Novel Ameloblastin Variants, Contrasting Amelogenesis Imperfecta Phenotypes.

Amelogenesis imperfecta (AI) comprises a group of rare, inherited disorders with abnormal enamel formation. Ameloblastin (AMBN), the second most abundant enamel matrix protein (EMP), plays a critical role in amelogenesis. Pathogenic biallelic loss-of-function AMBN variants are known to cause recessive hypoplastic AI. A report of a family with dominant hypoplastic AI attributed to AMBN missense change p.Pro357Ser, together with data from animal models, suggests that the consequences of AMBN variants in human AI remain incompletely characterized. Here we describe 5 new pathogenic AMBN variants in 11 individuals with AI. These fall within 3 groups by phenotype. Group 1, consisting of 6 families biallelic for combinations of 4 different variants, have yellow hypoplastic AI with poor-quality enamel, consistent with previous reports. Group 2, with 2 families, appears monoallelic for a variant shared with group 1 and has hypomaturation AI of near-normal enamel volume with pitting. Group 3 includes 3 families, all monoallelic for a fifth variant, which are affected by white hypoplastic AI with a thin intact enamel layer. Three variants, c.209C>G; p.(Ser70*) (groups 1 and 2), c.295T>C; p.(Tyr99His) (group 1), and c.76G>A; p.(Ala26Thr) (group 3) were identified in multiple families. Long-read AMBN locus sequencing revealed these variants are on the same conserved haplotype, implying they originate from a common ancestor. Data presented therefore provide further support for possible dominant as well as recessive inheritance for AMBN-related AI and for multiple contrasting phenotypes. In conclusion, our findings suggest pathogenic AMBN variants have a more complex impact on human AI than previously reported.

The burden of dental care in Amelogenesis Imperfecta paediatric patients in the UK NHS: a retrospective, multi-centred analysis.

PURPOSE: The burden of dental care in Amelogenesis Imperfecta (AI) has not been well described. This condition results in weak, discoloured and often sensitive teeth. Specialist paediatric care is available for AI patients in the UK, but treatment protocols and care provided are inconsistent. The aim of this study was therefore to analyse the provision of treatment and burden of care for children and families with AI across four Paediatric Dentistry centres in the UK. METHODS: A retrospective evaluation of AI patient clinical records across four UK consultant-led Paediatric Dentistry centres was completed. Frequency and duration of care were recorded along with treatment and experience of inhalation sedation, local and general anaesthetic. RESULTS: In total, 138 records were available for analysis. The average patient age at first referral was 7.7 years (range 1-16 years) and families travelled an average 21.8 miles per appointment (range 0.2-286 miles). Patients attended on average 4.5 appointments per year for 5.8 years. In total, 65.2% had experience of local anaesthetic, 27.5% inhalation sedation and 31.9% general anaesthetic. Dental treatment including restorations and extractions were commonly required on multiple teeth per patient. CONCLUSION: AI carries a high burden of specialist dental care to patients and families. Specialist centres are required to provide longitudinal, comprehensive care.

Phenotype and Variant Spectrum in the LAMB3 Form of Amelogenesis Imperfecta.

Amelogenesis imperfecta (AI) is a heterogeneous group of inherited disorders characterized by abnormal formation of dental enamel, either in isolation or as part of a syndrome. Heterozygous variants in laminin subunit beta 3 ( LAMB3) cause AI with dominant inheritance in the absence of other cosegregating clinical features. In contrast, biallelic loss-of-function variants in LAMB3 cause recessive junctional epidermolysis bullosa, characterized by life-threatening skin fragility. We identified 2 families segregating autosomal dominant AI with variable degrees of a distinctive hypoplastic phenotype due to pathogenic variants in LAMB3. Whole exome sequencing revealed a nonsense variant (c.3340G>T, p.E1114*) within the final exon in family 1, while Sanger sequencing in family 2 revealed a variant (c.3383-1G>A) in the canonical splice acceptor site of the final exon. Analysis of cDNA from family 2 revealed retention of the final intron leading to a premature termination codon. Two unerupted third molar teeth from individual IV:5 in family 2 were subject to computerized tomography and scanning electron microscopy. LAMB3 molar teeth have a multitude of cusps versus matched controls. LAMB3 enamel was well mineralized but pitted. The architecture of the initially secreted enamel was abnormal, with cervical enamel appearing much less severely affected than coronal enamel. This study further defines the variations in phenotype-genotype correlation for AI due to variants in LAMB3, underlines the clustering of nonsense and frameshift variants causing AI in the absence of junctional epidermolysis bullosa, and highlights the shared AI phenotype arising from variants in genes coding for hemidesmosome proteins.

