[Changes in epidemic intensity of influenza during 2014-2020 in Shanghai].

Objective: To evaluate the performance of the influenza surveillance network and compare the epidemic intensity of influenza during 2014-2020 in Shanghai. Methods: Based on the weekly reports of influenza-like illness (ILI) and laboratory-confirmed influenza cases from January 1, 2014 to December 31, 2020. This study first evaluated the data reporting and specimen collection of ILI cases for each sentinel hospital, and then calculated the percentage of ILI (ILI%), the proportion of specimens tested positive for influenza, and the incidence of influenza among all ILI outpatient and emergency visits to measure the epidemic intensity of influenza. Finally, seasonal autoregressive integrated moving average (ARIMA) model was applied to quantify the changes in epidemic intensity of influenza in 2020. Results: The proportion of influenza surveillance sentinel hospitals with a score of less than 5 in the evaluation of ILI data reporting and samples collection were 9.68% and 21.05% in 2020 in Shanghai, respectively. ILI% was estimated to be 1.51% (95%CI: 1.50%-1.51%) and 2.31% (95%CI: 2.30%-2.32%), respectively for 2014-2019 and 2020; the proportion of specimens tested positive was 24.27% (95%CI: 24.02%- 24.51%) and 7.15% (95%CI: 6.78%-7.54%), respectively; and the incidence of influenza was 3.66‰ (95%CI: 3.62‰-3.70‰) and 1.65‰ (95%CI: 1.57‰-1.74‰), respectively. ARIMA model showed that ILI% was increased by 45.25% in 2020 in Shanghai, and the proportion of specimens tested positive and the incidence of influenza were reduced by 78.45% and 51.80%, respectively. Conclusions: In 2020, the performance of influenza surveillance system has changed, ILI% has increased, the proportion of specimens tested positive and the incidence of influenza has decreased in Shanghai. The change in the quality of influenza surveillance is also a potential factor affecting the epidemic intensity of influenza. In the future, the quality control of influenza surveillance network still needs to be further strengthened.

[Practice and thinking of acute respiratory infection surveillance for the response of emerging respiratory diseases in Shanghai].

Shanghai Municipal Center for Disease Control and Prevention has implemented an active comprehensive surveillance project of acute respiratory infections in adults in Shanghai, including influenza like illness (ILI) and severe acute respiratory infection (SARI). By testing and identifying a variety of respiratory pathogens, it was found that influenza viruses were the main pathogens in 172 ILI cases in 2019. The positive rates of influenza A (H1N1) pdm09 virus, influenza A (H3N2) virus and influenza B virus Victoria lineage were 30.81%, 14.53% and 30.55%, respectively. The positive detection of influenza A (H1N1) pdm09 virus peaked in the first quarter. The positive rate of enterovirus/human rhinovirus was 6.40%, with a positive detection peak in the third quarter, while the positive rate of adenovirus was 4.65% with a positive detection peak in the second quarter of the year. Two human coronavirus (HCoV)-OC43 positive samples, 1 HCoV-HKU1 positive sample and 1 HCoV-NL63 positive sample were detected, respectively, and no HCoV-229E positive sample was detected. The detection rate of Staphylococcus aureus was 17.44%, and the detection rate of Klebsiellapneumoniae was 9.88%. Influenza viruses were also the main pathogens in 1 447 SARI cases. The positive rates of influenza A (H1N1) pdm09 virus, influenza A (H3N2) virus and influenza B virus Victoria lineage were 5.46%, 1.73% and 0.30%, respectively. The positive detection of influenza A (H1N1) pdm09 virus (17.50%) peaked in the first quarter. The total positive detection rate of enterovirus/human rhinovirus was 2.97%, the positive detection peaked in the first quarter. The positive rate of Mycoplasma pneumoniae was 3.25% and the positive rate of Legionella was 1.04%. 5 HCoV-229E positive samples, 10 HCoV-OC43 positive samples, 7 HCoV-HKU1 positive samples and 6 HCoV-NL63 positive samples were detected. Eight strains of Staphylococcus aureus, 4 strains of Pseudomonas aeruginosa and 3 strains of Klebsiella pneumoniae were detected after cultures. By implementing the active surveillance, we not only detected a case of human infection with avian influenza A(H7N9) virus in time, but also preliminary understood the pathogenic spectrum characteristics and seasonality of ILI and SARI in Shanghai. In recent years, the surveillance methods have been continuously improved and the number of sentinel hospitals has increased gradually. In particular, for the response to COVID-19, the Surveillance Information Reporting System of Acute Respiratory Infection based on HIS system has been promoted to cover the whole city, which might lay a foundation for the active surveillance and early warning of emerging infectious diseases in the future.

