A Newly Identified Missense Mutation in FARS2 Causes Autosomal‐Recessive Spastic Paraplegia

Hereditary spastic paraplegia (HSP) is a clinically and genetically heterogeneous group of neurodegenerative disorders characterized by spasticity of the lower limbs due to pyramidal tract dysfunction. Here, we report that a missense homozygous mutation c.424G>T (p.D142Y) in the FARS2 gene, which encodes a mitochondrial phenylalanyl tRNA synthetase (mtPheRS), causes HSP in a Chinese consanguineous family by using combination of homozygous mapping and whole‐exome sequencing. Immunohistochemical experiments were performed showing that the FARS2 protein was highly expressed in the Purkinje cells of rat cerebellum. The aminoacylation activity of mtPheRS was severely disrupted by the p.D142Y substitution in vitro not only in the first aminoacylation step but also in the last transfer step. Taken together, our results indicate that a missense mutation in FARS2 contributes to HSP, which has the clinical significance of the regulation of tRNA synthetases in human neurodegenerative diseases.     

A Dominant STIM1 Mutation Causes Stormorken Syndrome

Stormorken syndrome is a rare autosomal‐dominant disease with mild bleeding tendency, thrombocytopathy, thrombocytopenia, mild anemia, asplenia, tubular aggregate myopathy, miosis, headache, and ichthyosis. A heterozygous missense mutation in STIM1 exon 7 (c.910C>T; p.Arg304Trp) (NM_003156.3) was found to segregate with the disease in six Stormorken syndrome patients in four families. Upon sensing Ca²⁺ depletion in the endoplasmic reticulum lumen, STIM1 undergoes a conformational change enabling it to interact with and open ORAI1, a Ca²⁺ release‐activated Ca²⁺ channel located in the plasma membrane. The STIM1 mutation found in Stormorken syndrome patients is located in the coiled‐coil 1 domain, which might play a role in keeping STIM1 inactive. In agreement with a possible gain‐of‐function mutation in STIM1, blood platelets from patients were in a preactivated state with high exposure of aminophospholipids on the outer surface of the plasma membrane. Resting Ca²⁺ levels were elevated in platelets from the patients compared with controls, and store‐operated Ca²⁺ entry was markedly attenuated, further supporting constitutive activity of STIM1 and ORAI1. Thus, our data are compatible with a near‐maximal activation of STIM1 in Stormorken syndrome patients. We conclude that the heterozygous mutation c.910C>T causes the complex phenotype that defines this syndrome.     

A Mutation in the CASQ1 Gene Causes a Vacuolar Myopathy with Accumulation of Sarcoplasmic Reticulum Protein Aggregates

A missense mutation in the calsequestrin‐1 gene (CASQ1) was found in a group of patients with a myopathy characterized by weakness, fatigue, and the presence of large vacuoles containing characteristic inclusions resulting from the aggregation of sarcoplasmic reticulum (SR) proteins. The mutation affects a conserved aspartic acid in position 244 (p.Asp244Gly) located in one of the high‐affinity Ca²⁺‐binding sites of CASQ1 and alters the kinetics of Ca²⁺ release in muscle fibers. Expression of the mutated CASQ1 protein in COS‐7 cells showed a markedly reduced ability in forming elongated polymers, whereas both in cultured myotubes and in in vivo mouse fibers induced the formation of electron‐dense SR vacuoles containing aggregates of the mutant CASQ1 protein that resemble those observed in muscle biopsies of patients. Altogether, these results support the view that a single missense mutation in the CASQ1 gene causes the formation of abnormal SR vacuoles containing aggregates of CASQ1, and other SR proteins, results in altered Ca²⁺ release in skeletal muscle fibers, and, hence, is responsible for the clinical phenotype observed in these patients.     

