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Background COVID-19 vaccine supply shortages are causing concerns about compromised immunity in some countries as the interval between the first and second dose becomes longer. Conversely, countries with no supply constraints are considering administering a third dose. We assessed the persistence of immunogenicity after a single dose of ChAdOx1 nCoV-19 (AZD1222), immunity after an extended interval (44–45 weeks) between the first and second dose, and response to a third dose as a booster given 28–38 weeks after the second dose. Methods In this substudy, volunteers aged 18–55 years who were enrolled in the phase 1/2 (COV001) controlled trial in the UK and had received either a single dose or two doses of 5 × 1010 viral particles were invited back for vaccination. Here we report the reactogenicity and immunogenicity of a delayed second dose (44–45 weeks after first dose) or a third dose of the vaccine (28–38 weeks after second dose). Data from volunteers aged 18–55 years who were enrolled in either the phase 1/2 (COV001) or phase 2/3 (COV002), single-blinded, randomised controlled trials of ChAdOx1 nCoV-19 and who had previously received a single dose or two doses of 5 × 1010 viral particles are used for comparison purposes. COV001 is registered with ClinicalTrials.gov, NCT04324606, and ISRCTN, 15281137, and COV002 is registered with ClinicalTrials.gov, NCT04400838, and ISRCTN, 15281137, and both are continuing but not recruiting. Findings Between March 11 and 21, 2021, 90 participants were enrolled in the third-dose boost substudy, of whom 80 (89%) were assessable for reactogenicity, 75 (83%) were assessable for evaluation of antibodies, and 15 (17%) were assessable for T-cells responses. The two-dose cohort comprised 321 participants who had reactogenicity data (with prime-boost interval of 8–12 weeks: 267 [83%] of 321; 15–25 weeks: 24 [7%]; or 44–45 weeks: 30 [9%]) and 261 who had immunogenicity data (interval of 8–12 weeks: 115 [44%] of 261; 15–25 weeks: 116 [44%]; and 44–45 weeks: 30 [11%]). 480 participants from the single-dose cohort were assessable for immunogenicity up to 44–45 weeks after vaccination. Antibody titres after a single dose measured approximately 320 days after vaccination remained higher than the titres measured at baseline (geometric mean titre of 66·00 ELISA units [EUs; 95% CI 47·83–91·08] vs 1·75 EUs [1·60–1·93]). 32 participants received a late second dose of vaccine 44–45 weeks after the first dose, of whom 30 were included in immunogenicity and reactogenicity analyses. Antibody titres were higher 28 days after vaccination in those with a longer interval between first and second dose than for those with a short interval (median total IgG titre: 923 EUs [IQR 525–1764] with an 8–12 week interval; 1860 EUs [917–4934] with a 15–25 week interval; and 3738 EUs [1824–6625] with a 44–45 week interval). Among participants who received a third dose of vaccine, antibody titres (measured in 73 [81%] participants for whom samples were available) were significantly higher 28 days after a third dose (median total IgG titre: 3746 EUs [IQR 2047–6420]) than 28 days after a second dose (median 1792 EUs [IQR 899–4634]; Wilcoxon signed rank test p=0·0043). T-cell responses were also boosted after a third dose (median response increased from 200 spot forming units [SFUs] per million peripheral blood mononuclear cells [PBMCs; IQR 127–389] immediately before the third dose to 399 SFUs per milion PBMCs [314–662] by day 28 after the third dose; Wilcoxon signed rank test p=0·012). Reactogenicity after a late second dose or a third dose was lower than reactogenicity after a first dose. Interpretation An extended interval before the second dose of ChAdOx1 nCoV-19 leads to increased antibody titres. A third dose of ChAdOx1 nCoV-19 induces antibodies to a level that correlates with high efficacy after second dose and boosts T-cell responses. Funding UK Research and Innovation, Engineering and Physical Sciences Research Council, National Institute for Health Research, Coalition for Epidemic Preparedness Innovations, National Institute for Health Research Oxford Biomedical Research Centre, Chinese Academy of Medical Sciences Innovation Fund for Medical Science, Thames Valley and South Midlands NIHR Clinical Research Network, AstraZeneca, and Wellcome.

Since the end of the Pleistocene some 11,700 years ago, the landscape and vegetation of the Mongolian Gobi Desert has been profoundly changing, punctuated by the appearance of lakes, wetlands, and finally aridification. Vegetation communities have responded to these changes according to temperature shifts and northward to southward movements of the edges of East Asian monsoonal systems. Human groups have lived, foraged, and traveled through the landscape of the Gobi for millennia, adapting their technologies and systems of plant and animal use with the dramatic changes of flora and fauna, and likely contributed to the character of the vegetation communities in the region today. Pastoral nomads living in semi-arid regions are sometimes implicated as contributors to desertification. However, our research at the Ikh Nart Nature Reserve, Dornogovi Province, Mongolia has yielded geoarchaeological and phytolith data which show the opposite effect. Changing landscape and vegetation patterns from the Middle to Late Holocene suggest that early pastoralists might have contributed to a shift away from halophytic desert vegetation, and an increase in semi-arid desert-steppe grasses. We suggest that the halophytic succulents growing around saline ponds during the Mid-Holocene wet phase, were replaced by Stipa and other steppic grasses after pastoralists entered the region, increasing hillslope erosion which covered the saline sediments of the valley floor, and encouraged the growth of grass seeds carried in the dung of herd animals.

BACKGROUND: A safe and efficacious vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), if deployed with high coverage, could contribute to the control of the COVID-19 pandemic. We evaluated the safety and efficacy of the ChAdOx1 nCoV-19 vaccine in a pooled interim analysis of four trials. \ud \ud \ud METHODS: This analysis includes data from four ongoing blinded, randomised, controlled trials done across the UK, Brazil, and South Africa. Participants aged 18 years and older were randomly assigned (1:1) to ChAdOx1 nCoV-19 vaccine or control (meningococcal group A, C, W, and Y conjugate vaccine or saline). Participants in the ChAdOx1 nCoV-19 group received two doses containing 5 × 1010 viral particles (standard dose; SD/SD cohort); a subset in the UK trial received a half dose as their first dose (low dose) and a standard dose as their second dose (LD/SD cohort). The primary efficacy analysis included symptomatic COVID-19 in seronegative participants with a nucleic acid amplification test-positive swab more than 14 days after a second dose of vaccine. Participants were analysed according to treatment received, with data cutoff on Nov 4, 2020. Vaccine efficacy was calculated as 1 - relative risk derived from a robust Poisson regression model adjusted for age. Studies are registered at ISRCTN89951424 and ClinicalTrials.gov, NCT04324606, NCT04400838, and NCT04444674. \ud \ud \ud FINDINGS: Between April 23 and Nov 4, 2020, 23 848 participants were enrolled and 11 636 participants (7548 in the UK, 4088 in Brazil) were included in the interim primary efficacy analysis. In participants who received two standard doses, vaccine efficacy was 62·1% (95% CI 41·0-75·7; 27 [0·6%] of 4440 in the ChAdOx1 nCoV-19 group vs71 [1·6%] of 4455 in the control group) and in participants who received a low dose followed by a standard dose, efficacy was 90·0% (67·4-97·0; three [0·2%] of 1367 vs 30 [2·2%] of 1374; pinteraction=0·010). Overall vaccine efficacy across both groups was 70·4% (95·8% CI 54·8-80·6; 30 [0·5%] of 5807 vs 101 [1·7%] of 5829). From 21 days after the first dose, there were ten cases hospitalised for COVID-19, all in the control arm; two were classified as severe COVID-19, including one death. There were 74 341 person-months of safety follow-up (median 3·4 months, IQR 1·3-4·8): 175 severe adverse events occurred in 168 participants, 84 events in the ChAdOx1 nCoV-19 group and 91 in the control group. Three events were classified as possibly related to a vaccine: one in the ChAdOx1 nCoV-19 group, one in the control group, and one in a participant who remains masked to group allocation. \ud \ud \ud INTERPRETATION: ChAdOx1 nCoV-19 has an acceptable safety profile and has been found to be efficacious against symptomatic COVID-19 in this interim analysis of ongoing clinical trials. \ud \ud \ud FUNDING: UK Research and Innovation, National Institutes for Health Research (NIHR), Coalition for Epidemic Preparedness Innovations, Bill & Melinda Gates Foundation, Lemann Foundation, Rede D'Or, Brava and Telles Foundation, NIHR Oxford Biomedical Research Centre, Thames Valley and South Midland's NIHR Clinical Research Network, and AstraZeneca.