[Epidemiological analysis on bud-events of Norovirus-associated infectious diarrhea in Shanghai, 2018].

Objective: To analyze the epidemiological characteristics related to Norovirus-associated infectious diarrhea bud events in Shanghai, and to discuss the value of bud events surveillance, so as to help Norovirus-associated infectious diarrhea control, prevention and to improve the surveillance system on bud events. Methods: Data related to 142 Norovirus-associated infectious diarrhea bud events were gathered from 16 districts of Shanghai areas in 2018 and were analyzed. Results: There were 2 peaks, April and November for 142 Norovirus-associated infectious diarrhea bud events reported in 2018. 98.59% (140/142) of the bud events occurred in schools and kindergartens. 80.28% (114/142) of the bud events would involve 6-19 cases per each event. The median time duration between the onset date of the first case and the reporting date of the event was 2 days and the median event duration was 7 days. The bud event scale and regional incidence of bud events as well as the reporting timelines were influential factors on the duration of the bud events. Conclusions: Two peaks, spring and winter bud events of the Norovirus-associated infectious diarrhea were seen in Shanghai in 2018. Schools and kindergartens were to be prioritized locations for control and prevention of Norovirus-associated infectious diarrhea bud events. Surveillance system was proved to have contributed to the early detection, reporting and control of bud events.

[The first identification of epidemic clone of enterotoxic Escherichia coli O∶6 serogroup highly associated with azithromycin resistance in Shanghai].

Objective: To investigate the molecular characterization of adult diarrhea cases caused by enterotoxic Escherichia coli (ETEC) and explore the practical model of epidemiology for laboratory technique and data needs based on the surveillance network. Methods: Epidemiological design and sampling targeted adult cases ETEC caused diarrhea in epidemic season. The enterotoxin type, serogroup, resistance, colonization factor and molecular type of ETEC were identified. Multiple dynamic phenotypic characteristics of ETEC were indicated by multidimensional and multivariable data. Results: From 2016 to 2018, 84 eligible ETEC strains were detected. The dominant serums/toxins were O∶6 (STh), O∶25 (LT), O∶159 (STh), O∶153 (STh). O∶6 (STh+CS21), which replaced O∶25 and O∶159 as the popular clones in 2018. Six cases of O∶153 (STh+CFA/I+CS8+PT34) in outbreak in 2017 were imported ones. The resistance rates of ETEC strains detected in adults to sulfasoxazole, naproxinic acid, ampicillin and azithromycin were more than 30%, multidrug resistance (MDR) reached 58.3%. Serum/toxin types suggested that attenuated strains were more likely to become MDR. Molecular typing confirmed that the genetic similarity of the dominant clone of O∶6 serogroup (PT20-24) was higher than O∶25 and O∶159. There was a high correlation between the minimal inhibitory concentration (MIC) of azithromycin and the resistant gene mphA (87.5%, 28/32). O∶6 (STh+CS21+mphA) resistant clone was first detected in 2016. Conclusion: A new epidemic clone in adult ETEC diarrhea cases in Shanghai was O∶6 (STh+CS21+mphA). For the first time the association between azithromycin resistance gene mphA and a serum group of ETEC was observed. Multidimensional and multivariate analysis techniques based on epidemiology can help reveal the potential transmission pattern of ETEC for the accurate surveillance and early warning of outbreaks.

[Seasonality and etiological characteristics of infectious diarrhea in outpatients of 18 or above-year-old in Shanghai, 2014-2017].

