Science for the Next Century: Deep Phenotyping.

Our ability to unravel the mysteries of human health and disease have changed dramatically over the past 2 decades. Decoding health and disease has been facilitated by the recent availability of high-throughput genomics and multi-omics analyses and the companion tools of advanced informatics and computational science. Understanding of the human genome and its influence on phenotype continues to advance through genotyping large populations and using "light phenotyping" approaches in combination with smaller subsets of the population being evaluated using "deep phenotyping" approaches. Using our capability to integrate and jointly analyze genomic data with other multi-omic data, the knowledge of genotype-phenotype relationships and associated genetic pathways and functions is being advanced. Understanding genotype-phenotype relationships that discriminate human health from disease is speculated to facilitate predictive, precision health care and change modes of health care delivery. The American Association for Dental Research Fall Focused Symposium assembled experts to discuss how studies of genotype-phenotype relationships are illuminating the pathophysiology of craniofacial diseases and developmental biology. Although the breadth of the topic did not allow all areas of dental, oral, and craniofacial research to be addressed (e.g., cancer), the importance and power of integrating genomic, phenomic, and other -omic data are illustrated using a variety of examples. The 8 Fall Focused talks presented different methodological approaches for ascertaining study populations and evaluating population variance and phenotyping approaches. These advances are reviewed in this summary.

Rare bone diseases and their dental, oral, and craniofacial manifestations.

Hereditary diseases affecting the skeleton are heterogeneous in etiology and severity. Though many of these conditions are individually rare, the total number of people affected is great. These disorders often include dental-oral-craniofacial (DOC) manifestations, but the combination of the rarity and lack of in-depth reporting often limit our understanding and ability to diagnose and treat affected individuals. In this review, we focus on dental, oral, and craniofacial manifestations of rare bone diseases. Discussed are defects in 4 key physiologic processes in bone/tooth formation that serve as models for the understanding of other diseases in the skeleton and DOC complex: progenitor cell differentiation (fibrous dysplasia), extracellular matrix production (osteogenesis imperfecta), mineralization (familial tumoral calcinosis/hyperostosis hyperphosphatemia syndrome, hypophosphatemic rickets, and hypophosphatasia), and bone resorption (Gorham-Stout disease). For each condition, we highlight causative mutations (when known), etiopathology in the skeleton and DOC complex, and treatments. By understanding how these 4 foci are subverted to cause disease, we aim to improve the identification of genetic, molecular, and/or biologic causes, diagnoses, and treatment of these and other rare bone conditions that may share underlying mechanisms of disease.

Continuous infusion of local anaesthetic following laparoscopic hysterectomy--a randomised controlled trial.

OBJECTIVE: To estimate whether a continuous infusion of intraperitoneal local anaesthetic for 48 hours following laparoscopic hysterectomy reduced the need for opioids delivered with a patient-controlled analgesia pump. DESIGN: Double-blind randomised placebo-controlled trial. SETTING: District general hospital in the UK. POPULATION: Women undergoing a laparoscopic hysterectomy for a benign indication. METHODS: Women were randomised to receive either 0.5% levobupivicaine or 0.9% normal saline via an ON-Q elastomeric pump for 48 hours postoperatively. The amount of opioids used via the patient-controlled analgesia pump was recorded and pain was measured using an 11-point Box Scale. MAIN OUTCOME MEASURES: The primary outcome was the amount of patient-administered morphine used over the first 48 postoperative hours. Secondary outcomes were length of hospital stay, oral analgesia use and level of patient-reported pain. RESULTS: Sixty women participated and completed the trial. There was no difference (P = 0.59) in the median amount of patient-administered morphine used between the levobupivicaine (23 mg) and placebo (18.5 mg) groups; median group difference 3.0 (95% CI -7.0 to 14.0). There was also no difference in the length of hospital stay with 40% of the treatment group remaining in hospital >48 hours compared with 30% of the placebo group (P = 0.08). Pain scores at all postoperative time points remained similar, with a median group difference in pain scores of 1.0 (95% CI -1.0 to 2.0) at the end of the first postoperative day. CONCLUSIONS: Continuous infusion of 0.5% levobupivicaine into the peritoneal cavity following laparoscopic hysterectomy does not have any opioid-sparing effects.