Genetic testing for amelogenesis imperfecta: knowledge and attitudes of paediatric dentists.

Introduction: Genetic testing is increasingly applied across healthcare reflecting the value to diagnosis, clinical decision-making, service organisation and advancement of the research-informed evidence base. Patient expectations are changing. Genetic testing has not been part of dental practice. Introduction of an NHS-targeted gene panel test for amelogenesis imperfecta (AI), a heterogeneous genetic disorder affecting enamel appearance and function, represents a paradigm shift. This impacts on specialists in paediatric dentistry and other members of the dental team delivering longitudinal care for individuals with AI. Aim: To evaluate the opinions of paediatric dentists on genetic testing for dental conditions using AI as the exemplar. Method: Two focus groups of nine UK NHS paediatric dentists each were audio recorded (September 2016) and transcribed verbatim. Qualitative analysis was undertaken using Interpretative Phenomenological Analysis (IPA). Results: A wide range of views reflected existing insight and understanding. Three core concepts of justification, ownership and challenges emerged. The clinicians were generally open to involvement with genetic testing in paediatric dentistry, but required more support. Conclusion: Areas for clarification and professional development were identified as important in ensuring that genetic testing in dentistry, which is currently in its infancy, reaches translational potential and enhances patient care as this area of healthcare continues to advance rapidly.

Oral Medicine for undergraduate dental students in the United Kingdom and Ireland-A curriculum.

INTRODUCTION: Oral Medicine focuses on care for patients with chronic, recurrent and medically related disorders of the orofacial region that are distinct from diseases of the periodontal and tooth tissues, with an emphasis on non-surgical management. At present, there are no shared outcomes for Oral Medicine to define the standards to be achieved before new graduates become registered dentists engaged with ongoing professional development. CURRICULUM: We present a consensus undergraduate curriculum in Oral Medicine agreed by representatives from 18 Dental Schools in the United Kingdom and Republic of Ireland. The scope of Oral Medicine practice includes conditions involving the oral mucosa, salivary glands, neurological system or musculoskeletal tissues that are not directly attributable to dental (tooth and periodontium) pathology. Account is taken of the priorities for practice and learning opportunities needed to support development of relevance to independent clinical practice. The outcomes triangulate with the requirements set out by the respective regulatory bodies in the UK and Republic of Ireland prior to first registration and are consistent with the framework for European undergraduate dental education and greater harmonisation of dental education. CONCLUSIONS: This curriculum will act as a foundation for an increasingly shared approach between centres with respect to the outcomes to be achieved in Oral Medicine. The curriculum may also be of interest to others, such as those responsible for the training of dental hygienists and dental therapists. It provides a platform for future collective developments with the overarching goal of raising the quality of patient care.

Human Disease/Clinical Medical Sciences in Dentistry: Current state and future directions of undergraduate teaching in the UK and Ireland.

In March 2017, a group of teachers of human disease/clinical medical science (HD/CMSD) representing the majority of schools from around the UK and Republic of Ireland met to discuss the current state of teaching of human disease and also to discuss how the delivery of this theme might evolve to inform improved healthcare. This study outlines how the original teaching in medicine and surgery to dental undergraduate students has developed into the theme of HD/CMSD reflecting changing needs as well as guidance from the regulators, and how different dental schools have developed their approaches to reach their current state. Each school was also asked to share a strengths, weakness, opportunities and threats (SWOT) analysis of their programme and to outline how they thought their HD/CMSD programme may develop. The school representatives who coordinate the delivery and assessment of HD/CMSD in the undergraduate curriculum have extensive insight in this area and are well-placed to shape the HD/CMSD development for the future.

Development of a managed clinical network in oral medicine.

Oral medicine is concerned with the oral health care of patients with chronic, recurrent and medically related disorders of the oral and maxillofacial region, and with their diagnosis and non-surgical management. For historical reasons care for conditions falling within the scope of oral medicine practice has been inconsistent with limited planning of clinical services. Managed Clinical Networks (MCNs) bring advantages to all stakeholders with a positive impact on patient pathways and access to equitable and quality care across a network of providers working in a coordinated way to make best use of NHS resources. MCNs provide a framework to address the limitations of legacy arrangements and are very relevant to dentistry. Here we describe oral medicine MCN development in Yorkshire and the Humber within the framework of the Five year forward view NHS policy. A step-wise approach is being taken across the region to introduce an MCN model that reflects cooperative working between oral medicine, oral surgery, oral & maxillofacial surgery and other stakeholders. Preliminary data are already informing how a regional oral medicine MCN can be further developed with the potential for translation of the lessons learned to other regions.