Objective: To understand the seasonality and etiological characteristics of infectious diarrhea in adults from Shanghai. Methods: Adult patients with diarrhea who had visited the enteric disease clinics in 22 hospitals that carrying on the Diarrhea Comprehensive Surveillance sentinel programs in Shanghai during 2014-2017, were surveyed. Stool specimens were collected according to the different intervals of sampling and detected for 12 bacteria and 5 viruses. Concentration ratio and circular distribution method were used for data analysis. Results: From 2014 to 2017, a total of 9 573 stool specimens were collected from the targeted diarrhea patients ≥18 years old (n=96 067), through the Shanghai Diarrhea Comprehensive Surveillance program. The positive rate of detection was 46.44%. Seasonal peaks of infectious diarrhea were both seen in summer (bacteria peak, diarrheagenic Escherichia coli and Vibrio parahaemolyticus, etc.) and in winter (virus peak, Norovirus, etc.). Both bacterial and viral infections presented seasonal concentration (Raleigh's test P<0.001) but more obvious with bacterial infection. Viral infection accounted for 60.19% of the cause of infectious diarrhea. The top five predominant pathogens appeared as Norovirus, Rotavirus, diarrheagenic Escherichia coli, Vibrio parahaemolyticus, and Salmonella spp.. Conclusions: Among the adult outpatients with infectious diarrhea in Shanghai, obvious seasonality was seen, with peaks in both summer and winter. Viral infection with Norovirus in particular, appeared as the predominant source of infection. Active, continuous and comprehensive diarrhea-related surveillance programs would be able to monitor the changing dynamic of pathogen spectrum, and lead to the adoption of targeted preventive measures.

[Pathogenic characteristics of viral gastroenteritis among pediatric inpatients under five years old during 2014-2017].

Objective: To conduct a viral pathogen surveillance program on pediatric inpatients less than five years old with acute gastroenteritis in Shanghai and to better understand the pathogenic spectrum and molecular features in the target population, for setting up programs on control, prevention, medication and vaccine applications of the diseases. Methods: Fecal samples were collected from inpatients less than 5 years old who were admitted to a pediatric hospital for having acute gastroenteritis. Information related to demographic, clinical and epidemiological features of the patients was also collected. Laboratory assays including ELISA, real-time PCR and nested PCR, were performed to detect the presence of pathogens as rotavirus, calicivirus, astrovirus and adenovirus. Results: A total of 1 018 samples were collected (male 671 and 347 female), with the positive detection rate as 40.57% which peaked from autumn till winter, annually. Calicivirus and rotavirus A presented with the highest detection rates (24.75% and 13.95% respectively). The lowest detection rate was found in the 0-6 month-olds (32.20%). 65% of the patients with positive virus had received antibiotic treatment prior to the hospitalization. However, no statistically significant difference was seen, regarding the rates of antibiotic medication in the virus positive or negative populations (P>0.05). Data from the Rotavirus genotype analysis revealed that G9P[8] genotype was the predominant strain, and causing majority of rotavirus infections in all the age groups. Conclusions: Among the inpatients under 5 years of age in Shanghai, the positive detection rate for Calicivirus was higher than that for rotavirus group A, suggesting the necessity to carefully monitor the changes regarding the pathogenic spectrum and subtypes of the virus. Antibiotics should also be attentively administered, together with the development of suitable vaccine.

[Epidemiological characteristics and antibiotic resistance of Campylobacter spp. among diarrhea outpatients in Shanghai, 2013-2016].

Objective: To understand the epidemiological characteristics and antibiotic resistance profiles of Campylobacter spp. in Shanghai from 2013 to 2016. Methods: Stool samples collected from diarrhea outpatients were cultured for Campylobacter spp., using the membrane filter method in 23 hospitals under the sentinel programs, from 2013 to 2016. All the strains were identified by biochemical tests and PCR. Broth microdilution method was used to investigate the antibiotic resistance of 179 Campylobacter spp. strains that including azithromycin, ciprofloxacin, erythromycin, gentamicin, tetracycline, nalidixic acid, telycin, klinthromycin and flurbenicol. Results: A total of 179 Campylobacter spp. strains were isolated from 10 444 stool samples (1.7%). Campylobacter jejuni and Campylobacter coli appeared as the predominant ones (94.4% and 5.6%). The incidence rate was higher in children than that in adults, with peaks of infections mainly from April to June and October to December. Campylobacter jejuni strains seemed highly resistant to ciprofloxacin (96.4%), tetracycline (83.4%) and nalidixic acid (81.7%). The resistant rates appeared higher on Campylobacter coli strains that isolated from patients. Some strains were resistant to multi-drugs. Conclusions: Campylobacter spp. seemed one of the important pathogens that isolated from outpatients with diarrhea, in Shanghai. Both age and season related characteristics of Campylobacter spp. were seen. Campylobacter spp. isolated from patients was highly resistant to ciprofloxacin, tetracycline and nalidixic acid.