BACKGROUND: Older adults (aged ≥70 years) are at increased risk of severe disease and death if they develop COVID-19 and are therefore a priority for immunisation should an efficacious vaccine be developed. Immunogenicity of vaccines is often worse in older adults as a result of immunosenescence. We have reported the immunogenicity of a novel chimpanzee adenovirus-vectored vaccine, ChAdOx1 nCoV-19, in young adults, and now describe the safety and immunogenicity of this vaccine in a wider range of participants, including adults aged 70 years and older. METHODS: In this report of the phase 2 component of a single-blind, randomised, controlled, phase 2/3 trial (COV002), healthy adults aged 18 years and older were enrolled at two UK clinical research facilities, in an age-escalation manner, into 18-55 years, 56-69 years, and 70 years and older immunogenicity subgroups. Participants were eligible if they did not have severe or uncontrolled medical comorbidities or a high frailty score (if aged ≥65 years). First, participants were recruited to a low-dose cohort, and within each age group, participants were randomly assigned to receive either intramuscular ChAdOx1 nCoV-19 (2·2 × 1010 virus particles) or a control vaccine, MenACWY, using block randomisation and stratified by age and dose group and study site, using the following ratios: in the 18-55 years group, 1:1 to either two doses of ChAdOx1 nCoV-19 or two doses of MenACWY; in the 56-69 years group, 3:1:3:1 to one dose of ChAdOx1 nCoV-19, one dose of MenACWY, two doses of ChAdOx1 nCoV-19, or two doses of MenACWY; and in the 70 years and older, 5:1:5:1 to one dose of ChAdOx1 nCoV-19, one dose of MenACWY, two doses of ChAdOx1 nCoV-19, or two doses of MenACWY. Prime-booster regimens were given 28 days apart. Participants were then recruited to the standard-dose cohort (3·5-6·5 × 1010 virus particles of ChAdOx1 nCoV-19) and the same randomisation procedures were followed, except the 18-55 years group was assigned in a 5:1 ratio to two doses of ChAdOx1 nCoV-19 or two doses of MenACWY. Participants and investigators, but not staff administering the vaccine, were masked to vaccine allocation. The specific objectives of this report were to assess the safety and humoral and cellular immunogenicity of a single-dose and two-dose schedule in adults older than 55 years. Humoral responses at baseline and after each vaccination until 1 year after the booster were assessed using an in-house standardised ELISA, a multiplex immunoassay, and a live severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) microneutralisation assay (MNA80). Cellular responses were assessed using an ex-vivo IFN-γ enzyme-linked immunospot assay. The coprimary outcomes of the trial were efficacy, as measured by the number of cases of symptomatic, virologically confirmed COVID-19, and safety, as measured by the occurrence of serious adverse events. Analyses were by group allocation in participants who received the vaccine. Here, we report the preliminary findings on safety, reactogenicity, and cellular and humoral immune responses. This study is ongoing and is registered with ClinicalTrials.gov, NCT04400838, and ISRCTN, 15281137. FINDINGS: Between May 30 and Aug 8, 2020, 560 participants were enrolled: 160 aged 18-55 years (100 assigned to ChAdOx1 nCoV-19, 60 assigned to MenACWY), 160 aged 56-69 years (120 assigned to ChAdOx1 nCoV-19: 40 assigned to MenACWY), and 240 aged 70 years and older (200 assigned to ChAdOx1 nCoV-19: 40 assigned to MenACWY). Seven participants did not receive the boost dose of their assigned two-dose regimen, one participant received the incorrect vaccine, and three were excluded from immunogenicity analyses due to incorrectly labelled samples. 280 (50%) of 552 analysable participants were female. Local and systemic reactions were more common in participants given ChAdOx1 nCoV-19 than in those given the control vaccine, and similar in nature to those previously reported (injection-site pain, feeling feverish, muscle ache, headache), but were less common in older adults (aged ≥56 years) than younger adults. In those receiving two standard doses of ChAdOx1 nCoV-19, after the prime vaccination local reactions were reported in 43 (88%) of 49 participants in the 18-55 years group, 22 (73%) of 30 in the 56-69 years group, and 30 (61%) of 49 in the 70 years and older group, and systemic reactions in 42 (86%) participants in the 18-55 years group, 23 (77%) in the 56-69 years group, and 32 (65%) in the 70 years and older group. As of Oct 26, 2020, 13 serious adverse events occurred during the study period, none of which were considered to be related to either study vaccine. In participants who received two doses of vaccine, median anti-spike SARS-CoV-2 IgG responses 28 days after the boost dose were similar across the three age cohorts (standard-dose groups: 18-55 years, 20 713 arbitrary units [AU]/mL [IQR 13 898-33 550], n=39; 56-69 years, 16 170 AU/mL [10 233-40 353], n=26; and ≥70 years 17 561 AU/mL [9705-37 796], n=47; p=0·68). Neutralising antibody titres after a boost dose were similar across all age groups (median MNA80 at day 42 in the standard-dose groups: 18-55 years, 193 [IQR 113-238], n=39; 56-69 years, 144 [119-347], n=20; and ≥70 years, 161 [73-323], n=47; p=0·40). By 14 days after the boost dose, 208 (>99%) of 209 boosted participants had neutralising antibody responses. T-cell responses peaked at day 14 after a single standard dose of ChAdOx1 nCoV-19 (18-55 years: median 1187 spot-forming cells [SFCs] per million peripheral blood mononuclear cells [IQR 841-2428], n=24; 56-69 years: 797 SFCs [383-1817], n=29; and ≥70 years: 977 SFCs [458-1914], n=48). INTERPRETATION: ChAdOx1 nCoV-19 appears to be better tolerated in older adults than in younger adults and has similar immunogenicity across all age groups after a boost dose. Further assessment of the efficacy of this vaccine is warranted in all age groups and individuals with comorbidities. FUNDING: UK Research and Innovation, National Institutes for Health Research (NIHR), Coalition for Epidemic Preparedness Innovations, NIHR Oxford Biomedical Research Centre, Thames Valley and South Midlands NIHR Clinical Research Network, and AstraZeneca.

Background: The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) might be curtailed by vaccination. We assessed the safety, reactogenicity, and immunogenicity of a viral vectored coronavirus vaccine that expresses the spike protein of SARS-CoV-2. Methods: We did a phase 1/2, single-blind, randomised controlled trial in five trial sites in the UK of a chimpanzee adenovirus-vectored vaccine (ChAdOx1 nCoV-19) expressing the SARS-CoV-2 spike protein compared with a meningococcal conjugate vaccine (MenACWY) as control. Healthy adults aged 18–55 years with no history of laboratory confirmed SARS-CoV-2 infection or of COVID-19-like symptoms were randomly assigned (1:1) to receive ChAdOx1 nCoV-19 at a dose of 5 × 10 10 viral particles or MenACWY as a single intramuscular injection. A protocol amendment in two of the five sites allowed prophylactic paracetamol to be administered before vaccination. Ten participants assigned to a non-randomised, unblinded ChAdOx1 nCoV-19 prime-boost group received a two-dose schedule, with the booster vaccine administered 28 days after the first dose. Humoral responses at baseline and following vaccination were assessed using a standardised total IgG ELISA against trimeric SARS-CoV-2 spike protein, a muliplexed immunoassay, three live SARS-CoV-2 neutralisation assays (a 50% plaque reduction neutralisation assay [PRNT 50]; a microneutralisation assay [MNA 50, MNA 80, and MNA 90]; and Marburg VN), and a pseudovirus neutralisation assay. Cellular responses were assessed using an ex-vivo interferon-γ enzyme-linked immunospot assay. The co-primary outcomes are to assess efficacy, as measured by cases of symptomatic virologically confirmed COVID-19, and safety, as measured by the occurrence of serious adverse events. Analyses were done by group allocation in participants who received the vaccine. Safety was assessed over 28 days after vaccination. Here, we report the preliminary findings on safety, reactogenicity, and cellular and humoral immune responses. The study is ongoing, and was registered at ISRCTN, 15281137, and ClinicalTrials.gov, NCT04324606. Findings: Between April 23 and May 21, 2020, 1077 participants were enrolled and assigned to receive either ChAdOx1 nCoV-19 (n=543) or MenACWY (n=534), ten of whom were enrolled in the non-randomised ChAdOx1 nCoV-19 prime-boost group. Local and systemic reactions were more common in the ChAdOx1 nCoV-19 group and many were reduced by use of prophylactic paracetamol, including pain, feeling feverish, chills, muscle ache, headache, and malaise (all p80 and in 35 (100%) participants when measured in PRNT 50. After a booster dose, all participants had neutralising activity (nine of nine in MNA 80 at day 42 and ten of ten in Marburg VN on day 56). Neutralising antibody responses correlated strongly with antibody levels measured by ELISA (R 2=0·67 by Marburg VN; p

