de plantas, sem ou com cama-de-frango semidecomposta. O trabalho foi desenvolvido sob condições de campo, no Horto de Plantas Medicinais (HPM), da Universidade Federal da Grande Dourados (UFGD) em solo tipo Latossolo Vermelho distroférrico. Estudaram-se os arranjos em fileiras simples e duplas e o uso ou não de cama-de-frango de corte semidecomposta em cobertura, dispostos como fatorial 2 x 2, no delineamento experimental de blocos casualizados, com seis repetições. As maiores alturas máximas das plantas (61,3 cm planta-1) aos 555 dias após o transplante foram daquelas cultivadas sob fileira dupla com cama-de-frango. Os números médios de folhas e de ramificações por planta ao longo do ciclo foram de onze e duas, respectivamente. O diâmetro médio do caule de plantas cultivadas em fileiras simples foi de 8,3 mm, enquanto sob fileiras duplas, foi de 10,8 mm, ambos aos 552 dias de ciclo. As características avaliadas, na colheita, aos 555 dias após o transplante não foram influenciadas pela interação entre arranjos de plantas e o uso da cama-de-frango nem pelos fatores isoladamente. Dessa forma, concluiu-se que o cultivo em fileiras duplas foi favorável à produtividade da carobinha, por possibilitar maior produção por área.

The aim of this study was to evaluate the development and yield of "carobinha" (Jacaranda decurrens subsp. symmetrifoliolata) cultivated ex situ under two plant arrangements, with or without semi-decomposed chicken manure. This study was carried out under field conditions at the Medicinal Plant Garden (HPM), Federal University of Grande Dourados (UFGD), in Distroferric Red Latossol. Arrangements with simple and double rows and the use or not of semi-decomposed chicken manure as covering were studied as 2 x 2 factorial arrangement in a randomized block experimental design, with six replicates. The highest maximum heights (61.3 cm plant-1) at 555 days after transplant were obtained by plants cultivated under double rows with chicken manure. The mean numbers of leaves and branches per plant during the cycle were eleven and two, respectively. The mean stem diameter of plants that were cultivated in simple rows was 8.3 mm, while that of plants cultivated in double rows was 10.8 mm, both at 552 days of the cycle. The evaluated characteristics, in the harvest at 555 days after transplant, were not influenced by the interaction between plant arrangements and use of chicken manure or by factors in an isolated way. Thus, cultivation in double rows was favorable to "carobinha" yield for allowing the highest yield per area.

Osteoporosis in beta-thalassemia: Clinical and genetic aspects.

Osteoporosis and osteopenia are frequent complications of thalassemia major (TM) and intermedia (TI). Osteoporosis was found in 23/25 patients with TI and in 115/239 patients with TM. In TM, no association was found with specific polymorphisms in candidate genes (vitamin D receptor, estrogen receptor, calcitonin receptor, and collagen type 1 alpha 1). Osteoporosis in female patients with TM was strongly associated with primary amenorrhea (P < .0001), while in male patients with TM, hypogonadism was not significantly related to bone mineral density (BMD) (P = .0001). Low BMD was also associated with cardiomiopathy (P = .01), diabetes mellitus (P = .0001), chronic hepatitis (P = .0029), and increased ALT (P = .01).

Rapid and efficient genotype analysis of the COL1a1 Sp1 binding site dimorphism, a genetic marker for bone mineral density.

A biallelic G/T polymorphism within the first intron of COL1A1 gene at a recognition site for the transcription factor Sp1 has been shown to be significantly related to bone mass and osteoporotic fracture. To date this polymorphism has been detected by conventional genomic DNA amplification followed by restriction enzyme digestion and polyacrylamide gel electrophoresis. We have designed a rapid and efficient genotyping method based on allele-specific polymerase chain reaction

Relationship among VDR (BsmI and FokI), COLIA1, and CTR polymorphisms with bone mass, bone turnover markers, and sex hormones in men.

