Hyperostosis in siblings.

Infantile cortical hyperostosis - Caffey-Silverman disease - is a familial disorder manifesting in the late fetal period or infancy with excessive periosteal bone formation. Signs and symptoms regress spontaneously within months and result in expanded, deformed bones. The paucity of clinical symptoms may lead to delayed investigation and confusion of the remaining bone changes with those in other conditions. This problem is exemplified by two siblings misdiagnosed as osteogenesis imperfecta. The diagnosis of Caffey-Silverman disease was confirmed by molecular analysis showing the specific COL1A1 mutation in the patients and their clinically unaffected mother. Reduced penetrance rather than autosomal recessive inheritance explains multiple affected siblings born to healthy parents.

Medium-chain acyl-CoA dehydrogenase deficiency associated with a novel splice mutation in the ACADM gene missed by newborn screening.

BACKGROUND: Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common disorder of mitochondrial fatty acid ß-oxidation and a target disease of newborn screening in many countries. CASE PRESENTATION: We report on two siblings with mild MCAD deficiency associated with a novel splice site mutation in the ACADM gene. The younger sibling was detected by newborn screening, while the older sister was missed, but diagnosed later on by genetic family testing. Both children were found to be compound heterozygous for the common c.985A > G (p.K329E) mutation and a novel splice site mutation, c.600-18G > A, in the ACADM gene. To determine the biological consequence of the c.600-18G > A mutation putative missplicing was investigated at RNA level in granulocytes and monocytes of one of the patients. The splice site mutation was shown to lead to partial missplicing of the ACADM pre-mRNA. Of three detected transcripts two result in truncated, non-functional MCAD proteins as reflected by the reduced octanoyl-CoA oxidation rate in both patients. In one patient a decrease of the octanoyl-CoA oxidation rate was found during a febrile infection indicating that missplicing may be temperature-sensitive. CONCLUSIONS: Our data indicate that the c.600-18G > A variant activates a cryptic splice site, which competes with the natural splice site. Due to only partial missplicing sufficient functional MCAD protein remains to result in mild MCADD that may be missed by newborn screening.

Novel and recurrent TRPV4 mutations and their association with distinct phenotypes within the TRPV4 dysplasia family.

BACKGROUND: Mutations in TRPV4, a gene that encodes a Ca(2+) permeable non-selective cation channel, have recently been found in a spectrum of skeletal dysplasias that includes brachyolmia, spondylometaphyseal dysplasia, Kozlowski type (SMDK) and metatropic dysplasia (MD). Only a total of seven missense mutations were detected, however. The full spectrum of TRPV4 mutations and their phenotypes remained unclear. OBJECTIVES AND METHODS: To examine TRPV4 mutation spectrum and phenotype-genotype association, we searched for TRPV4 mutations by PCR-direct sequencing from genomic DNA in 22 MD and 20 SMDK probands. RESULTS: TRPV4 mutations were found in all but one MD subject. In total, 19 different heterozygous mutations were identified in 41 subjects; two were recurrent and 17 were novel. In MD, a recurrent P799L mutation was identified in nine subjects, as well as 10 novel mutations including F471del, the first deletion mutation of TRPV4. In SMDK, a recurrent R594H mutation was identified in 12 subjects and seven novel mutations. An association between the position of mutations and the disease phenotype was also observed. Thus, P799 in exon 15 is a hot codon for MD mutations, as four different amino acid substitutions have been observed at this codon; while R594 in exon 11 is a hotspot for SMDK mutations. CONCLUSION: The TRPV4 mutation spectrum in MD and SMDK, which showed genotype-phenotype correlation and potential functional significance of mutations that are non-randomly distributed over the gene, was presented in this study. The results would help diagnostic laboratories establish efficient screening strategies for genetic diagnosis of the TRPV4 dysplasia family diseases.

Trastuzumab therapy vs tetracycline controlled ERBB2 downregulation: influence on tumour development in an ERBB2-dependent mouse tumour model.