Since Naomi Miller’s first discussion of dung fuel within macro-botanical samples from Malyan, Iran, considerations of dung fuel across Southwest Asia have become commonplace, yet archaeobotanists remain divided on: (1) the extent to which dung fuel contributed to archaeobotanical assemblages relative to remnants of repeated crop processing and household activities; and (2) the plant-based, middle-range theories that should be used to infer the presence of dung within macro-botanical assemblages. Here we present a case-study integrating a simple, well-established geo-archaeological approach to assess the presence and relative abundance of dung spherulites within paired sediment and flotation samples from Ubaid period Tell Zeidan, Syria (5300–5100 bc). Spherulite data generated from “sediment smears” are integrated with macro-botanical and phytolith data to assess elevated concentrations of dung within samples. Our analyses demonstrate that plant-based depositional processes across a site are complex, reflecting the rich nature of plant use in antiquity. By using a multi-proxy approach, it is possible to differentiate between predominantly fuel-based deposits and those resulting from predominantly crop-processing processes with greater resolution. This study documents the use of wood fuels in hearths and dung fuel within pyrotechnic features and an oven during the Ubaid period, thereby contributing to discussions of fuel selection and the Secondary Products Revolution. When spherulites are preserved within sediment in abundance, they are also present in floated material, so it is possible to use this approach to consider the presence of dung within archived macro-botanical samples and resolve decade-old debates.

Renewed excavations at the Neolithic site of Beisamoun (Upper Jordan Valley, Israel) has resulted in the discovery of the earliest occurrence of an intentional cremation in the Near East directly dated to 7031–6700 cal BC (Pre-Pottery Neolithic C, also known as Final PPNB, which spans ca. 7100–6400 cal BC). The funerary treatment involved in situ cremation within a pyre-pit of a young adult individual who previously survived from a flint projectile injury. In this study we have used a multidisciplinary approach that integrates archaeothanatology, spatial analysis, bioanthropology, zooarchaeology, soil micromorphological analysis, and phytolith identification in order to reconstruct the different stages and techniques involved in this ritual: cremation pit construction, selection of fuel, possible initial position of the corpse, potential associated items and funerary containers, fire management, post-cremation gesture and structure abandonment. The origins and development of cremation practices in the region are explored as well as their significance in terms of Northern-Southern Levantine connections during the transition between the 8th and 7th millennia BC. The bones are distributed throughout the bottom of the pit, partly superimposed one on the other to a thickness of 40 cm. However, the density of remains was not very marked except at the centre of the pit (Fig 6). If there was an apparent anatomical disorder at first glance, by looking at the details some interesting patterning could be observed. Cranial and mandibular fragments were found only in the southern half of the structure. Next to the south wall on the upper level, we found the base of the skull (mandible reversed and occipital fragments); the rest of the cranial vault and face (frontal, maxillars, parietals and temporals) were found slightly lower down at the centre of the pit. Conversely, the cervical vertebrae were dispersed out from the centre to the northern half of the pit. The thoracic column and some of the ribs were concentrated in the centre, roughly following a west-east direction. The lumbar vertebrae were found in the middle and against the south-western wall of the structure with several vertebral fragments in close proximity to the sacrum, coccyx and the left coxal. The right coxal is found diametrically opposite to this coherent group, lying almost complete not far from the north-eastern wall of the pit. Altogether, despite an absence of articulated joints and dispersion of certain elements, the bones of the axial skeleton show some anatomical coherence.

With the growing complexity of health care, interprofessional communication and collaboration are essential to optimize the care of dental patients, including consideration of genetics. A dental case exemplifies the challenges and benefits of an interprofessional approach to managing pediatric patients with oligodontia and a family history of colon cancer. The interprofessional team includes dental, genetic, nutritional, and surgical experts.

This special issue examines new trends in phytolith scholarship and assesses the future direction of this field of research. The papers presented represent a broader shift in phytolith research into a new phase called the “Period of Expanding Applications”. It is characterized by 1) a rapid increase in the number of phytolith publications; 2) a diversification of research topics; 3) a reassessment of the use of radiocarbon and other isotopes in phytoliths; 4) the development of digital technologies for refining and sharing phytolith identifications; 5) renewed efforts for standardization of phytolith nomenclature and laboratory protocol; and 6) the development of the field of applied phytolith research. This paper argues that interdisciplinary collaborations and a continued effort to understand the basics of phytolith production patterns are essential for the growth of the discipline and its application in archaeological studies.

Early-onset periodontal diseases (EOP) are a heterogeneous group of diseases, sharing several characteristics including early onset and severe periodontal destruction. Although periodontopathogenic bacteria are primary etiologic agents in the disease process, familial aggregation of affected individuals suggests genetic factors also are important. Formal genetic studies suggest a pattern of disease transmission consistent with mendelian inheritance of a gene of major effect. This means that one or more genes could account for the observed familial pattern of some cases of early-onset periodontitis. Clearly, both genetic and environmental risk factors are important in EOP. Epidemiological studies can identify risk factors for periodontitis on the population level. Although the association of a suspected risk factor can be studied with classic epidemiological techniques, these techniques are generally not applicable to the assessment of risk in individuals. Now with the advent of molecular epidemiology, it is possible to begin development of individual susceptibility profiles that may incorporate both host genetic and environmental components. Evidence for a central role of genetic risk factors in EOP makes these diseases a practical model for such studies. This review presents the primary support for genetic risk factors for EOP diseases and presents some of the potential genetic factors that may be useful in developing molecular epidemiological risk profiles for EOP. J Periodontol 1996;67:355-366.

Background The caries lesion, the most commonly observed sign of dental caries disease, is the cumulative result of an imbalance in the dynamic demineralization and remineralization process that causes a net mineral loss over time. A classification system to categorize the location, site of origin, extent, and when possible, activity level of caries lesions consistently over time is necessary to determine which clinical treatments and therapeutic interventions are appropriate to control and treat these lesions. Methods In 2008, the American Dental Association (ADA) convened a group of experts to develop an easy-to-implement caries classification system. The ADA Council on Scientific Affairs subsequently compiled information from these discussions to create the ADA Caries Classification System (CCS) presented in this article. Conclusions The ADA CCS offers clinicians the capability to capture the spectrum of caries disease presentations ranging from clinically unaffected (sound) tooth structure to noncavitated initial lesions to extensively cavitated advanced lesions. The ADA CCS supports a broad range of clinical management options necessary to treat both noncavitated and cavitated caries lesions. Practical Implications The ADA CCS is available for implementation in clinical practice to evaluate its usability, reliability, and validity. Feedback from clinical practitioners and researchers will allow system improvement. Use of the ADA CCS will offer standardized data that can be used to improve the scientific rationale for the treatment of all stages of caries disease.

