CNS-Native Myeloid Cells Drive Immune Suppression in the Brain Metastatic Niche through Cxcl10.

Brain metastasis (br-met) develops in an immunologically unique br-met niche. Central nervous system-native myeloid cells (CNS-myeloids) and bone-marrow-derived myeloid cells (BMDMs) cooperatively regulate brain immunity. The phenotypic heterogeneity and specific roles of these myeloid subsets in shaping the br-met niche to regulate br-met outgrowth have not been fully revealed. Applying multimodal single-cell analyses, we elucidated a heterogeneous but spatially defined CNS-myeloid response during br-met outgrowth. We found Ccr2+ BMDMs minimally influenced br-met while CNS-myeloid promoted br-met outgrowth. Additionally, br-met-associated CNS-myeloid exhibited downregulation of Cx3cr1. Cx3cr1 knockout in CNS-myeloid increased br-met incidence, leading to an enriched interferon response signature and Cxcl10 upregulation. Significantly, neutralization of Cxcl10 reduced br-met, while rCxcl10 increased br-met and recruited VISTAHi PD-L1+ CNS-myeloid to br-met lesions. Inhibiting VISTA- and PD-L1-signaling relieved immune suppression and reduced br-met burden. Our results demonstrate that loss of Cx3cr1 in CNS-myeloid triggers a Cxcl10-mediated vicious cycle, cultivating a br-met-promoting, immune-suppressive niche.

A transcriptomic survey of the impact of environmental stress on response to dengue virus in the mosquito, Aedes aegypti.

Populations of Aedes aegypti naturally exhibit variable susceptibility to dengue viruses. This natural variation can be impacted by nutritional stress resulting from larval-stage crowding, indicating the influence of environment components on the adult mosquito immune response. In particular, larval crowding was previously shown to reduce the susceptibility of adult females of a Trinidad field isolate of A. aegypti to the dengue serotype 2 (JAM1409) virus. Here, we present the first whole transcriptome study to address the impact of environmental stress on A. aegypti response to dengue virus. We examined expression profiles of adult females resulting from crowded and optimum reared larvae from the same Trinidad isolate at two critical early time points-3 and 18 hours post dengue virus infected blood meal. We exposed specimens to either a dengue or naïve blood meal, and then characterized the response in ten gene co-expression modules based on their transcriptional associations with environmental stress and time. We further analyzed the top 30 hub or master regulatory genes in each of the modules, and validated our results via qRT-PCR. These hub genes reveal which functions are critical to the mechanisms that confer dengue virus refractoriness or susceptibility to stress conditioned A. aegypti, as well as the time points at which they are most important.

Mast cell activation test in the diagnosis of allergic disease and anaphylaxis.

BACKGROUND: Food allergy is an increasing public health issue and the most common cause of life-threatening anaphylactic reactions. Conventional allergy tests assess for the presence of allergen-specific IgE, significantly overestimating the rate of true clinical allergy and resulting in overdiagnosis and adverse effect on health-related quality of life. OBJECTIVE: To undertake initial validation and assessment of a novel diagnostic tool, we used the mast cell activation test (MAT). METHODS: Primary human blood-derived mast cells (MCs) were generated from peripheral blood precursors, sensitized with patients' sera, and then incubated with allergen. MC degranulation was assessed by means of flow cytometry and mediator release. We compared the diagnostic performance of MATs with that of existing diagnostic tools to assess in a cohort of peanut-sensitized subjects undergoing double-blind, placebo-controlled challenge. RESULTS: Human blood-derived MCs sensitized with sera from patients with peanut, grass pollen, and Hymenoptera (wasp venom) allergy demonstrated allergen-specific and dose-dependent degranulation, as determined based on both expression of surface activation markers (CD63 and CD107a) and functional assays (prostaglandin D2 and ß-hexosaminidase release). In this cohort of peanut-sensitized subjects, the MAT was found to have superior discrimination performance compared with other testing modalities, including component-resolved diagnostics and basophil activation tests. Using functional principle component analysis, we identified 5 clusters or patterns of reactivity in the resulting dose-response curves, which at preliminary analysis corresponded to the reaction phenotypes seen at challenge. CONCLUSION: The MAT is a robust tool that can confer superior diagnostic performance compared with existing allergy diagnostics and might be useful to explore differences in effector cell function between basophils and MCs during allergic reactions.

A sparse differential clustering algorithm for tracing cell type changes via single-cell RNA-sequencing data.