Trastuzumab (Herceptin) has improved therapy of breast cancer. Only patients overexpressing ERBB2 are treated with trastuzumab, whereas its use in tumours without ERBB2 expression is useless. This led to the concept that the subgroup of trastuzumab-sensitive tumours is 'ERBB2-dependent', meaning that ERBB2 signalling is indispensable for growth of these tumours. We used a mouse model that allows anhydrotetracycline (ATc)-controlled downregulation of ERBB2 in tumour tissue. ERBB2 mRNA and protein expression were downregulated below detection limit leading to a macroscopically complete tumour remission within 14 days. Tumour remission was accompanied by a strong decrease in proliferation, a moderate increase in apoptosis, as well as dephosphorylation of ERK1/2 and AKT/PKB. These data clearly indicate ERBB2 dependence. Therefore, a high sensitivity to trastuzumab may be suspected. Surprisingly, trastuzumab caused a much weaker effect compared to ATc-induced ERBB2 downregulation, although a decrease in ERBB2 membrane localisation was induced. Only a slight decrease in proliferation and a weak transient increase in apoptosis were observed. Interestingly, tumours responded to trastuzumab by a sharp fivefold increase in phosphorylated AKT/PKB as well as a 3.5- and 5.3-fold increase in AKT1 and AKT2 mRNA levels, respectively. In conclusion, 'ERBB2 dependence' is not sufficient to define trastuzumab-responsive tumours. The suboptimal effect of trastuzumab compared to the maximally possible effect induced by ATc demonstrates a high potential for improved ERBB2 blocking therapies.

Oncogene-blocking therapies: new insights from conditional mouse tumor models.

Identification of oncogene dependent signaling pathways controlling aggressive tumor growth has led to the emergence of a new era of oncogene-blocking therapies, including Herceptin and Gleevec. In the recent years conditional mouse tumor models have been established that allow switching-off the expression of specific oncogenes controlling tumor growth. The results may have two important implications for oncogene-blocking therapies: (i) downregulation of oncogenes, for instance HER2, MYC, RAS, RAF, BCR-ABL or WNT1, usually leads to a rapid tumor remission. However, it was observed that the initial remission was followed by recurrent tumor growth in most studies. Interestingly, different oncogenes controlled tumor growth in the recurrent than in the primary tumors. This could explain the astonishing clinical observation that inhibitors of a broader spectrum of protein kinases (so-called: "dirty inhibitors") may be superior over highly specific substances. Due to their additional "unspecific" inhibition of a broader spectrum of kinases, they may hamper the escape mechanisms by antagonizing also the pathways controlling recurrent tumor growth. (ii) Experiments with cell systems that allow switching-on oncogene expression point to a so far possibly underestimated cancer drug target: the dormant tumor cell. Oncogene expression (for instance: NeuT or RAS) led to a phenomenon named oncogene-induced senescence or dormancy. Dormant cells are unresponsive to mitogenic stimuli. Importantly, such cells are not at all ready to die, but can remain viable for extended periods of time. Recently, dormant tumor cells have been shown to be more resistant to stresses such as hypoxia or exposure to cytostatic drugs. It still is a matter of debate if and under which conditions dormant tumor cells can be "kissed to life". If these cells contribute to carcinogenesis, it will be important to identify substances specifically killing senescent cells. This review will focus on the possible relevance of senescence both as a pre-oncogenic condition and also for therapy.

Hepatitis B virus core promoter mutations in children with multiple anti-HBe/HBeAg reactivations result in enhanced promoter activity.