A Mutation that Creates a Pseudoexon in SOD1 Causes Familial ALS

Amyotrophic lateral sclerosis (ALS) is an adult onset neurodegenerative disease which targets motor neurons of the cortex, brainstem and spinal cord. About 5–10% of all amyotrophic lateral sclerosis cases are familial (FALS), and 15–20% of FALS cases are caused by mutations in the zinc-copper superoxide dismutase gene ( SOD1 ). We identified a large family from France with ten members affected with ALS. Linkage was established to the SOD1 locus on chromosome 21 and genomic and cDNA sequencing was performed for the SOD1 gene. This revealed an activated pseudoexon between exons 4 and 5 that was present in two tested members of the family. Translation of this 43 base pair exon results in the introduction of seven amino acids before a stop codon is present, leading to a prematurely truncated SOD1 protein product of 125 amino acids. Sequencing intron 4 in a patient revealed a heterozygous change 304 bp before exon 5 (c.358 – 304C > G), but only 5 bp after the cryptic exon, thus causing this alternative splice product. This mutation segregated in all affected individuals of the family. This adds an additional genetic mechanism for developing SOD1 -linked ALS and is one which can be more readily targeted by gene therapy.     

细胞色素P45A1基因变异与肺癌易患性

Individual susceptibility to cancer induced by environmental agents may be influenced by polymorphic metabolic genes responsible for the activation or detoxification of carcinogens．The association between genetic polymorphisms in cytochrome P4501A1 (CYP1A1) and lung cancer susceptibility has been extensively studied．The various CYP1A1 alleles exhibit population frequencies that depend on race and ethnicity．An increased risk of lung cancer in Asians was found to be associated with genetic polymorphisms in CYP1A1．However,reports from Caucasians are not consistant,probably suggesting the ethnic-sepecific effect of the polymorphisms in the locus on the cancer．Evidence also exists for the association between levels of carcinogen-DNA adducts or frequency of oncogene and tumor suppressor gene mutations and CYP1A1 polymorphisms．These findings provide a better understanding of the relationship between CYP1A1 and lung cancer susceptibility．     

Protein Phosphorylation Pattern and Role of Products of c-erbB-1 and c-abl Proto-oncogenes in Murine Preimplantation Embryonic Development

PROBLEM: To investigate the protein phosphorylation pattern and role of products of c-erbB-1 and c-abl proto-oncogenes with known tyrosine kinase activity in preimplantation embryonic development in mice. METHOD: The protein phosphorylation pattern was studied by in vitro ³²P metabolic labeling of murine ova/embryos as well as by in vitro kinase assay performed directly on various ova/embryos extracts. The role of products of c-erbB-1 (170 kDa, receptor for epidermal growth factor [EGF]) and c-abl proto-oncogenes (150 kDa) was examined by in vitro culturing murine embryos in the presence of monoclonal antibodies to respective protein products and by co-culturing with EGF, the ligand for EGF receptor (EGF-R). RESULTS: In vitro metabolic labeling of murine ova/embryos showed ³²P incorporation into at least two protein bands of murine ova (M_r 81 and 36 kDa), six protein bands of two-cell (M_r 81, 36; and 97, 52, 22 and 19 kDa, respectively), six protein bands of morula (M_r 81, 36; 97, 22, and 19; and 33 kDa, respectively), and eight protein bands of blastocyst (81, 36; 97, 22, 19; and 115, 58, and 15 kDa, respectively), stage embryos; there were some specific bands in each stage. Prolonged labeling from 2 to 4 h not only resulted in a relative increase in ³²P incorporation into these proteins but also revealed additional bands in morula (M_r 133 and 115 kD) and blastocyst (M_r 49, 33, and 31 kD) stage embryos. In vitro kinase assays performed directly on various ova/embryos extracts revealed at least three phosphoproteins (M_r 58, 36 and 33, respectively) that were common to ova, two-cell, morula, and early/late blastocyst stage embryos. Additionally, three protein bands each in murine ova and two-cell embryos (M_r 108, 81, 73 kDa, respectively), and four protein bands of late blastocyst (M_r 108, 73; 133 and 18 kDa, respectively) stage embryos were also revealed. Culture of two-cell embryos in the presence of EGF, the ligand for EGF-receptor, resulted in a concentration dependent increase (P< .001) in the number of cells per blastocyst. Monoclonal antibody to c-erbB-1 170 kDa protein (receptor for EGF) did not affect development of in vitro cultured murine embryos from two-cell to morula, but significantly (P<.001) inhibited the in vitro development of morula to late blastocyst stage. Monoclonal antibody to c-abl protein inhibited the development of murine embryos from two-cell to morula (P<.017), as well as, from morula to late blastocyst stage (P<.002 to .01). CONCLUSIONS: These results suggest that the stage-specific protein phosphorylation pattern and specific products of c-erbB-1 and c-abl proto-oncogenes may have a role in preimplantation embryonic development in mice.     