[Pathogenic characteristics of hospitalized severe acute respiratory infections in Shanghai, China, 2015-2017].

Objective: To understand the epidemiological and pathogenic characteristics of hospitalized severe acute respiratory infections (SARI) in Shanghai, China. Methods: From 2015 to 2017, one Tertiary hospital and one Secondary hospital were chosen as the surveillance sites. Two respiratory tract specimens per case were collected from SARI cases aged 15 years and older. One specimen was tested for 22 respiratory pathogens by RT-PCR, and the other specimen was cultured for 6 respiratory bacteria. Results: A total of 287 SARI cases were enrolled for sampling and lab testing. 70.73% of the cases were aged 60 years and older, with 41.46% (119/287) were positive for at least one pathogen. Influenza virus was the predominant pathogen, accounting for 17.77% (51/287) of all SARI cases. Human rhinovirus/Enterovirus and Coronavirus were both accounting for 7.32% (21/287), followed by Mycoplasma pneumoniae (5.57%, 16/287). The positive rates of parainfluenza virus, bocavirus, adenovirus, respiratory syncytial virus and human metapneumo virus were all less than 5%. Bacterial strains were identified in seven SARI cases, including Klebsiella pneumoniae (3 strains), Staphylococcus aureus (2 strains), Streptococcus pneumoniae (1 strain) and Pseudomonas aeruginosa (1 strain). Two or Three pathogens were co-detected from 40 cases, accounting for 33.61% of 119 positive cases. The most common co-detected pathogens were influenza virus and Mycoplasma pneumoniae (10 cases). Influenza cases peaked in winter-spring and summer. Mycoplasma pneumoniae peaked in winter-spring season and overlapped with influenza. The positive rates of pathogens were not significantly different between different age groups. Conclusions: Various respiratory pathogens can be detected from SARI cases aged 15 years and older. Influenza virus was the predominant pathogen and the co-detection of influenza virus with Mycoplasma pneumoniae the most common one.

[Epidemiological and etiological surveillance study of infectious diarrhea in Shanghai in 2013-2015].

Objective: To analyze the pathogen spectrum and epidemiological characters of infectious diarrhea cases in Shanghai from July 2013 to June 2015. Methods: From July 2013 to June 2015, using multi-stage sampling to select 22 hospitals as adult diarrhea surveillance sentinels, and 3 district central hospitals together with Shanghai municipal children's hospital as children diarrhea surveillance sentinels. A total of 6 267 diarrhea cases were sampled. The demographic characters, main clinical manifestations and signs of cases were investigated, and stool specimens were collected to conduct the bacteria culture and virus nucleic acid detection. The difference of positive rate of bacteria and virus in different seasons, and age difference of relevant pathogens were compared. Results: Among 6 267 cases, 3 262 (52.05%) were male, and most of them were in 50-69 years group (1 999 cases, 31.90%) and 30-49 years group (1 691 cases, 26.98%). Totally, 2 783 positive specimens were detected out of 6 267 specimens with the positive rate being 44.41%. A total of 7 kinds of bacteria and 5 kinds of viruses were detected. The positive rate for bacterium was 12.61%(790 cases), mainly being vibrio parahaemolyticus (256 cases), diarrheagenic E.coli (239 cases) and salmonella (209 cases). The positive rate for virus was 31.80% (1 993 cases), mainly being norovirus (1 172 cases) and rotavirus (546 cases). The positive rates of viruses in winter of 2013 and 2014 were 57.89%(429 cases) and 62.14%(522 cases) respectively, higher than those of bacterium in the same periods which were 2.43% (18 cases) and 2.86% (24 cases) respectively. The positive rates of bacterium in summer of 2013, 2014 and 2015 were 14.56% (97 cases), 25.29%(258 cases) and 21.21% (49 cases), also higher than those of viruses in the same periods which were 7.51% (50 cases), 8.43%(86 cases) and 11.69% (27 cases) respectively, all of the above differences were statistically significant (P<0.05). The cases of 50-69 years group had the highest positive rate of pathogen, which was 47.57% (951/1 999). Secondly was those of 30-49 years group with 47.13% (797/1 691). Positive rate of pathogen for cases of 0-2 years group was 35.74% (213/596). Conclusion: The pathogen spectrumes of infectious diarrhea in Shanghai are mainly vibrio parahaemolyticus, diarrheagenic E.coli, salmonella, norovirus and rotavirus. The summer and winter season peak was obvious. Specific etiological surveillance and control strategies should be strengthened aiming at focus groups including children and the elderly in different seasons.