The use of topical steroid preparations in oral medicine in the UK.

Topical corticosteroids (TCs) are the most commonly prescribed drugs in oral medicine practice. Use in management of immune-driven inflammatory oral mucosal disease is predominantly off-label and poorly supported by evidence, yet oral medicine specialists have considerable collective experience of prescribing, administering and managing these medications. TCs are also prescribed by others in healthcare including general dental practitioners. Successful TC use is influenced by accurate diagnosis, TC choice (potency and formulation), patient acceptability (including ease of use, taste and texture), frequency of application, duration of treatment, adverse effects, patient support and medication regulations and access. The aim of this review is to provide an overview of TC use in oral medicine practice. Recommendations are based on collective experience to help selection of the appropriate TC to maximise therapeutic efficacy and minimise the potential for adverse effects. This is within an understanding of medicines regulation and preparation availability when prescribing in both secondary and primary care.

Oral cancer - improving early detection and promoting prevention. Are you up to date?

Oral cancer is increasingly common. The need for early detection and promoting prevention is greater than ever and relevant to all who are responsible for the care of patients. Recent addition of oral cancer detection to the list of continuing professional development (CPD) topics recommended by the General Dental Council (GDC) reflects this importance.

Clinical medical sciences for undergraduate dental students in the United Kingdom and Ireland - a curriculum.

The technical aspects of dentistry need to be practised with insight into the spectrum of human diseases and illnesses and how these impact upon individuals and society. Application of this insight is critical to decision-making related to the planning and delivery of safe and appropriate patient-centred healthcare tailored to the needs of the individual. Provision for the necessary training is included in undergraduate programmes, but in the United Kingdom and Ireland there is considerable variation between centres without common outcomes. In 2009 representatives from 17 undergraduate dental schools in the United Kingdom and Ireland agreed to move towards a common, shared approach to meet their own immediate needs and that might also be of value to others in keeping with the Bologna Process. To provide a clear identity the term 'Clinical Medical Sciences in Dentistry' was agreed in preference to other names such as 'Human Disease' or 'Medicine and Surgery'. The group was challenged to define consensus outcomes. Contemporary dental education documents informed, but did not drive the process. The consensus curriculum for undergraduate Clinical Medical Sciences in Dentistry teaching agreed by the participating centres is reported. Many of the issues are generic and it includes elements that are likely to be applicable to others. This document will act as a focus for a more unified approach to the outcomes required by graduates of the participating centres and act as a catalyst for future developments that ultimately aim to enhance the quality of patient care.

A systematic review of medical interventions for oral submucous fibrosis and future research opportunities.

Oral Diseases (2011) 17 (Suppl. 1), 42-57 Oral submucous fibrosis (OSF) is a chronic, insidious disease caused by areca nut use, and is associated with both significant morbidity (including pain and reduced oral opening) and an increased risk for malignancy. This systematic review explored and updated the current medical (i.e., non-surgical) interventions available for the management of OSF. Of the 27 published medical interventions, there were four randomized controlled trials. The overall quality of these randomized controlled studies was assessed using the GRADE approach and significant limitations that challenged the conclusions were found. However, this review was valuable in terms of identifying opportunities to provide recommendations for future research, in terms of the populations to research, the types of interventions needed, the types of outcomes to be measured, the study designs needed, and the infrastructure required to conduct studies. The next step is to initiate a pathway for a low-cost research plan leading to the development of a brief protocol for future clinical trials in this field, with an emphasis on conducting studies in regions of the world where OSF is prevalent.

Hypomaturation amelogenesis imperfecta due to WDR72 mutations: a novel mutation and ultrastructural analyses of deciduous teeth.