α1-Antitrypsin Nonsense Mutation Associated with a Retained Truncated Protein and Reduced mRNA

α₁-Antitrypsin (α1AT) provides the major protection in the lung against neutrophil elastase-mediated proteolysis. Inheritance of α1AT deficiency alleles is associated with an increased risk of emphysema and liver disease. α1AT null alleles cause the total absence of serum α1AT and represent the ultimate in a continuum of alleles associated with α1AT deficiency. The molecular mechanisms responsible for absence of serum α1AT include splicing abnormalities, deletion of α1AT coding exons, and premature stop codons. We identified an Italian individual with asthma, emphysema, and a very low level of serum α1AT. DNA sequencing demonstrated theMprocidadeficiency allele and a novel null allele,QOtrastevere(c654 G → A, W194Z), a nonsense mutation near the intron 2 (IVS2) splice acceptor site. To determine the molecular basis ofQOtrastevereand specifically to evaluate whether this nonsense mutation interfered with mRNA processing by altered splicing, we used a Chinese hamster ovary cell line permanently transfected withQOtrastevereor normal M α1AT with and without IVS2. Northern blot analysis demonstrated that the normal M construct, with or without IVS2, expressed α1AT mRNA of a similar size. The nonsense mutation was associated with moderately reduced α1AT mRNA regardless of the presence or absence of IVS2. Reduction in α1AT mRNA regardless of the opportunity for splicing supports a translational-translocation model as the cause of reduced α1AT mRNA rather than the nuclear scanning model. Pulse–chase studies followed by immunoprecipitation demonstrated an endoplasmic reticulum-retained 31 kDaQOtrastevereα1AT, which was rapidly degraded. Although mRNA content was moderately reduced, retention and rapid intracellular degradation of the truncated form are the major mechanisms for the absence of secreted α1AT.     

A Novel Mutation in RPL10 (Ribosomal Protein L10) Causes X‐Linked Intellectual Disability,Cerebellar Hypoplasia,and Spondylo‐Epiphyseal Dysplasia

RPL10 encodes ribosomal protein L10 (uL16), a highly conserved multifunctional component of the large ribosomal subunit, involved in ribosome biogenesis and function. Using X‐exome resequencing, we identified a novel missense mutation (c.191C>T; p.(A64V)) in the N‐terminal domain of the protein, in a family with two affected cousins presenting with X‐linked intellectual disability, cerebellar hypoplasia, and spondylo‐epiphyseal dysplasia (SED). We assessed the impact of the mutation on the translational capacity of the cell using yeast as model system. The mutation generates a functional ribosomal protein, able to complement the translational defects of a conditional lethal mutation of yeast rpl10. However, unlike previously reported mutations, this novel RPL10 missense mutation results in an increase in the actively translating ribosome population. Our results expand the mutational and clinical spectrum of RPL10 identifying a new genetic cause of SED and highlight the emerging role of ribosomal proteins in the pathogenesis of neurodevelopmental disorders.     