Case-control study of risk factors for human infection with avian influenza A(H7N9) virus in Shanghai, China, 2013.

The first human infection with avian influenza A(H7N9) virus was reported in Shanghai, China in March 2013. An additional 32 cases of human H7N9 infection were identified in the following months from March to April 2013 in Shanghai. Here we conducted a case-control study of the patients with H7N9 infection (n = 25) using controls matched by age, sex, and residence to determine risk factors for H7N9 infection. Our findings suggest that chronic disease and frequency of visiting a live poultry market (>10 times, or 1-9 times during the 2 weeks before illness onset) were likely to be significantly associated with H7N9 infection, with the odds ratios being 4.07 [95% confidence interval (CI) 1.32-12.56], 10.61 (95% CI 1.85-60.74), and 3.76 (95% CI 1.31-10.79), respectively. Effective strategies for live poultry market control should be reinforced and ongoing education of the public is warranted to promote behavioural changes that can help to eliminate direct or indirect contact with influenza A(H7N9) virus.

Deaths of tuberculosis patients in urban China: a retrospective cohort study.

BACKGROUND: The case-fatality rate for tuberculosis (TB) remains high in China, whereas risk factors for deaths among TB cases are still unclear. DESIGN: This was a retrospective cohort study of all pulmonary TB patients registered in four districts of Shanghai from 2004 to 2008. Data were derived from the China National TB Surveillance System. A total of 4271 patients were followed up in communities. Data were analysed using Cox regression. RESULTS: The percentage of all-cause deaths in the study population was 15% 2-6 years after the most recent TB diagnosis. TB was responsible for only 17% of all deaths; male sex was significantly associated with all-cause deaths. After adjustment for sex, age and treatment history, four factors were significantly and independently associated with increased risk of death: psychopathy, chronic bronchitis, cancer and the presence of multiple diseases. TB was responsible for 7.2 potential years of life lost (PYLL); PYLL was higher in females (8.2) than males (5.3). CONCLUSIONS: TB remains a significant problem in urban China. Implementation of improved clinical management, prevention strategies and other public health programmes should target TB patients with chronic conditions, particularly those with multiple diseases.

The amelogenin C-terminus is required for enamel development.

The abundant amelogenin proteins are responsible for generating proper enamel thickness and structure, and most amelogenins include a conserved hydrophilic C-terminus. To evaluate the importance of the C-terminus, we generated transgenic mice that express an amelogenin lacking the C-terminal 13 amino acids (CTRNC). MicroCT analysis of TgCTRNC29 teeth (low transgene number) indicated that molar enamel density was similar to that of wild-type mice, but TgCTRNC18 molar enamel (high transgene number) was deficient, indicating that extra transgene copies were associated with a more severe phenotype. When amelogenin-null (KO) and TgCTRNC transgenic mice were mated, density and volume of molar enamel from TgCTRNCKO offspring were not different from those of KO mice, indicating that neither TgCTRNC18 nor TgCTRNC29 rescued enamel's physical characteristics. Because transgenic full-length amelogenin partially rescues both density and volume of KO molar enamel, it was concluded that the amelogenin C-terminus is essential for proper enamel density, volume, and organization.

Transgenic mice that express normal and mutated amelogenins.

Amelogenin proteins are secreted by ameloblasts within the enamel organ during tooth development. To better understand the function of the 180-amino-acid amelogenin (M180), and to test the hypothesis that a single proline-to-threonine (P70T) change would lead to an enamel defect similar to amelogenesis imperfecta (AI) in humans, we generated transgenic mice with expression of M180, or M180 with the proline-to-threonine (P70T) mutation, under control of the Amelx gene regulatory regions. M180 teeth had a relatively normal phenotype; however, P70T mineral was abnormally porous, with aprismatic regions similar to those in enamel of male amelogenesis imperfecta patients with an identical mutation. When Amelx null females were mated with P70T transgenic males, offspring developed structures similar to calcifying epithelial odontogenic tumors in humans. The phenotype argues for dominant-negative activity for the P70T amelogenin, and for the robust nature of the process of amelogenesis.