There are currently 67 dental schools in the United States and Puerto Rico. According to the 2014 edition of the Official Guide to Dental Schools For Students Entering in Fall 2015 published by the American Dental Education Association (www.adea.org), no dental schools currently require genetics prior to admission. Only one school, the University of Florida, requires one semester of molecular biology or genetics for admission. When last surveyed in 2001, only eight of 53 dental schools had a formal course in human genetics in their curricula (Dudlicek et al. 2004). Only one school did not respond to the survey. Despite calls from a variety of individuals and professional organizations that genetics should be an integral part of dental school curricula (Wright and Hart 2002; Behnke and Hassell 2004; Collins and Tabak 2004; Johnson et al. 2008; Slavkin 2014), little progress has been made to improve the teaching of human genetics to dental students. With this in mind, we once again call for dental schools to include human genetics as a formal course in their curricula. A search of the scientific literature reveals how the contribution of genetic factors to missing or misshapen teeth, cleft lip/palate, oral cancer, caries, periodontal disease and other oral pathologies and conditions continues to expand. The effects that systemic disorders can have on an individual's oral health are also well known. Gingival hyperplasia can be an isolated condition, part of a syndrome, or a side effect of certain medications. If a side effect of medication, it is reversible simply by stopping the drug. Inherited forms require surgical resection. Thus, a dentist needs to able to take a family and medical history to distinguish the forms. Associations have been made between enamel defects and kidney disease (Jaureguiberry et al. 2013), between missing teeth and colon cancer (Lammi et al. 2004), and between microdontia and deafness (Riazuddin et al. 2011). Knowing whether a dental phenotype is an isolated finding or is associated with other systemic manifestations with broader healthcare implications can lead to appropriate referrals. Dentists clearly have patients with genetic disorders in their practice. These genetic disorders may or may not have an impact on their oral health. These include Mendelian traits such as amelogenesis imperfecta and cystic fibrosis, cytogenetic disorders such as Smith–Magenis and Williams syndromes, as well as multifactorial traits such as cleft lip/cleft palate or diabetes. The last decade has seen an explosion in the number of genes associated with craniofacial development and diseases. Currently, mutations in at least seven genes are associated with amelogenesis imperfecta, a disorder of qualitative or quantitative defects of enamel (Table 1). Table 1 Genes that Cause Amelogenesis Imperfecta There are several reasons why genetics and genomics should be included in dental school criteria beyond the obvious value of making a dental diagnosis. Being able to take a family history to construct a three‐generation pedigree is crucial. While one should not discount the psychological or financial burden of missing or malformed teeth, genetic disorders may also have extraoral health consequences. For example, dentinogenesis imperfecta (DI) may occur as an isolated finding or as part of a syndrome such as osteogenesis imperfecta (OI), which is associated with bone fragility and hearing loss (Hart and Hart 2007). Mild type 1 OI may be mistaken for isolated DI (Pallos et al. 2001). Making a correct diagnosis is crucial for a discussion of phenotypic consequences, management, and genetic counseling for recurrence risks. Enamel renal syndrome is an autosomal‐recessive disorder due to mutations in the FAM20A gene. The combined dental features of amelogenesis imperfecta and gingival hyperplasia are highly suggestive of this disorder. The renal phenotype, nephrocalcinosis, is typically clinically asymptomatic in children. Dentists should refer individuals with the oral phenotype to nephrologists for evaluation (Jaureguiberry et al. 2013). Mutations in the ANIX2 gene have been associated with oligodontia and colorectal cancer (Lammi et al. 2004). Although not all patients with oligodontia have an increased risk of colorectal cancer, a history of colorectal cancer in the blood relatives of an oligodontia patient should prompt consideration of referral to either a genetic counselor or clinical geneticist for further evaluation. Dentists also need to have an understanding of genetics to appreciate the issues surrounding genetic testing. More than 3000 genetic tests currently available encompass a variety of genetic disease types. The majority of these tests for Mendelian and cytogenetic conditions are clinically valid and useful; however, they must be used properly. Unfortunately, some of the tests offered, particularly tests for small effect SNP variants marketed to predict risk for common, complex diseases are neither clinically valid nor clinically useful (SACGT 2000; Diehl et al. 2015; Ioannidis 2015). The dental provider will need to be able to evaluate these tests and decide whether or not to incorporate them into their clinical practice. In many cases, this will involve consultation with non‐dental health care providers, including geneticists and genetic counselors. As our understanding of genetics and the role of genetic factors in normal and abnormal development increases, it is imperative that dental students are taught more than just Mendelian and cytogenetic disorders. Dentists need to understand multifactorial conditions as well as appreciate how environmental exposure to microbial, viral, pharmacologic, diet, smoking, and other factors can affect the genetic and epigenetic landscape (Ambatipudi et al. 2016). This is particularly true for the dental care providers where smoking and the oral microbiome have a direct impact on the development of caries, periodontal diseases, and other oral pathologies. The patient‐educator model, in which patients tell their own stories to dental students, is a way to better engage learning and retention of knowledge. Although used extensively in the training of medical students and residents, this is a fairly new practice in dentistry (Renard et al. 2015). The authors of this study report that patient‐educators reinforce the importance of understanding basic science, including genetics, in the students’ future dental practice. Three years after experiencing patient‐educator teaching, 83% of dental students correctly diagnosed the genetic condition in a case‐based scenario. Another important facet of patient‐educators is bringing to light the psychosocial aspects of a particular disorder. Given the huge cosmetic side of dentistry, this psychosocial aspect should not be overlooked. Studies have shown that students often learn faster and are more empathetic when patient‐educators are involved (Renard et al. 2015). We believe that incorporation of patient‐educators should be adopted at some level in all dental schools. Health care providers cannot work in a vacuum. The dental practitioner needs to know where to go to look for genetic resources. When appropriate, the dentist should be part of the personalized medicine team. They need to be able to speak the language and understand basic concepts in order to interact with other members of the team in a meaningful way. This lack of genetics education limits the dentist's ability to interact with the larger health care team, further isolating dentistry from other health care disciplines. This isolation is confounded by separate health care records and distinct reimbursement systems (Regier and Hart 2016). In summary, dental graduates should have the basic genetics knowledge and skills to provide the educational foundations for understanding and applying genetics to clinical practice. While the genetic core competencies for such knowledge, skills, and attitudes will need to be developed for the dental profession, examples have been developed by others, including recommended core competencies in genetics for all health care professionals (Jenkins et al. 2001; NCHPEG 2007). The failure of dental schools to incorporate human genetics into their curricula is a failure to their students who will surely encounter patients with genetic disorders in their practice. Students without adequate training in genetics will not be prepared to effectively diagnose patients, adequately evaluate new therapies or tests based upon genomic information, nor work collaboratively with other members of the health care team. In the end, it will be the patients who suffer.

Although human gingival fibroblasts (hGFs) and human periodontal ligament fibroblasts (hPDLFs) exhibit numerous phenotypic similarities, it has been suggested that the secretory and behavioral differences, which exist between these cell types, are a result of the membrane protein composition of these cells.Four matched pairs of hGFs and hPDLFs were cultured. Before confluence, membrane-bound and -associated proteins from cells of the fourth passage were extracted. The processed protein samples were evaluated using capillary-liquid chromatography-nanospray tandem mass spectrometry. Global protein identification was performed on an orbitrap mass spectrometer equipped with a microspray source operated in positive ion mode. Proteome software was used to validate protein identifications derived from tandem mass spectrometry sequencing results.Four hundred fifty proteins were common to both hGFs and hPDLFs. Of the proteins identified, 214 were known membrane-bound or -associated proteins, and 165 proteins were known nuclear-associated proteins. Twenty-seven proteins, identified from the 450 proteins, common to both hGFs and hPDLFs, were detected in statistically significant greater quantities in either hGFs or hPDLFs. More specifically, 13 proteins were detected in significantly greater quantities in hGFs, whereas 14 proteins were detected in significantly greater quantities in hPDLFs.Distinct differences in the cellular protein catalog may reflect the dynamic role and high energy requirements of hGFs in extracellular matrix remodeling and response to inflammatory challenge as well as the role of hPDLFs in monitoring mechanical stress and maintaining tissue homeostasis during regeneration and remineralization.

Background/Aims: Calcium homeostasis requires regulated cellular and interstitial systems interacting to modulate the activity and movement of this ion. Disruption of these systems in the kidney results in nephrocalcinosis and nephrolithiasis, important medical problems whose pathogenesis is incompletely understood. Methods: We investigated 25 patients from 16 families with unexplained nephrocalcinosis and characteristic dental defects (amelogenesis imperfecta, gingival hyperplasia, impaired tooth eruption). To identify the causative gene, we performed genome-wide linkage analysis, exome capture, next-generation sequencing, and Sanger sequencing. Results: All patients had bi-allelic FAM20A mutations segregating with the disease; 20 different mutations were identified. Conclusions: This au-tosomal recessive disorder, also known as enamel renal syndrome, of FAM20A causes nephrocalcinosis and amelogenesis imperfecta. We speculate that all individuals with biallelic FAM20A mutations will eventually show nephrocalcinosis.