Microgenomics of ameloblastoma.

Gene expression profiles of human ameloblastoma microdissected cells were characterized with the purpose of identifying genes and their protein products that could be targeted as diagnostic and prognostic markers as well as for potential therapeutic interventions. Five formalin-fixed, decalcified, paraffin-embedded samples of ameloblastoma were subjected to laser capture microdissection, linear mRNA amplification, and hybridization to oligonucleotide human 41,000 RNA arrays and compared with universal human reference RNA, to determine the gene expression signature. Assessment of the data by Significance Analysis of Microarrays (SAM) and cluster analysis showed that 38 genes were highly expressed (two-fold increase) in all samples, while 41 genes were underexpressed (two-fold reduction). Elements of the sonic hedgehog pathway and Wingless type MMTV integration site family were validated by immunohistochemistry. We have identified the expression of multiple genes and protein products that could serve as potential diagnostic, prognostic, and therapeutic targets.

Enamel defects and salivary methylmalonate in methylmalonic acidemia.

INTRODUCTION AND OBJECTIVE: To characterize enamel defects in patients with methylmalonic acidemia (MMA) and cobalamin (cbl) metabolic disorders and to examine salivary methylmalonate levels in MMA. SUBJECTS AND METHODS: Teeth from patients (n = 32) were evaluated for enamel defects and compared with age- and gender-matched controls (n = 55). Complementation class (mut, cblA, cblB and cblC) and serum methylmalonate levels were examined. Primary teeth from two patients were examined by light and scanning electron microscopy and salivary methylmalonate levels from two patients were analyzed. RESULTS: Enamel defects were significantly more prevalent per tooth in the affected group than the control group, across complementation types (P < 0.0001). The mut MMA subgroup had a significantly higher prevalence per individual of severe enamel defects than controls (P = 0.021), and those with enamel defects exhibited higher serum methylmalonate levels than those without (P = 0.017). Salivary methylmalonate levels were extremely elevated and were significantly higher than controls (P = 0.002). Primary teeth were free of enamel defects except for two cblC patients who exhibited severe enamel hypoplasia. One primary tooth from a cblC patient manifested markedly altered crystal microstructure. CONCLUSION: Enamel anomalies represent a phenotypic manifestation of MMA and cbl metabolic disorders. These findings suggest an association between enamel developmental pathology and disordered metabolism.

Can symptomatology help in the diagnosis of endometriosis? Findings from a national case-control study--Part 1.

OBJECTIVE: To determine the value of patient-reported symptoms in diagnosing endometriosis. DESIGN: A national case-control study. SETTING: Data from the UK General Practice Research Database for years 1992-2001. SAMPLE: A total of 5540 women aged 15-55 years, diagnosed with endometriosis, each matched to four controls without endometriosis. METHODS: Data were analysed to determine whether specific symptoms were highly indicative of endometriosis. Odds ratios for these symptoms were derived by conditional logistic regression analysis. MAIN OUTCOME MEASURES: Symptoms associated with endometriosis. RESULTS: The prevalence of diagnosed endometriosis was 1.5%. A greater proportion of women with endometriosis had abdominopelvic pain, dysmenorrhoea or menorrhagia (73%) compared with controls (20%). Compared with controls, women with endometriosis had increased risks of abdominopelvic pain (OR 5.2 [95% CI: 4.7-5.7]), dysmenorrhoea (OR 8.1 [95% CI: 7.2-9.3]), menorrhagia (OR 4.0 [95% CI: 3.5-4.5]), subfertility (OR 8.2 [95% CI: 6.9-9.9]), dyspareunia and/or postcoital bleeding (OR 6.8 [95% CI: 5.7-8.2]), and ovarian cysts (OR 7.3 [95% CI: 5.7-9.4]), and of being diagnosed with irritable bowel syndrome (IBS) (OR 1.6 [95% CI: 1.3-1.8]) or pelvic inflammatory disease (OR 3.0 [95% CI: 2.5-3.6]). Women with endometriosis were also found to consult the doctor more frequently than the controls and were twice as likely to have time off work. CONCLUSIONS: Specific symptoms and frequent medical consultation are associated with endometriosis and appear useful in the diagnosis. Endometriosis may coexist with or be misdiagnosed as pelvic inflammatory disease or IBS.