Osteoporosis is a disease characterized by low bone mineral density (BMD) and up to 80% of its variance is under genetic control. Although osteoporosis is more frequent in women, one-third of hip fractures also occur in men. Much information on genetic factors and bone density has been obtained in women, but only a few studies have been performed in osteoporotic men. We have evaluated the relationship between polymorphisms for several candidate genes such as vitamin D receptor (VDR), collagen type Ia1 (COLIA1), and calcitonin receptor (CTR) in a sample of unrelated Italian men (n = 253, mean age 58.41 +/- 15.64 SD). We found no significant differences in BMD when subjects were stratified for their VDR (BsmI and FokI) and COLIA1 genotypes. BMD both at the lumbar spine and at the femoral neck were associated with polymorphism of CTR gene. The CC genotype of CTR gene had the lowest BMD value (P <0.05 and P <0.01 at the spine and hip, respectively) and its prevalence was significantly over-represented in the subgroup of men with prior hip or vertebral fracture as compared with controls (P = 0.004% c2 = 11.10). The men with the CC genotype also showed significantly lower body mass index (BMI), serum sex hormone binding globulin (SHBG), estradiol, total alkaline phosphatase-(total AP) and bone alkaline phosphatase (bone AP) levels and significantly higher free androgen index (FAI). In conclusion, the polymorphism of CTR gene but not VDR and COLIA1 is associated with osteoporosis incidence and the levels of alkaline phosphatase and estradiol. The lower BMD in CC genotype is apparently associated in males with depressed bone formation and lower estradiol levels.

Stickler syndrome and vitreoretinal degeneration: correlation between locus mutation and vitreous phenotype. Apropos of a case.

BACKGROUND: Autosomal dominant vitreoretinopathies are characterized by genetic heterogeneity. Structural mutations in COL2A1 are the most frequent cause of Stickler syndrome with ocular involvement. The affected patients have a characteristic vitreous alteration, so-called membranous vitreous, or type 1 vitreous phenotype. Recently a novel mutation in the gene encoding the alpha 1 chain of type XI collagen (COL11A1) was reported in rare Stickler pedigrees, with a different, so-called beaded or type 2 vitreous phenotype. METHODS: Five patients of an Italian family affected by high myopia, high frequency of retinal detachment, and other systemic stigmata evocative of Stickler syndrome (flat midface, depressed nasal bridge, short nose, spondyloepiphyseal dysplasia and osteoarthritis) were studied. Genetic investigations were also performed, considering three candidate loci for Stickler syndrome and Wagner syndrome (COL2A1, COL11A1, WGN1). RESULTS: Segregation analysis was performed utilizing polymorphic markers. COL2A1 and WGN1 segregations were excluded; COL11A1 showed concordance with the disease. The vitreous phenotype of the family was a typical type 1 or "membranous" vitreous, although all the previously reported COL11A1-related Stickler syndromes had always shown the type 2 or "beaded" vitreous phenotype. CONCLUSIONS: The clear presence of the type 1 or "membranous" vitreous phenotype in our family, despite the probable mutation in the COL11A1 gene, suggests greater phenotypical heterogeneity and a more extensive mutation spectrum, even of the COL11A1 gene, than previously thought, explaining the basis for the different vitreous phenotypes seen in Stickler syndrome.

Autosomal dominant benign recurrent intrahepatic cholestasis (BRIC) unlinked to 18q21 and 2q24.

Benign recurrent intrahepatic cholestasis (BRIC) is an autosomal recessive liver disease characterized by multiple episodes of cholestasis without progression to chronic liver disease. On the basis of recent evidence of locus heterogeneity, we studied 19 subjects (7 affected members) of a BRIC family. Male-to-male transmission and the presence of affected females suggested autosomal dominant inheritance. Blood samples were collected after informed consent. Subjects were genotyped by using markers mapping to 18q and 2q24 region, respectively, where the genes FIC1 and FIC2 have been mapped. Segregation of haplotypes excluded the two regions in our family. These findings suggest further genetic heterogeneity of the origin of BRIC.