A Histoplasma capsulatum-Specific IgG1 Isotype Monoclonal Antibody,H1C,to a 70-Kilodalton Cell Surface Protein Is Not Protective in Murine Histoplasmosis

Monoclonal antibodies to Histoplasma capsulatum can modify pathogenesis. We now show that monoclonal antibody H1C to a 70-kDa antigen increases intracellular fungal growth and reduces macrophage nitric oxide release but has no effect on fungal burden or survival in murine infection. This further demonstrates the complexities of host-pathogen interactions.Histoplasma capsulatum is a dimorphic fungal pathogen that is the causative agent of histoplasmosis. After Candida species, H. capsulatum is the most common causative agent of systemic mycoses in North America and either the first- or second-most-common cause in Central and South America (4, 7). We previously identified an IgG1 isotype monoclonal antibody (MAb), H1C, that reacts with a 70-kDa H. capsulatum cell wall antigen (6). The MAb is highly specific for H. capsulatum, as it did not react with yeast cell antigens from related microbes. An inhibition enzyme-linked immunosorbent assay (ELISA) system using H1C is also highly specific for the detection of antigenemia and antigenuria in patients with histoplasmosis (6). Furthermore, the inhibition ELISA may be useful for monitoring treatment responses in patients with histoplasmosis (5).We have shown that MAbs to the H. capsulatum cell surface proteins histone 2B (H2B) and heat shock protein 60 (Hsp60) can alter the intracellular fate of the fungus, modify the inflammatory response to infection, and prolong the survival of lethally infected mice (8, 9). In particular, we demonstrated the marked protective efficacy of IgG1 and IgG2a MAbs to Hsp60. In the present study, we assessed the capacity of H1C to modify host-pathogen interactions.H1C was purified from cell culture supernatants using protein G-agarose beads (Pierce Biotechnology) according to the manufacturer''s instructions. Aliquots of H1C were screened to ensure the absence of endotoxin with the Limulus amebocyte lysate assay (BioWhittaker Inc.). An irrelevant, isotype-matched mouse IgG1 MAb (SouthernBiotech) was used as a control for all experiments. H. capsulatum G217B was used as the reference strain for all the studies (gift from G. Deepe, University of Cincinnati). H. capsulatum yeast cells were grown in Ham''s F-12 medium at 37°C with rotary shaking and collected as described previously (9). Enumeration of the yeast cells was accomplished with a hemacytometer.Immunofluorescence and immunoblotting were performed as described previously (8), except that H1C labeled with Alexa 488 was used in lieu of MAbs to Hsp60. Fluorescence analysis revealed that H1C diffusely labeled the fungal cell surface in a punctuate manner (Fig. ​(Fig.11 A), and H1C reacted with a 70-kDa protein, as shown by immunoblotting (Fig. ​(Fig.1B).1B). A whole-cell H. capsulatum yeast ELISA was also used to examine the binding of H1C to H. capsulatum, as described previously (9). Significant binding occurred at concentrations >1 μg/ml H1C (Fig. ​(Fig.1C).1C). Hence, H1C readily interacted with the 70-kDa protein at the H. capsulatum cell surface.Open in a separate windowFIG. 1.MAb H1C binds to a 70-kDa protein on the cell surface of H. capsulatum. (A) Immunofluorescence analysis demonstrates that H1C diffusely reacts with the fungal cell surface. (B) H1C reacts with a 70-kDa protein in cell wall extracts from H. capsulatum. (C) Yeast cell ELISA demonstrates the reactivity of H1C with intact fungal cells. Experiments were repeated at least three times, with consistent results.Phagocytosis and killing assays were accomplished as described by confocal microscopy and fluorescence-activated cell sorting (9) using J774.16 macrophagelike cells. H1C increased the association of H. capsulatum with the J774.16 cells (Fig. ​(Fig.22 A). Although increases in yeast cell attachment occurred at 25 and 50 μg/ml of H1C (Fig. ​(Fig.2B),2B), phagocytosis of H. capsulatum increased at all the concentrations tested (Fig. ​(Fig.2C).2C). Further, H. capsulatum opsonized with H1C had significantly increased intracellular survival after 24 h compared to controls (Fig. ​(Fig.2D).2D). GraphPad Prism version 5.00 for Windows (GraphPad Software) was used for statistical analyses. Kruskal-Wallis nonparametrical tests were used for one-way analysis of variance to compare differences between groups, and individual comparisons of groups were performed with the Bonferroni posttest.Open in a separate windowFIG. 