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.

mRNA expression and phenotype of odontogenic tumours in the v-Ha-ras transgenic mouse.

UNLABELLED: Ameloblastomas are the most common odontogenic neoplasia in humans, and although typically considered locally invasive and benign, frequently recur subsequent to surgical resection. The Tg.AC transgenic mouse carrying the v-Ha-ras oncogene has been found to spontaneously develop ameloblastoma-like tumours (35% by 1 year of age) that are rare in the wild type FVB background strain. OBJECTIVE: The purpose of this study was to characterise the mRNA expression of genes in the mouse tumours that are either expressed in human ameloblastomas or essential for normal odontogenesis and to correlate the expression to the histological phenotype. STUDY METHODS: Histological, immunohistochemical and RT-PCR studies were used to evaluate clinically demonstrable odontogenic tumours occurring spontaneously in seven Tg.AC v-Ha-ras transgenic mice (homozygous, at 7 months of age or heterozygous at 11 months of age). RESULTS: Most genes profiled were expressed in all tumour samples, however three (amelogenin, matrix metalloproteinase-20 (MMP-20) and Dlx7) displayed differential expression. In addition, only the most highly differentiated tumour stained positively for collagen. In most cases, the variable expression could be explained by reference to the histological phenotype, although differences in gene expression were apparent within the Type 2 and the mixed phenotype tumours. CONCLUSIONS: These data confirm that many of the genes thought to be important in odontogenesis and odontogenic tumour formation in humans are also expressed in these murine ameloblastoma-like tumours however genes associated with terminal differentiation of ameloblasts demonstrate differential expression between the tumour phenotypes.

Characterization and mRNA expression in an unusual odontogenic lesion in a patient with tricho-dento-osseous syndrome.

UNLABELLED: Odontogenic lesions are rare, but can be associated with significant morbidity. While their molecular determinants are unknown, they likely express many genes common to normal odontogenesis. This study evaluated the histology and mRNA expression of an unusual odontogenic lesion in a patient with a confirmed history of tricho-dento-osseous syndrome. METHODS: Decalcified, frozen 8 micro m sections of the lesion were cut and mounted on glass slides and stained with hematoxylin/eosin for analysis. The expression of multiple genes associated with normal odontogenesis and related pathologies were evaluated by RT-PCR, where possible in samples of the hard and soft tissue components of the lesion. RESULTS: Histological examination showed the lesion had large areas of irregular, dentine-like material, enamel matrix, areas of woven immature bone and multiple fully mineralised tooth crowns. Although most of the gene transcripts were amplified from both samples, some, including DLX3/7 and Collagen I demonstrated differential expression. CONCLUSIONS: This study shows the gene expression profile of aberrant odontogenesis with associated odontoma formation is similar to that of normal tooth and the genes expressed in other odontogenic lesions. While the role of altered gene expression in the development of such lesions has previously been postulated from transgenic models, this is the only report of an odontogenic lesion in a patient with TDO, and begins to elucidate possible gene interactions key to its development.

Altered pH regulation during enamel development in the cystic fibrosis mouse incisor.