Sera of two children were examined to determine whether specific hepatitis B virus (HBV) mutants may contribute to anti-hepatitis B e/hepatitis B e antigen (anti-HBe/HBeAg) reactivations during the course of chronic hepatitis B. The full-length HBV genome isolated from sera of patient 1 and the basic core promoter (BCP) from patient 2 were amplified and sequenced before and after several reactivations. The functional significance of the mutant BCP from patient 1 was studied using the luciferase assay. In both patients, rare mutations were found in the BCP at nucleotides 1764(G-->T)/1766(C-->G) and 1766(C-->T)/1768(T-->A) in case 1 and 2, respectively. In the BCP from patient 1, a putative new binding site for the transcription factor hepatocyte nuclear factor 3 (HNF3) was generated. The functional analyses of the mutant showed a 2.8-fold increase of core promoter activity, whereas the BCP variant of patient 2 was also identified to result in enhanced promoter activity. The alignment of full-length genomes from child 1 to the reference sequence showed 61 nucleotide substitutions. Furthermore, the time of reactivations from child 1 was always accompanied by selection of a precore mutation at nucleotide position 1899. In liver tissue of patient 1 before development of hepatocellular carcinoma only free viral sequences were found, whereas a single site integration of HBV was detected in hepatocytes after activation of carcinogenesis. Specific mutations in the HBV BCP of the two patients that are rarely present in chronic carriers were identified to increase the core promoter activity possibly by altering transcription factor binding, suggesting that these variants may be involved in the pathogenesis of frequent HBV reactivations.

A senescence-like phenotype distinguishes tumor cells that undergo terminal proliferation arrest after exposure to anticancer agents.

Exposure of human tumor cell lines to different chemotherapeutic drugs, ionizing radiation, and differentiating agents induced morphological, enzymatic, and ploidy changes resembling replicative senescence of normal cells. Moderate doses of doxorubicin induced this senescence-like phenotype (SLP) in 11 of 14 tested cell lines derived from different types of human solid tumors, including all of the lines with wild-type p53 and half of p53-mutated cell lines. SLP induction seemed to be independent from mitotic cell death, the other major effect of drug treatment. Among cells that survived drug exposure, SLP markers distinguished those cells that became terminally growth-arrested within a small number of cell divisions from the cells that recovered and resumed proliferation. SLP induction in breast carcinoma cells treated with retinoids in vitro or in vivo was found to correlate with permanent growth inhibition under the conditions of minimal cytotoxicity, suggesting that this response may be particularly important for the antiproliferative effect of differentiating agents. The senescence-like program of terminal proliferation arrest may provide an important determinant of treatment outcome and a target for augmentation in cancer therapy.

Mutations in the basic core promotor and the precore region of hepatitis B virus and their selection in children with fulminant and chronic hepatitis B.

The involvement of precore stop codon 1896-A and base exchanges in the AT-rich region at positions 1762 and 1764 of the hepatitis B core promotor has been controversely discussed in adults with fulminant hepatitis B. Because no data are currently available on children, we analyzed the basic core promotor (BCP) and precore region in children with chronic and fulminant hepatitis B. The BCP and precore region were sequenced directly and after cloning from mothers and infants. Thirteen children suffered from chronic liver disease, 6 of whom were treated with interferon alfa (IFN-alpha). All 13 patients seroconverted from hepatitis B e antigen (HBeAg) to hepatitis B e antigen antibodies (anti-HBe), and sera were analyzed before and after seroconversion. Nine vertically infected infants developed a fulminant course of hepatitis B. The occurrence of BCP (1762-T/1764-A, 7.7%) and precore (1896-A, 7.7%; 1899-A, 15%) mutations in chronic hepatitis B was rare. A genotype shift from D to A was observed in 3 patients after development of anti-HBe. A high number of base exchanges was detected in those infants with fulminant hepatitis B. Eight of nine showed a G-A exchange at positions 1896/97 (89%), 1899 (56%), and/or mutations at nucleotide (nt) positions 1762 (56%) and 1764 (78%). All virus strains belonged to genotype D, whereas in the only surviving infant, a D-to-A shift was detected. Hepatitis B virus (HBV) DNA clones were examined from 3 babies and 5 mothers. Our results showed a heterogeneous virus population in 4 of 5 mothers. In contrast, a homogeneous virus population emerged in the infants. According to our data, the analysis in children with fulminant and chronic hepatitis B revealed a striking presence of BCP and precore mutants in infants with fulminant hepatitis (FH) when compared with clinically inapparent anti-HBe-positive children (P <.002), which could be one factor in the pathogenesis of fulminant hepatitis B in children.