2.MAb H1C alters H. capsulatum-macrophage interactions. (A) H1C alters H. capsulatum association with J774.16 macrophage-like phagocytosis. H1C opsonization affects H. capsulatum attachment (B) and phagocytosis (C). (D) H1C-opsonized H. capsulatum has significantly improved intracellular survival. (E) Opsonization of H. capsulatum with H1C reduces nitric oxide production. (F) Opsonization of H. capsulatum with H1C increases macrophage death. Macrophage toxicity data are at 2 h of coculture, and similar results were present at 4 h. For each graph, three independent experiments were performed, with each condition tested in triplicate, except for the nitric oxide experiments, which were performed twice with triplicates. Graphs were generated with pooled data from the experiments. The bars are the mean values, with error bars representing the standard deviations. *, P < 0.05, which indicates a comparison between the condition and H. capsulatum in the absence of antibody. “Hc” represents yeast without antibody in the presence of macrophages, “irrelevant” indicates an IgG1 isotype control antibody with yeast cells and macrophages, and the concentrations listed represent the specific amounts of H1C MAb present with H. capsulatum yeast and macrophages.Since intracellular growth increased in the presence of H1C, we examined the production of nitric oxide and superoxide by J774.16 cells cocultured with H. capsulatum exposed to H1C, irrelevant antibody, or phosphate-buffered saline (PBS), as described previously (10), and also assessed J774.16 viability. Nitric oxide formation was assessed with a commercial Griess reagent kit (Promega), and superoxide dismutase activity was quantified with a kit (Cayman Chemical). H1C-opsonized H. capsulatum induced significantly less nitric oxide release than controls at concentrations ≥25 μg/ml after 2 h of coculture (Fig. ​(Fig.2E).2E). In contrast, there were no differences in superoxide radicals (data not shown). J774.16 viability after exposure to H. capsulatum was measured using an MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay (Sigma-Aldrich). H1C concentrations ≥25 μg/ml in the presence of H. capsulatum yeast cells resulted in a dose-dependent toxicity on the J774.16 cells at 2 h of coculture (Fig. ​(Fig.2D).2D). H1C alone in the presence of the J774.16 cells had no effect on the macrophages (data not shown). Given the damage to the macrophages caused by the opsonized H. capsulatum yeast cells, it is possible that the reduction in nitric oxide was a direct effect of the reduction in viable macrophages.C57BL/6 mice (6 to 8 weeks old, female; NCI) were used for survival studies, which were approved by the Animal Institute Committee of the Albert Einstein College of Medicine. C57BL/6 mice were injected intraperitoneally with 250 μg of H1C, an isotype-matched control MAb, or PBS. Two hours later, the mice were intranasally challenged with either 5 × 10⁶ CFU (sublethal dose) or 1.25 × 10⁷ CFU (lethal dose for survival studies) of H. capsulatum yeast. The mice were closely monitored. CFU studies revealed that there was a slight trend toward an increase in numbers of CFU at day 3 after infection in animals given H1C, but the differences at days 3 and 7 in the fungal burdens between H1C-treated animals and controls were not significant (Fig. ​(Fig.33 A). Lethally challenged mice receiving H1C died at day 10 ± 1, whereas control mice died between days 10 and 12, which results are not statistically different by Kaplan-Meier analysis (P > 0.05). We also tested the efficacy of 100 and 500 μg of H1C in the lethal-challenge model, but these doses similarly failed to alter survival (data not shown).Open in a separate windowFIG. 3.Pretreatment with intraperitoneal injections of MAb H1C. A dose of 100 μg does not alter the pulmonary fungal burden in sublethally infected mice (A) or prolong survival in mice receiving lethal challenge (B). CFU studies were preformed with three mice per condition for each day assessed. Survival studies were performed using groups of 10 mice, and the experiment was repeated, with similar results.MAbs have been shown to protect against several fungal diseases, including candidiasis, cryptococcosis, aspergillosis, and pneumocystosis (2, 3). We have previously demonstrated that IgM, IgG2a, and IgG1 MAbs to H. capsulatum H2B or Hsp60 modify histoplasmosis (8, 9), but an IgG2b to Hsp60 is disease enhancing (8, 9). Hence, this report presents the second nonprotective MAb to H. capsulatum. Isotype has been shown to influence the outcome of cryptococcosis with human and murine MAbs to the major polysaccharide of Cryptococcus neoformans; in addition, a recent study showed that human IgG1-treated mice had accelerated death (1). Our findings do not rule out the possibility that a protective antibody to the 70-kDa protein could be generated, either one with a different isotype or one corresponding to a different epitope on the protein. Notably, although we found no differences in fungal burden or survival in our murine infection model, our results nevertheless suggest a possible role for the 70-kDa molecule in the virulence of the fungus, since H1C-opsonized H. capsulatum had increased intracellular survival, modified nitric oxide production, and increased macrophage damage. Our data further demonstrate the complexities involved in antibody-pathogen interactions.(The data in this paper are from a thesis to be submitted by A. J. Guimarães in partial fulfillment of the requirements for a Ph.D. degree from the Sue Golding Graduate Division of Medical Science, Albert Einstein College of Medicine, Yeshiva University, Bronx, NY.)     