BACKGROUND: Mutations in WDR72 have been identified in autosomal recessive hypomaturation amelogenesis imperfecta (AI). OBJECTIVE: to describe a novel WDR72 mutation and report the ultrastructural enamel phenotype associated with a different WDR72 mutation. METHODS: A family segregating autosomal recessive hypomaturation AI was recruited, genomic DNA obtained and WDR72 sequenced. Four deciduous teeth from one individual with a previously published WDR72 mutation, extracted as part of clinical care, were subjected to scanning electron microscopy, energy-dispersive X-ray analysis and transverse microradiography. RESULTS: A novel homozygous nonsense mutation, R897X, was identified in WDR72 in a family originating from Pakistan. Ultrastructural analysis of enamel from the deciduous teeth of an AI patient with the WDR72 mutation S783X revealed energy-dispersive X-ray analysis spectra with normal carbon and nitrogen peaks, excluding retention of enamel matrix protein. However, transverse microradiography values were significantly lower for affected teeth when compared to normal teeth, consistent with reduced mineralisation. On scanning electron microscopy the enamel rod form observed was normal, yet with inter-rod enamel more prominent than in controls. This appearance was unaltered following incubation with either α-chymotrypsin or lipase. CONCLUSIONS: The novel WDR72 mutation described brings the total reported WDR72 mutations to four. Analyses of deciduous tooth enamel in an individual with a homozygous WDR72 mutation identified changes consistent with a late failure of enamel maturation without retention of matrix proteins. The mechanisms by which intracellular WDR72 influences enamel maturation remain unknown.

Ultrastructural analyses of deciduous teeth affected by hypocalcified amelogenesis imperfecta from a family with a novel Y458X FAM83H nonsense mutation.

BACKGROUND: Nonsense mutations in FAM83H are a recently described underlying cause of autosomal dominant (AD) hypocalcified amelogenesis imperfecta (AI). OBJECTIVE: This study aims to report a novel c.1374C>A p.Y458X nonsense mutation and describe the associated ultrastructural phenotype of deciduous teeth. METHODS: A family of European origin from the Iberian Peninsula with AD-inherited AI was ascertained. Family members were assessed through clinical examination and supporting investigations. Naturally exfoliated deciduous teeth from 2 siblings were investigated by scanning electron microscopy (SEM), energy dispersive X-ray analysis (EDX) and transverse microradiography (TMR). RESULTS: On clinical and radiographic investigation the appearances of the affected deciduous and permanent teeth were consistent with hypocalcified AI with small focal areas of more normal looking enamel. DNA sequencing identified a novel c.1374C>A p.Y458X FAM83H nonsense mutation in affected, but not in either unaffected family members or unrelated controls. Exfoliated teeth were characterised by substantial post-eruptive enamel loss on gross examination. Irregular, poor quality enamel prisms were observed on SEM. These were coated in amorphous material. TMR and EDX confirmed reduced mineral and increased organic content in enamel, respectively. CONCLUSIONS: FAM83H nonsense mutations have recently been recognised as a cause of AD hypocalcified AI. We report a novel nonsense FAM83H mutation and describe the associated preliminary ultrastructural phenotype in deciduous teeth. This is characterised by poorly formed enamel rods with inappropriate retention of amorphous material, which is likely to represent retained organic matrix that contributes to the overall hypomineralised phenotype.

Vertebrate pseudogenes.

Pseudogenes are commonly encountered during investigation of the genomes of a wide range of life forms. This review concentrates on vertebrate, and in particular mammalian, pseudogenes and describes their origin and subsequent evolution. Consideration is also given to pseudogenes that are transcribed and to the unusual group of genes that exist at the interface between functional genes and non-functional pseudogenes. As the sequences of different genomes are characterised, the recognition and interpretation of pseudogene sequences will become more important and have a greater impact in the field of molecular genetics.

Histological identification of carcinoma in 21 gauge needle tracks after fine needle aspiration biopsy of head and neck carcinoma.

Six cancer resection specimens were thoroughly sectioned and microscopically examined at areas known to have been around 21 gauge fine needle aspiration (FNA) biopsy sites, in an attempt to identify needle tracks. All cases had an interval of not less than 10 days between FNA biopsy and surgery. Foci of tumour were identified histologically in needle tracks from two patients with carcinoma. This is the first instance, outside of experimental animal models, of histologically confirmed, viable tumour spread in FNA biopsy tracks. Although this complication is not common and is of unknown clinical significance, it is one that all clinicians who undertake FNA of malignant neoplasms should be aware of.

An overview of the complexities and subtleties of immunohistochemistry.

Immunohistochemistry has the potential to be a powerful research tool. However, immunohistochemical studies are frequently undertaken without regard to the complexities and subtleties of these useful techniques. This review aims to address the problems and limitations that are often encountered, and the procedures that should be considered in both the planning and interpretation of immunohistochemical studies. Particular reference is made to the generation of functionally different protein isoforms from a single gene by alternative splicing and post-translational modifications, primary antibody selection, the effects of tissue manipulation such as fixation and antigen retrieval, the need for appropriate controls and interpretation of staining patterns.

Alu sequences.