Regulation of pH is necessary to the production of an environment conducive to enamel growth and mineralization. We hypothesize that abnormal extracellular pH in the enamel matrix of mice with the cystic fibrosis gene knocked out (CF mice) results in altered enamel mineralization. The enamel matrix pH during amelogenesis was studied in 10 normal and 10 CF mice. Freshly dissected incisors were immersed in pH indicator or glyoxal bis (2-hydro-xyanil) (GBHA). The normal mouse enamel matrix pH was generally higher and modulated differently than did the CF mouse enamel. GBHA staining showed that normal mice had 2 well-demarcated bands in the maturation zone that correlated to the neutral pH zones, while CF mice showed no staining. These results indicate that CFTR plays a role in pH regulation during enamel development and that a reduced pH results in a lack of calcium influx during enamel maturation and hypomineralization of the CF incisor enamel.

Cystic fibrosis transmembrane regulator gene (CFTR) is associated with abnormal enamel formation.

Cystic fibrosis (CF), a chloride ion transport disorder, is caused by mutations of the cftr gene and is the most common autosomal-recessive heritable disease in Caucasians. CFTR knockout mice have enamel with crystallite defects, retained protein, and hypomineralization, suggesting a role for CFTR in enamel formation and mineralization. This investigation examined CFTR expression and elemental composition in developing murine incisor teeth. RT-PCR showed cftr mRNA expression in the normal mouse apical incisor tissue but not in the CFTR knockout tissue. Elemental analysis by energy-dispersive x-ray spectroscopy showed relatively decreased chloride in secretory-stage CF enamel. Iron and potassium were significantly increased, and calcium was significantly decreased (p value = 0.05) in the CF mature enamel. Abnormal enamel mineralization, ion concentrations, and molecular evidence of cftr mRNA expression by odontogenic cells strongly suggest that CFTR plays an important role in enamel formation.

The genome projects: implications for dental practice and education.

Information from the Human Genome Project (HGP) and the integration of information from related areas of study and technology will dramatically change health care for the craniofacial complex. Approaches to risk assessment and diagnosis, prevention, early intervention, and management of craniofacial conditions are and will continue to evolve through the application of this new knowledge. While this information will advance our health care abilities, it is clear that the dental profession will face challenges regarding the acquisition, application, transfer, and effective and efficient use of this knowledge with regards to dental research, dental education, and clinical practice. Unraveling the human genomic sequence now allows accurate diagnosis of numerous craniofacial conditions. However, the greatest oral disease burden results from dental caries and periodontal disease that are complex disorders having both hereditary and environmental factors determining disease risk, progression, and course. Disease risk assessment, prevention, and therapy, based on knowledge from the HGP, will likely vary markedly for the different complex conditions affecting the head and neck. Integration of Information from the human genome, comparative and microbial genomics, proteomics, bioinformatics, and related technologies will provide the basis for proactive prevention and intervention and novel and more efficient treatment approaches. Oral health care practitioners will increasingly require knowledge of human genetics and the application of new molecular-based diagnostic and therapeutic technologies.

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.

Race and the decision to refer for coronary revascularization: the effect of physician awareness of patient ethnicity.

OBJECTIVES: We sought to assess whether there were differences, relative to racial ethnicity, in coronary revascularization recommendations made by a panel that had no knowledge of the patients' ethnicity. BACKGROUND: Coronary revascularization is employed less frequently in African American than in white patients. It is unclear whether this utilization pattern is driven by clinical differences between the two populations or by nonclinical factors. METHODS: Data were reviewed from 938 (26.5% African American, 73.5% white) consecutive cardiac catheterizations done between 1993 and 1995. Revascularization recommendations were made by cardiologists and cardiothoracic surgeons provided with the patients' clinical and angiographic data, but without knowledge of their ethnicity. Revascularization recommendations were compared between African American and white patients and correlated with clinical characteristics. RESULTS: No difference was noted in the percentage of African American and white patients recommended for revascularization, without reference to whether the recommendation was for percutaneous transluminal coronary angioplasty (PTCA) or for coronary artery bypass graft surgery (CABG) 40 vs. 46%, p = NS). African Americans were recommended more frequently for PTCA (22 vs. 18%, p = NS), whereas CABG was recommended for more white patients (28 vs. 18%, p = 0.002). Significantly fewer African Americans had disease in the left main or left anterior descending coronary artery or in multiple arteries. After adjusting for age, co-morbidity, left ventricular dysfunction and the extent of coronary disease, African Americans were more likely to have a recommendation for PTCA (odds ratio [OR] 1.42, 95% confidence interval [CI] 0.96 to 2.11, p = 0.08) and less likely to have a recommendation for CABG (OR 0.59, 95% CI 0.37 to 0.94, p = 0.02). CONCLUSIONS: This study suggests that when only clinical factors are considered, the rates of recommendations for revascularization will be similar for white and African American patients; but the type of revascularization procedure may differ by ethnicity and may depend, in part, on clinical factors.