Tooth enamel defects in mice with a deletion at the Arhgap 6/Amel X locus.

The amelogenin proteins regulate enamel mineral formation in the developing tooth. The human AMELX gene, which encodes the amelogenin proteins, is located within an intron of the Arhgap 6 gene. ARHGAP 6 encodes a Rho GAP, which regulates activity of Rho A, a small G protein involved in intracellular signal transduction. Mice were generated in which the entire ARHGAP 6 gene was deleted by Cre-mediated recombination, which also removed the nested Amel X gene. Enamel from these mice appeared chalky white, and the molars showed excessive wear. The enamel layer was hypoplastic and non-prismatic, whereas other dental tissues had normal morphology. This phenotype is similar to that reported for Amel X null mice, which have a short deletion that removed the region surrounding the translation initiation site, and resembles some forms of X-linked amelogenesis imperfecta in humans. Analysis of the enamel from the Arhgap 6/Amel X-deleted mice verifies that the Amel X gene is nested within the murine Arhgap 6 gene and shows that removal of the entire Amel X gene leads to a phenotype similar to the earlier Amel X null mouse results, in which no amelogenin protein was detected. However, an unusual layer of aprismatic enamel covers the enamel surface, which may be related to the 1.1-Mb deletion, which included Arhgap 6 in these mice.

The small bovine amelogenin LRAP fails to rescue the amelogenin null phenotype.

Amelogenins are the most abundant secreted proteins in developing dental enamel. These evolutionarily-conserved proteins have important roles in enamel mineral formation, as mutations within the amelogenin gene coding region lead to defects in enamel thickness or mineral structure. Because of extensive alternative splicing of the primary RNA transcript and proteolytic processing of the secreted proteins, it has been difficult to assign functions to individual amelogenins. To address the function of one of the amelogenins, we have created a transgenic mouse that expresses bovine leucine-rich amelogenin peptide (LRAP) in the enamel-secreting ameloblast cells of the dental organ. Our strategy was to breed this transgenic mouse with the recently generated amelogenin knockout mouse, which makes none of the amelogenin proteins and has a severe hypoplastic and disorganized enamel phenotype. It was found that LRAP does not rescue the enamel defect in amelogenin null mice, and enamel remains hypoplastic and disorganized in the presence of this small amelogenin. In addition, LRAP overexpression in the transgenic mouse (wildtype background) leads to pitting in the enamel surface, which may result from excess protein production or altered protein processing due to minor differences between the amino acid compositions of murine and bovine LRAP. Since introduction of bovine LRAP into the amelogenin null mouse does not restore normal enamel structure, it is concluded that other amelogenin proteins are essential for normal appearance and function.

A new frameshift mutation encoding a truncated amelogenin leads to X-linked amelogenesis imperfecta.

The amelogenin proteins are the most abundant organic components of developing dental enamel. Their importance for the proper mineralization of enamel is evident from the association between previously identified mutations in the X-chromosomal gene that encodes them and the enamel defect amelogenesis imperfecta. In this investigation, an adult male presenting with a severe hypoplastic enamel phenotype was found to have a single base deletion at the codon for amino acid 110 of the X-chromosomal 175-amino acid amelogenin protein. The proband's mother, who also has affected enamel, carries the identical deletion on one of her X-chromosomes, while the father has both normal enamel and DNA sequence. This frameshift mutation deletes part of the coding region for the repetitive portion of amelogenin as well as the hydrophilic tail, replacing them with a 47-amino acid segment containing nine cysteine residues. While greater than 60% of the protein is predicted to be intact, the severity of this phenotype illustrates the importance of the C-terminal region of the amelogenin protein for the formation of enamel with normal thickness.

Amelogenin-deficient mice display an amelogenesis imperfecta phenotype.