A combination of genetic suppressor elements produces resistance to drugs inhibiting DNA replication.

Many anticancer drugs inhibit DNA replication. To investigate the mechanism of permanent growth inhibition after transient arrest of DNA replication, we selected genetic suppressor elements (GSEs) conferring resistance to replication inhibitor Aphidicolin. Starting from a retroviral expression library carrying normalized fragments of human cell cDNA, we isolated four GSEs which, when introduced as a combination, produced resistance to Aphidicolin, doxorubicin and hydroxyurea in HT1080 fibrosarcoma cells. The four GSEs were derived from ORFX bromodomain protein gene, WIZ zinc finger protein gene, the gene for subunit 3 of cytochrome c oxidase, and the gene corresponding to an EST with no known function. A cell line carrying all four GSEs showed a weaker induction of the senescence-like phenotype after treatment with Aphidicolin or doxorubicin; the resistance of this cell line was not associated with decreased doxorubicin accumulation. These results indicate that combined effects of GSEs derived from these four genes increase cellular resistance to replication-inhibiting drugs, possibly by inhibiting drug-induced senescence.

Alpha-interferon treatment in HBeAg positive children with chronic hepatitis B and associated hepatitis D.

The main problem of children with HBeAg positive hepatitis B and associated hepatitis D is progression to liver cirrhosis with decompensation of liver function and need for liver replacement therapy within 15-20 years after infection. To determine whether interferon-alpha (IFN-alpha) therapy has a positive effect on HBV replication and inflammatory activity, we evaluated clinical and serological data of 8 children treated with IFN-alpha and 6 historic control patients without treatment. 4 of the nontreated patients seroconverted from HBeAg to anti-HBe between 7 to 17 years after initial diagnosis and showed decreased inflammatory activity in the liver. In the treatment group, the rate of seroconversion to anti-HBe (3 early, 2 late seroconverters) corresponded well to former trial results obtained in patients exclusively infected by HBV. Serum aminotransferase levels decreased or normalized in seroconverted children. In chronic HBV infection with associated hepatitis D (HDV) infection--compared to the spontaneous course of the disease--IFN-alpha therapy reduced inflammatory activity by earlier seroconversion to anti-HBe in responding patients. Moreover, viral replication and infectivity of hepatitis B was markedly reduced, but no effect on replication of HDV could be documented. Although long-term effects cannot be exactly estimated, at present IFN-alpha remains the only available treatment for HBeAg and anti-HDV positive children and seems to be of benefit for responding patients.

The hepatitis B virus seroconversion to anti-HBe is frequently associated with HBV genotype changes and selection of preS2-defective particles in chronically infected children.

In order to investigate the generation and selection of hepatitis B virus mutants and the influence of interferon on their evolution, a longitudinal study including 22 patients was performed. The complete preS1/S2 open reading frame was analyzed by direct sequencing from serum samples obtained before and after seroconversion to anti-HBe in 11 children without alpha-interferon treatment. Furthermore, in 11 cases with therapy additional samples obtained during interferon therapy were investigated. The comparison of each patient's preS sequences analyzed before and during therapy did not show any nucleotide change, while in both groups numerous silent and missense mutations were found immediately after seroconversion. Surprisingly, in 7 cases the hepatitis B virus changed genotype from A to D (subtype adw to ayw) after seroconversion. Additional rearrangements were observed in 4 patients. In 3 cases the selection of preS2 start codon mutants was detected after seroconversion and in 1 individual a 183-nucleotide deletion was found during and after HBeAg positivity. In conclusion, the emergence of preS rearrangements and numerous base exchanges provide evidence for a strong selection process focused against the preS region. Moreover, the appearance of genotype changes after anti-HBe seroconversion reveales a thus far unrecognized event during the natural course of HBV infection in childhood.