Genetic factors play an important etiologic role in destructive periodontal diseases. There have been reports that sex chromosomes, especially disorders associated with the X chromosome, affect periodontal health. Although numerous X-linked diseases have been reported to be associated with various periodontal diseases, the association of gingivitis and/or periodontitis with these genetic syndromes should be considered tenuous and raises the question of whether the periodontal manifestation truly arises from an underlying X-linked genetic etiology. A brief overview of genetics in relation to sex chromosomes and putative X-linked genetic periodontal diseases is given.

With the growing complexity of health care, interprofessional communication and collaboration are essential to optimize the care of dental patients, including consideration of genetics. A dental case exemplifies the challenges and benefits of an interprofessional approach to managing pediatric patients with oligodontia and a family history of colon cancer. The interprofessional team includes dental, genetic, nutritional, and surgical experts.

Agricultural origins and dispersals are subjects of fundamental importance to archaeology as well as many other scholarly disciplines. These investigations are world-wide in scope and require significant amounts of paleobotanical data attesting to the exploitation of wild progenitors of crop plants and subsequent domestication and spread. Accordingly, for the past few decades the development of methods for identifying the remains of wild and domesticated plant species has been a focus of paleo-ethnobotany. Phytolith analysis has increasingly taken its place as an important independent contributor of data in all areas of the globe, and the volume of literature on the subject is now both very substantial and disseminated in a range of international journals. In this paper, experts who have carried out the hands-on work review the utility and importance of phytolith analysis in documenting the domestication and dispersals of crop plants around the world. It will serve as an important resource both to paleo-ethnobotanists and other scholars interested in the development and spread of agriculture.

Juvenile hyaline fibromatosis (JHF) and infantile systemic hyalinosis (ISH) are rare, autosomal recessive disorders of the connective tissue caused by mutations in the gene encoding the anthrax toxin receptor 2 protein (ANTXR2) located on chromosome 4q21. Characteristically, these conditions present with overlapping clinical features, such as nodules and/or pearly papules, gingival hyperplasia, flexion contractures of the joints, and osteolytic bone defects. The present report describes a pair of sibs and three other JHF/ISH patients whose diagnoses were based on typical clinical manifestations and confirmed by histopathologic analyses and/or molecular analysis. A comparison of ISH and JHF, additional thoughts about new terminology (hyaline fibromatosis syndrome) and a modified grading system are also included.

Archaeologists use survey artifacts to study any number of interesting topics. The focus of this study is to test the usefulness of starch grains and phytoliths found on artifacts recovered during archaeological survey. Phytolith and starch grain analysis was used to determine the level of environmental contamination on three types of medieval ceramics collected during survey work from plowed fields in the parish of Wicken, Northamptonshire, England. The plant residues found on these artifacts were compared with their surrounding soil and with contemporaneous excavated artifacts recovered from under the floorboards of a medieval house in nearby Wyton, Cambridgeshire, England. Through the use of the “piggyback” approach pioneered by Chandler-Ezell and Pearsall, phytoliths and starch grains were systematically removed from the artifacts surface. Residues associated with contamination were removed from the outermost layer of the artifact surface while potentially uncontaminated residues were removed from the innermost layer of the artifact surface. Matching phytoliths and starch grains were found on the outermost surface of the survey artifacts and in the surrounding soils. No phytoliths and starch grains were found on the innermost surface of the survey artifacts suggesting that residues from the surrounding soils did not penetrate into the pores and crevices of the artifacts. The overall results suggest that, although they are preliminary, there is potential for plant residue analysis of survey artifacts.

Sahar Alrayyes, DDS, MS, and Thomas C. Hart, DDS, MS, PhD eriodontal disease among children and adolescents consists mainly of ingivitis. The prevalence of marked periodontal destruction is low in oung individuals. In the USA, the prevalence of severe periodontal ttachment loss on multiple teeth among children and young adults is etween 0.2% and 0.5%. Although periodontitis is more common in dults, it is more aggressive when present in children and adolescents. eriodontal diseases in young individuals can develop as a consequence f a local or a systemic factor. Local factors include plaque, calculus, rthodontic appliances, orthodontic appliances, and dental anomalies (ie, namel projections, enamel pearls). Systemic factors include malnutriion, systemic diseases, gender, race, hormones, and smoking. Most periodontitis cases among children and adolescents occur as a anifestation of certain systemic diseases with an impaired immune system hat compromises their response to microbial plaque and increases the ikelihood of periodontal bone loss and premature loss of their teeth. eutrophils play a major role in the human defense system against bacterial nfections and any depletion or dysfunction in its adherence and chemotaxis s seen in neutropenia, Down syndrome (DS), Papillon-LeFevre syndrome PLS), and leukocyte adhesion deficiency (LAD) can result in oral disease. he oral manifestations associated with neutrophils dysfunction are well ocumented and include oral mucous membrane infection, gingivitis, perioontitis, and premature loss of teeth manifestation. This review focuses n these disorders.

Tricho-dento-osseous (TDO) syndrome is an autosomal dominant disorder characterized by abnormalities in the thickness and density of bones and teeth. A 4-bp deletion mutation in the Distal-Less 3 (DLX3) gene is etiologic for most cases of TDO. To investigate the in vivo role of mutant DLX3 (MT-DLX3) on dentin development, we generated transgenic (TG) mice expressing MT-DLX3 driven by a mouse 2.3 Col1A1 promoter. Dentin defects were radiographically evident in all teeth and the size of the nonmineralized pulp was enlarged in TG mice, consistent with clinical characteristics in patients with TDO. High-resolution radiography, microcomputed tomography, and SEM revealed a reduced zone of mineralized dentin with anomalies in the number and organization of dentinal tubules in MT-DLX3 TG mice. Histological and immunohistochemical studies demonstrated that the decreased dentin was accompanied by altered odontoblast cytology that included disruption of odontoblast polarization and reduced numbers of odontoblasts. TUNEL assays indicated enhanced odontoblast apoptosis. Expression levels of the apoptotic marker caspase-3 were increased in odontoblasts in TG mice as well as in odontoblastic-like MDPC-23 cells transfected with MT-DLX3 cDNA. Expression of Runx2, Wnt 10A, and TBC1D19 colocalized with DLX3 expression in odontoblasts, and MT-DLX3 significantly reduced expression of all three genes. TBC1D19 functions in cell polarity and decreased TBC1D19 expression may contribute to the observed disruption of odontoblast polarity and apoptosis. These data indicate that MT-DLX3 acts to disrupt odontoblast cytodifferentiation leading to odontoblast apoptosis, and aberrations of dentin tubule formation and dentin matrix production, resulting in decreased dentin and taurodontism.In summary, this TG model demonstrates that MT-DLX3 has differential effects on matrix production and mineralization in dentin and bone and provides a novel tool for the investigation of odontoblast biology.