Transduced fibroblasts and metachromatic leukodystrophy lymphocytes transfer arylsulfatase A to myelinating glia and deficient cells in vitro.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease, caused by deficiency of arylsulfatase A (ASA), that manifests primarily in the white matter of the nervous system. Currently, no specific treatment exists that will reverse its fatal outcome. Replacement therapy has been hampered by the blood-brain barrier (BBB). To circumvent this problem we designed an ex vivo gene therapy strategy that includes the retrovirus-mediated ASA transduction of cells, such as activated lymphocytes, that are able to traverse the BBB or other membranes of the CNS. For this purpose, two recombinant retroviruses based on the pLXSN vector were produced, containing the wild-type ASA cDNA or a pseudodeficiency ASA cDNA, which encodes a smaller enzyme with normal activity. After transduction, ASA activity increased more than 100-fold in fibroblasts from an MLD patient. Furthermore, ASA-transduced MLD PBLs expressed 30 times higher ASA activity when compared with control PBLs. Moreover, cell culture experiments demonstrated that transduced fibroblasts could efficiently transfer ASA to deficient cells across a transwell barrier, whereas transduced MLD lymphocytes could transfer ASA to deficient fibroblasts only by direct cell-to-cell contact. Finally, ASA was taken up by normal oligodendrocytes and Schwann cells, the target myelinating glial cells for therapy in MLD. These data suggest possible short-term strategies for transfer of ASA into the CNS via transduced autologous cells while long-term strategies, related to autologous transduced bone marrow transplant, take effect in patients.

A common beta hexosaminidase gene mutation in adult Sandhoff disease patients.

beta-Hexosaminidase gene mutations were analyzed in two adult-onset Sandhoff disease Italian patients by PCR analysis of a common known mutation (delta 5') and by heteroduplex analysis of genomic and RT-PCR DNA fragments, covering the whole gene. The patients' genotypes were delta 5'/C1214%, and G890A/C1214T, respectively. As mutation C1214T (Pro405Leu) is also present in the other two late-onset cases so far described, we suggest that C1214T is a common mutation in this type of Sandhoff disease. Mutation G890A (Cys297Tyr) is a novel mutation which presumably causes altered processing of the pro beta chain.

Determination of a new collagen type I alpha 2 gene point mutation which causes a Gly640 Cys substitution in osteogenesis imperfecta and prenatal diagnosis by DNA hybridisation.

The molecular defect responsible for a sporadic case of extremely severe (type II/III) osteogenesis imperfecta was investigated. The mutation site was localised in the collagen type I pro alpha 2 mRNA molecules produced by the proband's skin fibroblasts by chemical cleavage of mismatch in heteroduplex nucleic acids. Reverse transcription-polymerase chain reaction DNA amplification, followed by cloning and sequencing, showed heterozygosity for a G to T transversion in the first nucleotide of exon 37 of the COL1A2 gene, which led to a cysteine for glycine substitution at position 640 of the triple helical domain. This newly characterised mutation is localised in a domain which contains several milder mutations, confirming that glycine substitutions within the alpha 2(I) chain do not follow a linear gradient pattern for genotype to phenotype correlations. In a subsequent pregnancy, absence of the G2327T mutation in the fetus was shown by allele specific oligonucleotide hybridisation to the trophoblast derived fibroblast mRNA after reverse transcription and in vitro amplification. (The nucleotide number assigned to the mutant base was inferred from the numbering system devised by the Osteogenesis Imperfecta Analysis Consortium (The OIAC Newsletter, 1 April 1994).)

A base substitution at IVS-19 3'-end splice junction causes exon 20 skipping in pro alpha 2(I) collagen mRNA and produces mild osteogenesis imperfecta.

Molecular investigations on a young patient and her family were undertaken to identify the molecular defect responsible for a mild form of osteogenesis imperfecta (OI) with blue sclerae, dentinogenesis imperfecta and joint laxity. Analysis of collagenous proteins from the proband's fibroblasts showed the presence of two populations of alpha 2(I) chains, one normal and one migrating faster on SDS gels, thereby suggesting deletion of amino acid sequences. The faster migrating chains were retained mainly in the cell layer and not found in the extracellular matrix deposited by cultured fibroblasts. Chemical cleavage of mismatch (CCM) analysis on the patient's pro alpha 2(I) mRNA: normal cDNA heteroduplexes localized the molecular defect. cDNA sequencing revealed a deletion of exon 20 (54 bp) in about half of the molecules. Genomic DNA sequencing revealed heterozygosity for a G-to-C transversion of the last nucleotide of intron 19, which changed the 3' consensus splicing site. As a consequence pro alpha 2(I)mRNA was abnormally spliced from the last codon of exon 19 to the first codon of exon 21. To our knowledge, this is the first acceptor site mutation so far described in an OI patient. Restriction analysis indicated that the mutation was present also in three other affected family members. The full sequence of COL1A2 introns 19 and 20 are reported.