A Correlative Study of p53 Protein Alteration and p53 Gene Mutation in Glioblastoma Multiforme

The p53 tumor suppressor gene is frequently mutated in glioblastomas. Mutations within the p53 gene often result in aberrant expression of the p53 protein leading to protein accumulation within the nucleus of the cells which can be detected by immunochemistry. Many studies have correlated alterations of p53 protein expression with p53 gene mutations. Positive staining of tumor cells for p53 protein has been widely assumed, perhaps incorrectly, to signify the presence of p53 gene mutations. This study compared the immunostaining patterns for p53 protein in 37 glioblastomas with the molecular genetic data obtained by the single strand conformation polymorphism assay. p53 gene mutations were detected in 46% (17 of 37) of glioblastomas, while 65% (24 of 37) of glioblastomas were positive for protein accumulation by immunohistochemistry. Although 30 of 37 glioblastomas analyzed showed concordance for p53 protein expression and p53 gene mutations, a subset of seven glioblastomas showed discordant accumulation of the p53 protein in the absence of any detectable p53 gene mutations. The mdm-2 gene was assessed in 17 glioblastomas for gene rearrangements or amplification, but none were found. This result suggests that a mechanism other than p53 gene mutation can result in altered p53 protein expression.     

Missense and silent mutations in COL2A1 result in Stickler syndrome but via different molecular mechanisms

Stickler syndrome due to mutations in COL2A1 is usually the result of premature termination codons and nonsense mediated decay resulting in haploinsufficiency of type II collagen. Here we present two missense mutations and one apparently silent mutation that each result in Stickler syndrome, but via different molecular mechanisms. One alters the translation initiating ATG codon. The second mutation is a unique glycine substitution in the minor collagen helix of the procollagen. To our knowledge a glycine substitution has not previously been reported in this region of fibrillar procollagens. The third mutation appears to be a silent change altering a GGC codon to GGT both for glycine, but use of a splicing reporter assay demonstrates that it results in missplicing and a shift in the reading frame.     