Relationship between 24-H blood pressure and sleep disordered breathing in a normotensive community sample.

Sleep disordered breathing (SDB) and hypertension are commonly associated. In this study, we assessed how longitudinal measures of SDB predict a 24-h ambulatory blood pressure monitoring (ABPM) profile. Participants (n = 82) were recruited from a community-based urban (26% African American) sample and included family members of patients with laboratory diagnosed SDB (cases) and family members of neighborhood control subjects evaluated at baseline and at 5 years. Nearly all participants were normotensive and were not receiving therapy for SDB. During both examinations, the respiratory distress index (RDI) was assessed with overnight in-home polysomnography. Seated blood pressure (BP) was assessed at a baseline examination (t,) and after a 5-year follow-up period (t5), when 24-h ABPM also was performed. The change in RDI (t5-t1) over 5 years was significantly associated with 24-h mean systolic blood pressure (SBP) (P = .04), 24-h maximum diastolic blood pressure (DBP) (P = .03), sleep mean SBP (P = .05), sleep mean DBP (P < .05), and sleep maximum SBP (P = .02). Regression analysis revealed that average 24-h mean arterial pressure (MAP) and mean 24-h DBP were each best predicted by change in RDI, explaining 5% of the variance in these 24-h BP readings, and by current smoking status. After accounting for these variables, BP was not predicted by any of the other potential confounders (all P > .10). Mean RDI (averaged between t5 and t1) was associated with mean MAP, mean SBP, and maximal SBP measured during sleep. This study documents for the first time the association between changes in sleep apnea activity and BP and in a community-based normotensive sample. Further long-term evaluation of the effects of these findings and the long-term consequences of hypertension are needed.

Laparoscopic treatment of complete obliteration of the cul-de-sac associated with endometriosis: long-term follow-up of en bloc resection.

OBJECTIVE: To evaluate symptom relief following a laparoscopic technique designed for treatment of complete obliteration of the cul-de-sac associated with endometriosis, with fertility preserved. DESIGN: Preoperative and postoperative questionnaire study of a cohort of patients with complete obliteration of the cul-de-sac undergoing a standardized laparoscopic surgical treatment. SETTING: American tertiary referral center for the surgical treatment of endometriosis. PATIENTS: Eighty-four consecutive patients undergoing laparoscopic treatment of endometriosis with complete cul-de-sac obliteration with 67 replying to a postoperative questionnaire. INTERVENTIONS: Laparoscopic excision of all endometriosis including treatment of complete obliteration of the cul-de-sac by en bloc resection and bowel resection as needed. MAIN OUTCOME MEASURES: Symptom relief as measured on a 5-point ranked ordinal scale administered before and after surgery, as well as perioperative complications, postoperative fertility, and prognostic value of preoperative findings on pelvic examination. RESULTS: Symptom reduction was obtained for all symptoms related to cul-de-sac disease, particularly for patients with severe or debilitating symptoms preoperatively. There was no significant complication, and the postoperative fertility rate was 43%. Seventy-three percent of patients with obliteration of the cul-de-sac had histologically proved rectal endometriosis. Nodularity and tenderness on examination were predictive of symptom improvement. CONCLUSIONS: Aggressive laparoscopic excision of endometriosis carried out in a specialist center offers good symptom relief, especially for those with severe or debilitating symptoms. To ensure complete removal of all disease, intestinal surgery is required in most patients with complete obliteration of the cul-de-sac.