Substitution of an aspartic acid for glycine 700 in the alpha 2(I) chain of type I collagen in a recurrent lethal type II osteogenesis imperfecta dramatically affects the mineralization of bone.

We describe a new dominant mutation of type I collagen responsible for a recurrent lethal osteogenesis imperfecta. Dermal cultured fibroblasts of the proband produced both normal and overmodified type I collagen chains. Previous results (Cohen-Solal, L., Bonaventure, J., and Maroteaux, P. (1991) Hum. Genet. 87, 297-301) and cyanogen bromide peptide mapping after non-equilibrium pH gradient gel electrophoresis indicated that the anomaly was a charge mutation localized in the alpha 2CB3-5A. The mutation was identified as a G to A transition in the COL1A2 gene, which converts glycine 700 to aspartic acid in the alpha 2I chain. This mutation caused the abolition of a ScrFI site, which was also absent in the suspected mosaic father. Pulse-chase experiment showed intracellular retention and increase of the degradation of the synthesized collagen. To understand more directly the tissue defect in osteogenesis imperfecta, skin and especially bone were studied with biochemical and transmission electron microscopy techniques. Collagen matrix of both tissues was dramatically decreased and presented a retarded migration, showing that abnormal molecules were incorporated during the fibrillogenesis. The abnormal collagen mostly remained within the fibroblasts and osteoblasts, which presented typical features of intracellular retention. We observed the presence of spheritic aggregates of mineral, unrelated to the scarce and thin collagen fibrils, in bone. Such abnormal mineralization could be the consequence not only of the decrease of the collagen content but more importantly of the inability of the abnormal molecules to form an organized network necessary to the deposition of apatite crystallites.

Osteogenesis imperfecta and type-I collagen mutations. A lethal variant caused by a Gly910-->Ala substitution in the alpha 1 (I) chain.

In this study we describe a new dominant point mutation in COL1A1 causing a lethal form of Osteogenesis imperfecta (type II B). Dermal cultured fibroblasts from the proband were shown to produce both normal and heavily overmodified type-I collagen. The mutation introduced a local conformational perturbation, which causes abnormal exposure of arginine residues; the triple helical domain was susceptible to trypsin digestion even at 30 degrees C. The chains bearing the point mutation were poorly secreted and short-term pulse experiments showed that the extensive intracellular retention of mutant trimers also impaired the secretion of normal chains. The molecular defect was localized in a COL1A1 allele by cloning and sequencing a cDNA region corresponding to the CB6 peptide. A G to C transversion which causes the substitution in the triple helical region of Gly910 with alanine was found. The mutation also causes the disappearance of a MspI-recognition site at nucleotide 3263 of the pro alpha 1 (I) coding sequence. Restriction analysis, along with the biochemical screening of collagens, allowed us to perform prenatal diagnosis on cells from chorionic-villus sampling and to exclude the recurrence of the mutation in the sibling.

Haplotype analysis of collagen type I genes in the general population and in osteogenesis imperfecta families.

The allele frequencies of 2 new polymorphic markers of collagen type I proalpha 1 (COL1A1) and proalpha 2 (COL1A2) genes were determined in a random sample of chromosomes by polymerase chain reaction. The minor allele frequencies were 0.27 for COL1A1/+88Mn1I, and 0.39 for COL1A2/1446 PvuII RFLPs, respectively. These 2 polymorphisms increased the combined (PIC) values we previously determined in the Italian population with Southern blotting procedures, from 0.71 at the COL1A1 locus to 0.81, and from 0.73 at the COL1A2 locus to 0.88, respectively. With a combination of these markers, we have carried out the segregation analysis of 4 new families in which osteogenesis imperfecta (OI) segregated as a dominant trait. The disease segregated with COL1A1 in 2 OI type I families, and with COL1A2 in one OI type IV family. In one OI type I family the concordant locus was uncertain. This analysis was extended to the 7 dominant OI families we previously reported: in 3 out of 11 pedigrees either locus still could not be excluded, indicating the need for more genetic markers. COL1A1 and COL1A2 haplotype frequencies were compared in normal and OI chromosomes: no preferential association of the disease with a given haplotype was detected. The correlation between affected locus and clinical aspects is discussed.