A 70-Kilodalton Recombinant Heat Shock Protein of Candida albicans Is Highly Immunogenic and Enhances Systemic Murine Candidiasis

The 70-kDa recombinant Candida albicans heat shock protein (CaHsp70) and its 21-kDa C-terminal and 28-kDa N-terminal fragments (CaHsp70-Cter and CaHsp70-Nter, respectively) were studied for their immunogenicity, including proinflammatory cytokine induction in vitro and in vivo, and protection in a murine model of hematogenous candidiasis. The whole protein and its two fragments were strong inducers of both antibody (Ab; immunoglobulin G1 [IgG1] and IgG2b were the prevalent isotypes) and cell-mediated immunity (CMI) responses in mice. CaHsp70 preparations were also recognized as CMI targets by peripheral blood mononuclear cells of healthy human subjects. Inoculation of CaHsp70 preparations into immunized mice induced rapid production of interleukin-6 (IL-6) and tumor necrosis factor alpha, peaking at 2 to 5 h and declining within 24 h. CaHsp70 and CaHsp70-Cter also induced gamma interferon (IFN-γ), IL-12, and IL-10 but not IL-4 production by CD4⁺ lymphocytes cocultured with splenic accessory cells from nonimmunized mice. In particular, the production of IFN-γ was equal if not superior to that induced in the same cells by whole, heat-inactivated fungal cells or the mitogenic lectin concanavalin A. In immunized mice, however, IL-4 but not IL-12 was produced in addition to IFN-γ upon in vitro stimulation of CD4⁺ cells with CaHsp70 and CaHsp70-Cter. These animals showed a decreased median survival time compared to nonimmunized mice, and their mortality was strictly associated with organ invasion by fungal hyphae. Their enhanced susceptibility was attributable to the immunization state, as it did not occur in congenitally athymic nude mice, which were unable to raise either Ab or CMI responses to CaHsp70 preparations. Together, our data demonstrate the elevated immunogenicity of CaHsp70, with which, however, no protection against but rather some enhancement of Candida infection seemed to occur in the mouse model used.     

Mutation analysis of COL1A1 and COL1A2 in patients diagnosed with osteogenesis imperfecta type I-IV

Osteogenesis Imperfecta (OI) is a heterogeneous group of inherited disorders characterized by increased bone fragility, with clinical severity ranging from mild to lethal. To date, seven types of OI have been described, based on clinical phenotype and histological findings. Most patients with a clinical diagnosis of OI type I-IV have a mutation in the COL1A1 or COL1A2 genes which encode the two alpha chains of type I collagen, the major component of the bone matrix. Analysis of COL1A1 and COL1A2 in a cohort of 83 unrelated patients with OI type I-IV identified a total of 62 mutations. Thirty-eight appear novel, 26 in COL1A1, and 12 in COL1A2, and these are described here. The largest group consists of point mutations affecting glycine residues in the triple helical domain of the two alpha chains, predicted to disrupt protein folding and structure. This is in accordance with previously published data. A doublet GC deletion, an unusual 398 base deletion predicted to completely remove exon 20 of COL1A2, and a point mutation resulting in substitution of a conserved cysteine in the C-terminal propeptide are described. In addition rare mutations at the cleavage sites of the C-propeptide and the N-terminal signal peptide are described.     

A Novel Mutation in SURF1 Causes Skipping of Exon 8 in a Patient with Cytochrome c Oxidase-Deficient Leigh Syndrome and Hypertrichosis

Leigh syndrome is a rare pediatric neurodegenerative disorder attributed to impaired mitochondrial energy metabolism. Mutations in SURF1 have been described in several patients with Leigh syndrome associated with cytochrome c oxidase deficiency. We report a new 18-bp deletion (821del18), spanning the splice donor junction of exon 8 of SURF1, in an infant presenting with cytochrome c oxidase-deficient Leigh syndrome and hypertrichosis. cDNA sequencing demonstrated that this deletion results in a messenger lacking exon 8. RT-PCR experiments suggested a rapid degradation of the aberrant mRNA species from the 5′-end.