Epidemiologic studies of rare diseases may produce surprising findings and raise ethical issues. This is illustrated in this study performed in 171,977 consecutive Taiwanese newborns (including 90,288 boys) from July 2006 through June 2008 by measuring dry blood spot and then leukocyte -galactosidase A ( -Gal A) activities and finally by detecting mutations in the GLA gene involved in Fabry disease. Schematically, two phenotypes of this disease are known: the classic form, with systemic involvement and very low -Gal activity in males; and the later-onset form ( 40 years of age), with some residual -Gal A activity, which is dominated by cardiac involvement. All 11 newborns who had 5% of normal mean -Gal A activity were boys who had GLA mutations. In the group of 66 newborns (64 boys and 2 girls) with -Gal A activities between 5% and 30%, 61 hemizygous boys and 2 heterozygous girls had GLA mutations. Among the group of 12 newborns (11 boys and 1 girl) with -Gal A 30%, only 1 boy had a previously reported mutation, identified in a family with later-onset renal disease. In total, 72 male and 2 female newborns had GLA mutations, an overall frequency of approximately 1 in 1250 boys and approximately 1 in 40,840 girls. Four boys were “predicted” to have the classic phenotype, a frequency of about 1 in 22,570 newborn boys. In contrast, the estimated frequency of the lateronset phenotype is approximately 1 in 1390 male newborns mutations. Three families provided information on other members of the kindreds, two with classic and one with later-onset phenotype. All three families had previously undiagnosed symptomatic family members, including one heterozygous female with ESRD and two males with renal involvement. This is undoubtedly the positive side of such studies. A second study was performed more recently in 110,027 Taiwanese newborns between January 2008 and January 2009 by using a similar protocol (plasma -Gal A activity was measured) (1). The results of this study confirmed those of the previous screening. A high prevalence of the cardiac variant Fabry mutation IVS4 919G3A, first discovered in Japanese patients, was found among newborns (approximately 1 in 1600 boys) in both studies. This splicing mutation was most common (82% of patients). The alternatively spliced transcript was normally present in small amount ( 5% of normal transcript) in most human tissues. However, the G3A transversion enhanced the percent expression of the alternatively spliced -Gal A variant and included a 57-nucleotide intronic sequence that caused a frameshift mutation, resulting in a truncated enzyme polypeptide that had no detectable enzyme activity. The clinical significance of this splicing mutation remains to be fully clarified. Of interest, Lin et al. (1) have investigated 9 grandfathers and 11 grandmothers carrying this mutation, as do their respective grandsons. Among the 9 maternal grandfathers, only 3 had hypertrophic cardiomyopathy, compared with none of the 11 grandmothers. These results should be compared with those reported in 2006 from Torino, Piedmont, Italy, by Spada et al. (2). They screened 37,000 consecutive newborns with similar methods and identified 12 infants with GLA mutations, including 11 who had molecular lesions that expressed residual activity consistent with the later-onset phenotype. The overall frequency of Fabry mutations was approximately 1 in 3100 Caucasian boys. Mutation analysis predicted that one of the newborns had the classic phenotype (1 in approximately 37,000), whereas 11 of the newborns were predicted to have the later-onset phenotype (1 in approximately 3400). The prevalence of the classic form is close to that found in previous estimations, whereas that of the later-onset phenotype seems to be higher than commonly thought. In Taiwan, the frequency was 2.5 times more frequent than in the Italian population. These studies raise many ethical and clinical issues. What can be said to the parents of neonates harboring a GLA mutation suggestive of a later-onset disease? The clinical consequences of some mutations, if any, cannot be predicted. The ethnic background should be taken into account. If clinical consequences can be expected, when to start evaluating the cardiac condition and when to consider enzyme replacement therapy, if necessary? What are the psychologic consequences of the screening on the mutation carrier and his/her family? The ethical issues raised by early detection and prediction of later-onset genetic Published online ahead of print. Publication date available at www.cjasn.org.

OBJECTIVES: Cystinosis is a rare autosomal recessive lysosomal storage disorder with developmental and mineralization anomalies as part of its clinical presentation. The objective of this study was to provide the first systematic assessment of the craniofacial and dental characteristics associated with cystinosis. STUDY DESIGN: Oral and radiographic evaluations were performed on 73 patients with cystinosis. Analyses of cephalometry (n = 20), taurodontism (n = 47), caries (n = 47), enamel defects (n = 48), soft tissue anomalies (n = 48), and dental age (n = 41) were performed on the cystinosis group, and compared with age- and sexcomparable controls or standards. RESULTS: Cystinosis patients manifested relative mandibular deficiency, an increased facial height, and a reduced airway space. Taurodontism and enamel defects were significantly more prevalent in cystinosis patients compared with controls (P < 0.0001 and P = 0.027, respectively). Children (aged

Through linkage analysis and candidate gene sequencing, we identified three unrelated families with the autosomal-dominant inheritance of early onset anemia, hypouricosuric hyperuricemia, progressive kidney failure, and mutations resulting either in the deletion (p.Leu16del) or the amino acid exchange (p.Leu16Arg) of a single leucine residue in the signal sequence of renin. Both mutations decrease signal sequence hydrophobicity and are predicted by bioinformatic analyses to damage targeting and cotranslational translocation of preprorenin into the endoplasmic reticulum (ER). Transfection and in vitro studies confirmed that both mutations affect ER translocation and processing of nascent preprorenin, resulting either in reduced (p.Leu16del) or abolished (p.Leu16Arg) prorenin and renin biosynthesis and secretion. Expression of renin and other components of the renin-angiotensin system was decreased accordingly in kidney biopsy specimens from affected individuals. Cells stably expressing the p.Leu16del protein showed activated ER stress, unfolded protein response, and reduced growth rate. It is likely that expression of the mutant proteins has a dominant toxic effect gradually reducing the viability of renin-expressing cells. This alters the intrarenal renin-angiotensin system and the juxtaglomerular apparatus functionality and leads to nephron dropout and progressive kidney failure. Our findings provide insight into the functionality of renin-angiotensin system and stress the importance of renin analysis in families and individuals with early onset hyperuricemia, anemia, and progressive kidney failure.