Mild dominant osteogenesis imperfecta with intrafamilial variability: the cause is a serine for glycine alpha 1(I) 901 substitution in a type-I collagen gene.

The molecular defect responsible for a case of mild osteogenesis imperfecta (OI) with repeated femoral fractures was investigated. The proband and his mother, who presented minor OI signs but no bone fractures, were shown to produce normal and abnormal type-I procollagen molecules in their dermal fibroblasts. The molecular defect was localized in about half of the proband's pro alpha 1(I) mRNA molecules by chemical cleavage with piperidine of hydroxylamine-reacted mRNA:cDNA heteroduplexes. The corresponding region was reverse-transcribed and amplified by polymerase chain reaction (PCR). Cloning and sequencing of the amplified products revealed in both subjects a G-to-A transition in the first base of codon 901 of the alpha 1(I) triple helical domain, which led to a serine for glycine substitution. Allele-specific oligonucleotide hybridization to amplified genomic DNA from fibroblasts and leukocytes confirmed the heterozygous nature of both patients and proved the absence of mosaicism. The presence of the mutation was excluded in other healthy family members, who were reported to have bluish selerae. The mild phenotypic outcome of this newly characterized mutation contradicts previous findings on glycine substitutions in the C-terminal region of collagen triple helix, most of which caused lethal OI.

A de novo G to T transversion in a pro-alpha 1 (I) collagen gene for a moderate case of osteogenesis imperfecta. Substitution of cysteine for glycine 178 in the triple helical domain.

Cultured fibroblasts from a patient affected with a moderate form of osteogenesis imperfecta were defective for the synthesis of type I collagen molecules; about half of the alpha 1(I) chains contained a cysteine residue in the triple helical domain and a disulfide link formed when two mutant alpha 1(I) chains were incorporated into a type I collagen heterotrimer. The proband's parents were clinically and biochemically normal. The cysteine was localized within peptide alpha 1(I)CB8 between residues 170 and 200 of the triple helical domain using a chemical procedure with 2-nitro-5-thiocyanobenzoic acid (Tenni, R., Rossi, A., Valli, M., Mottes, M., Pignatti, P. F., and Cetta, G. (1990) Matrix 10, 20-26). Type I procollagen heterotrimers containing either one or two mutant chains showed (i) a slight abnormality in secretion from cells; (ii) a low degree of post-translational overmodifications; (iii) the same, but lower than normal, thermal stability. Total RNA was isolated from the proband's dermal fibroblast cultures, and cDNAs for pro-alpha 1(I) were prepared d using total RNA. A portion of cDNA, coding for the region encompassing residues 119-193 of alpha 1(I) triple helical domain, was amplified by polymerase chain reaction. A single base pair mismatch was identified by chemical cleavage of DNA.DNA heteroduplexes, indicating a possible substitution of a guanine in the triplet coding for glycine 178 or 181. The same unique mismatch was detected by chemical cleavage in about one-half of the molecules in heteroduplexes formed between patient's pro-alpha 1(I) mRNAs and a normal cDNA probe. The amplified products were cloned and sequenced, confirming the heterozygous nature of the patient and demonstrating the presence and the location of a missense mutation; a single T for G substitution was found in the first base of the triplet coding for residue 178 of alpha 1(I) triple helical domain, leading to a cysteine for glycine substitution. Allele-specific oligonucleotide hybridization to amplified DNA confirmed a de novo point mutation in the proband's genome. The findings in this patient are in accord with the phenotypic gradient model, which correlates the localization of the structural defect with the clinical outcome of osteogenesis imperfecta. The mutant protein has some properties that differ from the caused by the cysteine for glycine 175 substitution, suggesting a direct influence of the neighboring amino acids on the effects of the mutation.