Alu sequences are frequently encountered during study of human genomic nucleic acid and form a major component of repetitive DNA. This review describes the origin of Alu sequences and their subsequent amplification and evolution into distinct subfamilies. In recent years a number of different functional roles for Alu sequences have been described. The multiple influences of Alu sequences on RNA polymerase II-mediated gene expression and the presence of Alu sequences in RNA polymerase III-generated transcripts are discussed.

Human tenascin-C: identification of a novel type III repeat in oral cancer and of novel splice variants in normal, malignant and reactive oral mucosae.

Tenascin-C is a mosaic, linear glycoprotein that is up-regulated during many normal and pathological processes involving either cell migration or tissue morphogenesis, such as invasion of malignant cells and wound healing. Human tenascin-C contains 8 consecutive type III fibronectin (TNCfn) domains that are involved in alternative splicing and potentially generate a large number of isoforms that code for tenascin-C proteins with subtly different functions. Human tenascin-C splice variants were investigated by RT-PCR in a range of normal and pathological oral mucosal tissues. A novel, 9th human TNCfn domain involved in alternative splicing was identified. It shares 70% nucleic acid and 55% protein sequence homology with chicken TNCfn-ad2. As in avians, this novel repeat was located between TNCfn-B and TNCfn-ad1 and accordingly was designated human TNCfn-ad2. Human TNCfn-ad2 was detected in only 2 of 10 oral cancers. However, TNCfn-ad2 was absent from 40 normal, reactive, pre-malignant and other oral mucosal specimens investigated. Previous studies have described 8 splice variant transcripts for human tenascin-C. By systematic investigation we identified further novel splice variants for human tenascin-C. Furthermore, our results indicate that many potential splice variants probably do not exist in the tissues investigated. Thus, we have demonstrated that human tenascin-C transcripts generate a complex but selected repertoire of different alternative splice products.

RT-PCR investigation of fibronectin mRNA isoforms in malignant, normal and reactive oral mucosa.

This study aimed to establish patterns of cellular fibronectin mRNA splice variants in normal oral mucosa, oral squamous cell carcinoma, oral leukoplakias with and without atypia, and focal reactive overgrowths of oral mucosa. Particular emphasis was placed on evaluation of either the EDA or EDB domains as markers of malignancy. Total RNA was extracted from normal oral mucosa, oral squamous cell carcinoma, oral leukoplakias with and without atypia, reactive epulides, fibroepithelial polyps and denture-related hyperplasia. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to identify different fibronectin transcripts at three splice sites (EDA, EDB and IIICS). All the tissues investigated produced EDA+, EDA-, EDB+ and EDB- splice variants, and this study did not support RT-PCR-based detection of either EDA or EDB domains as markers of malignancy in oral tissues. Variations in IIICS splice patterns were observed, although these were not specific to any lesion group. In particular, there were differences in either the inclusion or omission of the domain coding for the CS-5 binding site for alpha 4 beta 1 integrin, whereas the CS-1 binding site for alpha 4 beta 1 integrin was typically present when additional domains were included at the IIICS splice site. In conclusion, complex patterns of fibronectin splice variant transcripts exist in normal and pathological oral mucosa. This may reflect the multiple biological functions identified for fibronectin proteins, although the significance of different specific fibronectin splice variants has yet to be fully elucidated.

Histochemical and immunohistochemical localisation of elastic system fibres in focal reactive overgrowths of oral mucosa.

Eight specimens each of the following groups were investigated: gingival pyogenic granuloma, fibrous epulis, calcifying fibrous epulis, peripheral giant cell granuloma, giant cell fibroma (four gingival, four non-gingival), denture-irritation hyperplasia and fibroepithelial polyp. These lesions have diverse histopathological appearances but the composition of their connective tissue is poorly defined. The elastic system consists of a complex mixture of glycoproteins that in normal oral mucosa form three differentially distributed fibre types; oxytalan, elaunin and elastic. The elastic system was investigated by Verhoeff's haematoxylin stain, aldehyde fuchsin staining and an anti-elastin monoclonal antibody. Elastin was identified in all fibroepithelial polyps and denture-irritation hyperplasias, but in none of the other lesions. In particular, this identified a distinct difference in the extracellular matrix between the giant cell fibroma and fibroepithelial polyp. Many of the epulides included only oxytalan fibres, but the presence of oxytalan fibres did not follow any pattern within either a single lesion group, or between different lesions. However, the presence of oxytalan fibres in the absence of elastin does not necessarily support a periodontal ligament origin for reactive epulides.