To the Editor: Amelogenesis imperfecta (AI) are a heterogeneous group of disorders that perturb enamel development. Historically, 14 subtypes have been recognized based on clinical phenotype and mode of inheritance (1). To date, mutations in AMELX, ENAM, KLK4, and MMP20 have been documented in hypoplastic and hypomaturation AI (2–6). However, the genetic basis for most cases of autosomal dominant AI in the Turkish population remained unknown. Recently, mutations underlying a hypocalcified form of AI were reported (7, 8). In the present study, we evaluated eight Turkish kindreds with autosomal dominant hypocalcified AI (ADHCAI). Genetic linkage studies were consistent with linkage to chromosome 8q24.3 in seven families. Sequence analysis identified FAM83H nonsense mutations in all eight families. Eight probands were identified from Dental School Clinics at Ege University, the University of Istanbul and Yeditepe University, Istanbul, Turkey, in accordance with Institutional Review Board approval from the corresponding University and the National Institutes of Health. Available family members received an oral examination and dental radiographs to determine affection status. The presence of AI was established by generalized yellow–brown discoloration of the teeth, hypoplastic appearing enamel and pathological loss of enamel. Affected individuals were diagnosed with ADHCAI based on previously proposed criteria (1) (Fig. 1). Seven kindreds were consistent for autosomal dominant transmission of AI. In an eighth family, only the proband was affected, but the phenotype was consistent with hypocalcified AI and the known AI genes AMELX, ENAM, KLK4, and MMP20 had been excluded based on sequence analysis. A total of 50 individuals were clinically examined, including 26 affected individuals. DNA extraction from peripheral venous blood was performed as previously described (6). DNA extraction from saliva was performed according to the Oragene protocol (DNA Genotek Inc., Ottawa, Ontario, Canada). Fig. 1 Pedigrees, clinical photographs, mutations and haplotype analysis for Turkish families segregating autosomal dominant hypocalcified amelogenesis imperfecta. (a) Family 1 segregating a previously identified mutation, c.1192C>T (p.Q398X). (b) Family ... Primers and conditions used to amplify the FAM83H gene are shown in Table 1. The large approximately 4.8 kb exon 5 was split into eight overlapping amplicons. Polymerase chain reaction (PCR) products were purified using Exo-Sap and sequenced in both directions to minimize sequencing artifacts using ABI Big Dye Terminator chemistry, version 3.1, and an ABI 3730 (Applied Biosystems, Foster City, CA). Table 1 Primers and conditions to amplify FAM83H Six short tandem repeat polymorphism (STRP) markers (D8S1836, D8S15018mg, D8S373, D8S2334, D8S1925 and D8S1926) spanning a 2.35-Mb interval that includes the FAM83H locus were genotyped. Markers were amplified from genomic DNA using fluorescently labeled primers and AmpliTaq Gold PCR Master Mix (Applied Biosystems) according to the manufacturer’s protocol. PCR products were electrophoresed using an ABI 3130 DNA analyzer with size standard GeneScan 400HD Rox. Data were analyzed with GeneMapper ID software, v3.7. Haplotypes were generated based on allele transmission, incorporating sequence variants identified by sequencing. Genotyping of STRP loci surrounding the FAM83H locus in families 1–7 were consistent with genetic linkage to this interval of chromosome eight. As we were unable to amplify the large exon 5 using the published primers (7), primers were redesigned as shown in Table 1 and required a variety of conditions to successfully amplify this region. Three mutations were identified (Fig. 1, Table 2). Affected individuals from family 1 were found to have a C to T substitution at nucleotide position 1192 of the complementary DNA reference sequence (Gen-Bank accession number: {"type":"entrez-nucleotide","attrs":{"text":"NM_198488.3","term_id":"157311634","term_text":"NM_198488.3"}}NM_198488.3), a mutation previously identified in a Korean family (7). A novel c.1366C>T mutation was identified in family 2. This substitution is predicted to be a nonsense mutation, p.Q444X (Fig. 1b). Affected individuals from the remaining six families all had a novel nonsense mutation, p.Q456X, due to a c.1366C>T mutation (Fig. 1c). The finding of a common c.1366C>T mutation in six families (families 3–8) raised the possibility of a mutational hot spot or a founder effect. Based on haplotype analysis, the c.1366C>T mutation appears to have been inherited ‘identical by descent’ from a common ancestor in five of these families with a shared haplotype extending over an interval of 1.8 Mb spanning the FAM83H locus (Fig. 1d). In family 8, the c.1366C>T mutation appears on a different haplotype background. Additionally, the proband was the only affected family member. Mutational analysis confirmed neither parent has a FAM83H mutation. Thus, consistent with the haplotype analysis, this mutation appears to have arisen de novo, but germline mosaicism in one of the parents cannot be excluded. Table 2 Mutations in FAM83H FAM83H mutations were identified in affected individuals from all eight Turkish families studied. With these results, eight FAM83H mutations are now reported (Table 2). Only two mutations, c.1192C>T and c.1366C>T, have been found in more than one kindred. The c.1192C>T mutation reported previously in a Korean family was identified in one Turkish family (family 1) in the current study (7). Based on haplotype analysis, the c.1366C>T mutation identified in six Turkish families reported here appears to have occurred as a founder mutation in five Turkish families (families 3–7) and as an apparent de novo mutation in another Turkish family (family 8, Fig. 1d). Lee et al. (8) also reported a de novo p.E415X FAM83H mutation. Identification of two de novo mutations from a total of eight mutations suggests that the FAM83H gene may have a high mutation rate, which is surprising given that the gene is relatively small (98,126 bp). Consistent with previous findings, all mutations found in these Turkish families were nonsense mutations (7, 8). The finding of only nonsense mutations raises the question of whether the underlying molecular mechanism is haploinsufficiency or a dominant negative effect. Although all reported mutations are in the terminal exon and the corresponding messenger RNAs might be expected to escape nonsense-mediated decay (NMD), there are examples in humans of terminal exon nonsense mutations triggering NMD, notably in COL10A1 (9). If mutant transcripts undergo NMD then haploinsufficiency of FAM83H leads to ADHCAI. If the transcripts escape NMD then the truncated products may interfere with the normal product and produce a phenotype through a dominant negative effect. The finding that all mutations are nonsense mutations that cluster within a 380 amino acid region is more supportive of a dominant negative effect. As mutations are identified in more families, the underlying molecular mechanism may become clearer. FAM83H represents the first gene widely expressed outside of the developing tooth to be associated with AI. Kim et al. (7) demonstrated expression in mouse eye, liver and kidney. We verified expression in human kidney, liver and gingiva (data not shown). The phenotype observed in association with FAM83H mutations indicates that FAM83H functions in mineralization, but its expression in non-mineralized tissues suggests that either it does not directly function in mineralization or it has another function in non-mineralized tissues. Enamel matrix proteins are almost completely removed as enamel crystallites grow and mineralization progresses (10). In both hypomaturation and hypocalcified forms of AI, significant amounts of protein are detected in fully developed enamel (11). It is not surprising, therefore, that mutation in the known enamel proteases, kallikrein 4 and matrix metalloproteinase 20, result in hypomaturation phenotypes (4–6). MMP20 and KLK4 mutations result in autosomal recessive phenotypes, suggesting that haploinsufficiency of these proteinases is sufficient for normal enamel development. Analysis of the predicted protein sequence of FAM83H does not identify obvious homology outside of the FAM83 family. Given that FAM83H mutations cluster within a 380 amino acid region, this region appears to be critical for normal enamel development and calcification. FAM83H mutations were identified in all eight of the Turkish families segregating ADHCAI, suggesting that FAM83H mutations may be responsible for the majority of ADHCAI in Turkey. The generality of the founder effect in the Turkish population remains to be seen.

Distal-less 3 (DLX3) gene mutations are etiologic for Tricho-Dento-Osseous syndrome. To investigate the in vivo impact of mutant DLX3 on bone development, we established transgenic (TG) mice expressing the c.571_574delGGGG DLX-3 gene mutation (MT-DLX3) driven by a mouse 2.3 Col1A1 promoter. Microcomputed tomographic analyses demonstrated markedly increased trabecular bone volume and bone mineral density in femora from TG mice. In ex vivo experiments, TG mice showed enhanced differentiation of bone marrow stromal cells to osteoblasts and increased expression levels of bone formation markers. However, TG mice did not show enhanced dynamic bone formation rates in in vivo fluorochrome double labeling experiments. Osteoclastic differentiation capacities of bone marrow monocytes were reduced in TG mice in the presence of osteoclastogenic factors and the numbers of TRAP(+) osteoclasts on distal metaphyseal trabecular bone surfaces were significantly decreased. TRACP 5b and CTX serum levels were significantly decreased in TG mice, while IFN-γ levels were significantly increased. These data demonstrate that increased levels of IFN-γ decrease osteoclast bone resorption activities, contributing to the enhanced trabecular bone volume and mineral density in these TG mice. These data suggest a novel role for this DLX-3 mutation in osteoclast differentiation and bone resorption.

Human linkage and association studies suggest a gene(s) for nonsyndromic cleft lip with or without cleft palate (CL/P) on chromosome 4q31-q32 at or near the platelet-derived growth factor-C (PDGF-C) locus. The mouse Pdgfc(-/-) knockout shows that PDGF-C is essential for palatogenesis. To evaluate the role of PDGF-C in human clefting, we performed sequence analysis and SNP genotyping using 1048 multiplex CL/P families and 1000 case-control samples from multiple geographic origins. No coding region mutations were identified, but a novel -986 CT SNP (rs28999109) was significantly associated with CL/P (P=0.01) in cases from Chinese families yielding evidence of linkage to 4q31-q32. Significant or near-significant association was also seen for this and several other PDGF-C SNPs in families from the United States, Spain, India, Turkey, China, and Colombia, whereas no association was seen in families from the Philippines, and Guatemala, and case-controls from Brazil. The -986T allele abolished six overlapping potential transcription regulatory motifs. Transfection assays of PDGF-C promoter reporter constructs show that the -986T allele is associated with a significant decrease (up to 80%) of PDGF-C gene promoter activity. This functional polymorphism acting on a susceptible genetic background may represent a component of human CL/P etiology.

Dentinogenesis imperfecta (DGI) and dentin dysplasia (DD) are allelic disorders due to mutations in DSPP. Typically, the phenotype breeds true within a family. Recently, two reports showed that 3 different net -1 bp frameshift mutations early in DSPP’s repeat domain caused DD, whereas 6 more 3′ frameshift mutations were associated with DGI. Here we identify a DD kindred with a novel -1 bp frameshift (c.3141delC) that falls within the portion of the DSPP repeat domain previously associated solely with the DGI phenotype. This new frameshift mutation shows that overlapping DSPP mutations can give rise to either DGI or DD phenotypes. Furthermore, the consistent kindred presentation of the DD or DGI phenotype appears to be dependent on an as-yet-undescribed genetic modifier closely linked to DSPP.