Dental enamel is the hardest tissue in the body and cannot be replaced or repaired, because the enamel secreting cells are lost at tooth eruption. X-linked amelogenesis imperfecta (MIM 301200), a phenotypically diverse hereditary disorder affecting enamel development, is caused by deletions or point mutations in the human X-chromosomal amelogenin gene. Although the precise functions of the amelogenin proteins in enamel formation are not well defined, these proteins constitute 90% of the enamel organic matrix. We have disrupted the amelogenin locus to generate amelogenin null mice, which display distinctly abnormal teeth as early as 2 weeks of age with chalky-white discoloration. Microradiography revealed broken tips of incisors and molars and scanning electron microscopy analysis indicated disorganized hypoplastic enamel. The amelogenin null phenotype reveals that the amelogenins are apparently not required for initiation of mineral crystal formation but rather for the organization of crystal pattern and regulation of enamel thickness. These null mice will be useful for understanding the functions of amelogenin proteins during enamel formation and for developing therapeutic approaches for treating this developmental defect that affects the enamel.

Model system for evaluation of alternative splicing: exon skipping.

Alternative splicing of the primary RNA transcript is a common mechanism for generating protein diversity. A model system was developed to study this process in vitro that is useful for evaluation of splicing of transcripts expressed in cells that do not grow well in culture. The system was used to analyze skipping of exon 4 of the amelogenin message, normally expressed in ameloblast cells for a short interval during tooth enamel development. Amelogenins are highly conserved proteins resulting from extensive alternative splicing, with domains involved in a range of functions, including mineral formation and intercellular signaling. In the bovine gene, the very short intron 4 was predicted to inhibit inclusion of exon 4, because in murine ameloblasts, exon 4 is detectably included in mRNA, and intron 4 is longer than the bovine counterpart. Bovine intron 4 was lengthened, and this size increase enhanced exon 4 inclusion sixfold to eightfold, although splice site selection was inaccurate. Intron length, therefore, is not the sole determinant controlling amelogenin exon 4 inclusion, and cis-acting inhibitory elements may also be involved in exon skipping. This vector system allows evaluation of splicing of a tissue-specific RNA by focusing on exons of interest through transfection of heterologous cultured cells without complications attributable to background transcription of the gene being evaluated.

Comparison of upstream regions of X- and Y-chromosomal amelogenin genes.

The amelogenin genes encode abundant enamel proteins that are required for the development of normal tooth enamel. These genes are active only in enamel-forming ameloblasts within the dental organ of the developing tooth, and are part of a small group of genes that are active on both sex chromosomes. The upstream regions of the bovine X- and Y-chromosomal and the sole murine X-chromosomal amelogenin genes have been cloned and sequenced, and conservation at nearly 60% is found in the 300 bp upstream of exon 1 for the 3 genes. A region of the bovine X-chromosomal gene that has inhibitory activity when assayed by gene transfer into heterologous cells includes motifs that have a silencing activity in other genes, and may be important to the mechanism that represses amelogenin expression in non-ameloblast cells in vivo. A comparison of sequences from three genes has led to the identification of several regions with conserved motifs that are strong candidates for having positive or negative regulatory functions, and these regions can now be tested further for interaction with nuclear proteins, and for their ability to regulate expression in vivo.

DNA sequences of amelogenin genes provide clues to regulation of expression.

The amelogenins are a heterogeneous group of enamel proteins, which have an important function in enamel formation, as mutations in the amelogenin gene result in the enamel defect amelogenesis imperfecta. The cDNAs that encode murine, bovine, human, porcine, rat and opossum amelogenins have been cloned, and as many as nine alternatively spliced messages can be produced from a single primary transcript, explaining some of the protein heterogeneity. Bovine and human amelogenin genes are found on both X and Y chromosomes, and the sexually dimorphic proteins would have 87-93% identity. A comparison of genes from human, bovine and mouse indicates that they are organized into seven exons, and sequences are highly homologous among species. Bovine, murine and human upstream regions also have similarities, with consensus sequences for potential binding of transcription factors, such as AP1 and CTF/NF1. Transgenic mouse studies have shown that 2300-3500 bp of upstream region are sufficient for expression, while 900 bp are insufficient. Analysis of DNA sequence has identified (a) major homology between species for coding exons with the exception of exon 4, (b) similarities in upstream regions likely involved in tissue specific regulation of expression, and (c) sequences at the RNA splice sites which may determine exon inclusion or skipping.