Novel COL1A1 mutation (G559C) [correction of G599C] associated with mild osteogenesis imperfecta and dentinogenesis imperfecta.

A genotype-phenotype analysis of a three-generation family segregating for an autosomal-dominant osteogenesis imperfecta (OI) variant is reported here. The family was ascertained through the presentation of a proband concerned about discoloration of her teeth, found to be dentinogenesis imperfecta (DGI). Examination of 36 family members identified 15 individuals with DGI. Linkage studies were performed for genetic markers from candidate intervals known to contain genes responsible for DGI on chromosomes 4q, 7q, and 17q. Conclusive evidence for linkage of DGI was obtained to genetic markers on chromosome 17q21-q22 (DLX-3, Z(max) = 5.34, theta = 0.00). All DGI-affected family members shared a common haplotype, which was not present in individuals without DGI. Haplotype analysis sublocalized the gene to a 5-cM genetic interval that contained the collagen 1 alpha 1 (COL1A1) gene. More than 150 different COL1A1 gene mutations have been associated with various forms of OI, and five of these have been associated with DGI and type IV OI. After excluding these five mutations, mutational analysis was performed on the remaining exons including intron--exon boundaries, which resulted in identification of a Gly559Cys mutation in exon 32, present in all DGI-affected family members. Clinical features segregating with this G559C mutation included hyperextensible joints, joint pain and an increased propensity for bone fractures with moderate trauma. This is the first report of joint pain associated with a COL1A1 mutation and DGI. The mild skeletal features and reduced penetrance of the non-dental findings illustrate the importance of genetic evaluations for families with a history of DGI.

Clinical evaluation of and parental satisfaction with resin-faced stainless steel crowns.

PURPOSE: This study evaluated the clinical success and parental acceptance of anterior primary dentition caries treatment with prefabricated resin-faced stainless steel crowns. METHODS: A retrospective analysis of maxillary anterior primary dentition caries treatment using Whiter Biter II Crowns was performed. Each crown was evaluated for retention, fracture, interface failure, color match, marginal integrity, and surface texture. Parental satisfaction regarding the esthetics of the crowns was evaluated by survey. RESULTS: Thirty-eight crowns were evaluated in 12 children. The average crown age at time of examination was 20.7 months. Three teeth were lost to trauma with all other crowns remaining intact. Twelve crowns (32%) showed loss of at least some facial resin. Nine crowns (24%) had complete loss of the resin facing. Overall parental satisfaction with the treatment was excellent, however, satisfaction with crown esthetics received the lowest rating. CONCLUSIONS: While parental satisfaction with treatment of anterior primary dentition caries with prefabricated resin-faced stainless steel crowns is excellent, the high failure rate of the resin facings is problematic.

Coronary heart disease in African Americans.

African Americans have the highest overall mortality rate from coronary heart disease (CHD) of any ethnic group in the United States, particularly out-of-hospital deaths, and especially at younger ages. Although all of the reasons for the excess CHD mortality among African Americans have not been elucidated, it is clear that there is a high prevalence of certain coronary risk factors, delay in the recognition and treatment of high-risk individuals, and limited access to cardiovascular care. The clinical spectrum of acute and chronic CHD in African Americans is similar to that in whites. However, African Americans have a higher risk of sudden cardiac death and present more often with unstable angina and non-Q-wave myocardial infarction than whites. African Americans have less obstructive coronary artery disease on angiography, but may have a similar or greater total burden of coronary atherosclerosis. Ethnic differences in the clinical manifestations of CHD may be explained largely by the inherent heterogeneity of the coronary syndromes, and the disproportionately high prevalence and severity of hypertension and type 2 diabetes in African Americans. Identification of high-risk individuals for vigorous risk factor modification-especially control of hypertension, regression of left ventricular hypertrophy, control of diabetes, treatment of dyslipidemia, and smoking cessation--is key for successful risk reduction.