Within nine dentin dysplasia (DD) (type II) and dentinogenesis imperfecta (type II and III) patient/families, seven have 1 of 4 net –1 deletions within the ∼2-kb coding repeat domain of the DSPP gene while the remaining two patients have splice-site mutations. All frameshift mutations are predicted to change the highly soluble DSPP protein into proteins with long hydrophobic amino acid repeats that could interfere with processing of normal DSPP and/or other secreted matrix proteins. We propose that all previously reported missense, nonsense, and splice-site DSPP mutations (all associated with exons 2 and 3) result in dominant phenotypes due to disruption of signal peptide-processing and/or related biochemical events that also result in interference with protein processing. This would bring the currently known dominant forms of the human disease phenotype in agreement with the normal phenotype of the heterozygous null Dspp (–/+) mice. A study of 188 normal human chromosomes revealed a hypervariable DSPP repeat domain with extraordinary rates of change including 20 slip-replication indel events and 37 predominantly C-to-T transition SNPs. The most frequent transition in the primordial 9-basepair (bp) DNA repeat was a sense-strand CpG site while a CpNpG (CAG) transition was the second most frequent SNP. Bisulfite-sequencing of genomic DNA showed that the DSPP repeat can be methylated at both motifs. This suggests that, like plants and some animals, humans methylate some CpNpG sequences. Analysis of 37 haplotypes of the highly variable DSPP gene from geographically diverse people suggests it may be a useful autosomal marker in human migration studies. Hum Mutat 0, 1–13, 2008. Published 2008, Wiley-Liss, Inc.

Amelogenesis imperfecta (AI) is caused by AMEL, ENAM, MMP20 and KLK4 gene mutations. Mice lacking expression of the AmelX, Enam and Mmp20 genes have been generated. These mouse models provide tools for understanding enamel formation and AI pathogenesis. This study describes the AI phenotypes and relates them to their mouse model counterparts. Human AI phenotypes were determined in a clinical population of AI families and published cases. Human and murine teeth were evaluated using light and electron microscopy. A total of 463 individuals from 54 families were evaluated and mutations in the AMEL, ENAM and KLK4 genes were identified. The majority of human mutations for genes coding enamel nonproteinase proteins (AMEL and ENAM) resulted in variable hypoplasia ranging from local pitting to a marked, generalized enamel thinning. Specific AMEL mutations were associated with abnormal mineralization and maturation defects. Amel and Enam null murine models displayed marked enamel hypoplasia and a complete loss of prism structure. Human mutations in genes coding for the enamel proteinases (MMP20 and KLK4) cause variable degrees of hypomineralization. The murine Mmp20 null mouse exhibits both hypoplastic and hypomineralized defects. The currently available Amel and Enam mouse models for AI exhibit enamel phenotypes (hypoplastic) that are generally similar to those seen in humans. Mmp20 null mice have a greater degree of hypoplasia than humans with MMP20 mutations. Mice lacking expression of the currently known genes associated with the human AI conditions provide useful models for understanding the pathogenesis of these conditions.

Oral microbes that colonize in the mouths of humans contribute to disease susceptibility, but it is unclear if host genetic factors mediate colonization. We therefore tested the hypothesis that the levels at which oral microbes colonize in the mouth are heritable. Dental plaque biofilms were sampled from intact tooth surfaces of 118 caries-free twins. An additional 86 caries-active twins were sampled for plaque from carious lesions and intact tooth surfaces. Using a reverse capture checkerboard assay the relative abundance of 82 bacterial species was determined. An integrative computational predictive model determined microbial abundance patterns of microbial species in caries-free twins as compared to caries-active twins. Heritability estimates were calculated for the relative microbial abundance levels of the microbial species in both groups. The levels of 10 species were significantly different in healthy individuals than in caries-active individuals, including,A. defectiva, S. parasanguinis, S. mitis/oralis, S. sanguinis, S. cristatus, S. salivarius, Streptococcussp. clone CH016,G. morbillorumandG. haemolysans.Moderate to high heritability estimates were found for these species (h2= 56%–80%,p< .0001). Similarity of the overall oral microbial flora was also evident in caries-free twins from multivariate distance matrix regression analysis. It appears that genetic and/or familial factors significantly contribute to the colonization of oral beneficial species in twins.

Objective Allogeneic hematopoietic stem cell transplantation (allo-HCT) is frequently complicated by severe infections and graft-vs-host disease (GVHD). Saliva contains many components of adaptive and innate immune response crucial for local host defenses. Changes in salivary constituents could reflect systemic processes such as immune reconstitution and development of GVHD that occur posttransplant. This study was an initial evaluation of salivary protein changes that occur after allo-HCT. Patients and Methods Serially collected saliva samples from 41 patients undergoing allo-HCT were evaluated. Changes in salivary proteome were initially examined by SELDI-TOF mass spectrometry. Individual protein changes were identified by 2-dimensional differential in-gel electrophoresis (2D-DIGE) with subsequent MS/MS sequencing and ELISA. Results Significant increases and decreases in multiple salivary proteins that lasted at least 2 months posttransplant were detected by SELDI-TOF mass spectrometry. Lactoferrin and secretory leukocyte protease inhibitor demonstrated elevations 1 month post-HCT that persisted at least 6 months. Secretory IgA (sIgA) levels were decreased 1 month posttransplant, with recovery at approximately 6 months. Levels of salivary β 2 -microglobulin were elevated at 6 months and correlated with sIgA levels. Conclusion Allo-HCT is associated with long-term changes in several salivary proteins important for innate immune responses. These results support further studies on the association of salivary proteins with posttransplant complications including infections and GVHD.

Muenke syndrome is an autosomal dominant disorder characterized by coronal suture craniosynostosis, hearing loss, developmental delay, carpal and tarsal fusions, and the presence of the Pro250Arg mutation in the FGFR3 gene. Reduced penetrance and variable expressivity contribute to the wide spectrum of clinical findings in Muenke syndrome. To better define the clinical features of this syndrome, we initiated a study of the natural history of Muenke syndrome. To date, we have conducted a standardized evaluation of nine patients with a confirmed Pro250Arg mutation in FGFR3. We reviewed audiograms from an additional 13 patients with Muenke syndrome. A majority of the patients (95%) demonstrated a mild-to-moderate, low frequency sensorineural hearing loss. This pattern of hearing loss was not previously recognized as characteristic of Muenke syndrome. We also report on feeding and swallowing difficulties in children with Muenke syndrome. Combining 312 reported cases of Muenke syndrome with data from the nine NIH patients, we found that females with the Pro250Arg mutation were significantly more likely to be reported with craniosynostosis than males (P < 0.01). Based on our findings, we propose that the clinical management should include audiometric and developmental assessment in addition to standard clinical care and appropriate genetic counseling.

Dentin, the most abundant tissue in teeth, is produced by odontoblasts, which differentiate from mesenchymal cells of the dental papilla. Dentinogenesis is a highly controlled process that results in the conversion of unmineralized predentin to mineralized dentin. By weight, 70% of the dentin matrix is mineralized, while the organic phase accounts for 20% and water constitutes the remaining 10%. Type I collagen is the primary component (>85%) of the organic portion of dentin. The non-collagenous part of the organic matrix is composed of various proteins, with dentin phosphoprotein predominating, accounting for about 50% of the non-collagenous part. Dentin defects are broadly classified into two major types: dentinogenesis imperfectas (DIs, types I–III) and dentin dysplasias (DDs, types I and II). To date, mutations in DSPP have been found to underlie the dentin disorders DI types II and III and DD type II. With the elucidation of the underlying genetic mechanisms has come the realization that the clinical characteristics associated with DSPP mutations appear to represent a continuum of phenotypes. Thus, these disorders should likely be called DSPP-associated dentin defects, with DD type II representing the mild end of the phenotypic spectrum and DI type III representing the severe end.

Kallikrein-4 is known to be highly expressed during the maturation stage of enamel formation and is thought to be critical for the final phase of crystallite growth. The purpose of this study was to evaluate the enamel phenotype in humans with a known KLK-4 mutation (g.2142G>A). Primary teeth from two individuals with a known KLK-4 mutation were evaluated using amino acid analysis and light and electron microscopy. Light microscopy showed the enamel was of normal thickness but opaque throughout its width compared with normal enamel. Electron microscopy showed enamel affected by the KLK-4 mutation had a normal prismatic structure and generally had a well-organized and discernable crystallite composition. In some areas, globular structures were present where crystallites were not discernable or appeared to have an altered morphology. The KLK-4 mutant enamel had an increased protein content compared with normal enamel. Human enamel formed with a lack of functioning KLK-4 proteinase is altered primarily in the completeness of crystallite growth, while enamel thickness and prism structure remains essentially normal. Collectively, these studies suggest that the KLK-4 proteinase is essential for the final crystallite growth of enamel but is not critical for crystallite orientation, prism formation or enamel thickness.