Cell types in cell populations change as the condition changes: some cell types die out, new cell types may emerge and surviving cell types evolve to adapt to the new condition. Using single-cell RNA-sequencing data that measure the gene expression of cells before and after the condition change, we propose an algorithm, SparseDC, which identifies cell types, traces their changes across conditions and identifies genes which are marker genes for these changes. By solving a unified optimization problem, SparseDC completes all three tasks simultaneously. SparseDC is highly computationally efficient and demonstrates its accuracy on both simulated and real data.

The Unfolded Protein Response in Amelogenesis and Enamel Pathologies.

During the secretory phase of their life-cycle, ameloblasts are highly specialized secretory cells whose role is to elaborate an extracellular matrix that ultimately confers both form and function to dental enamel, the most highly mineralized of all mammalian tissues. In common with many other "professional" secretory cells, ameloblasts employ the unfolded protein response (UPR) to help them cope with the large secretory cargo of extracellular matrix proteins transiting their ER (endoplasmic reticulum)/Golgi complex and so minimize ER stress. However, the UPR is a double-edged sword, and, in cases where ER stress is severe and prolonged, the UPR switches from pro-survival to pro-apoptotic mode. The purpose of this review is to consider the role of the ameloblast UPR in the biology and pathology of amelogenesis; specifically in respect of amelogenesis imperfecta (AI) and fluorosis. Some forms of AI appear to correspond to classic proteopathies, where pathological intra-cellular accumulations of protein tip the UPR toward apoptosis. Fluorosis also involves the UPR and, while not of itself a classic proteopathic disease, shares some common elements through the involvement of the UPR. The possibility of therapeutic intervention by pharmacological modulation of the UPR in AI and fluorosis is also discussed.

Amelogenesis imperfecta caused by N-terminal enamelin point mutations in mice and men is driven by endoplasmic reticulum stress.

'Amelogenesis imperfecta' (AI) describes a group of inherited diseases of dental enamel that have major clinical impact. Here, we identify the aetiology driving AI in mice carrying a p.S55I mutation in enamelin; one of the most commonly mutated proteins underlying AI in humans. Our data indicate that the mutation inhibits the ameloblast secretory pathway leading to ER stress and an activated unfolded protein response (UPR). Initially, with the support of the UPR acting in pro-survival mode, Enamp.S55I heterozygous mice secreted structurally normal enamel. However, enamel secreted thereafter was structurally abnormal; presumably due to the UPR modulating ameloblast behaviour and function in an attempt to relieve ER stress. Homozygous mutant mice failed to produce enamel. We also identified a novel heterozygous ENAMp.L31R mutation causing AI in humans. We hypothesize that ER stress is the aetiological factor in this case of human AI as it shared the characteristic phenotype described above for the Enamp.S55I mouse. We previously demonstrated that AI in mice carrying the Amelxp.Y64H mutation is a proteinopathy. The current data indicate that AI in Enamp.S55I mice is also a proteinopathy, and based on comparative phenotypic analysis, we suggest that human AI resulting from the ENAMp.L31R mutation is another proteinopathic disease. Identifying a common aetiology for AI resulting from mutations in two different genes opens the way for developing pharmaceutical interventions designed to relieve ER stress or modulate the UPR during enamel development to ameliorate the clinical phenotype.

Identifying and removing the cell-cycle effect from single-cell RNA-Sequencing data.

Single-cell RNA-Sequencing (scRNA-Seq) is a revolutionary technique for discovering and describing cell types in heterogeneous tissues, yet its measurement of expression often suffers from large systematic bias. A major source of this bias is the cell cycle, which introduces large within-cell-type heterogeneity that can obscure the differences in expression between cell types. The current method for removing the cell-cycle effect is unable to effectively identify this effect and has a high risk of removing other biological components of interest, compromising downstream analysis. We present ccRemover, a new method that reliably identifies the cell-cycle effect and removes it. ccRemover preserves other biological signals of interest in the data and thus can serve as an important pre-processing step for many scRNA-Seq data analyses. The effectiveness of ccRemover is demonstrated using simulation data and three real scRNA-Seq datasets, where it boosts the performance of existing clustering algorithms in distinguishing between cell types.

Entry mechanisms of herpes simplex virus 1 into murine epidermis: involvement of nectin-1 and herpesvirus entry mediator as cellular receptors.

UNLABELLED: Skin keratinocytes represent a primary entry site for herpes simplex virus 1 (HSV-1) in vivo. The cellular proteins nectin-1 and herpesvirus entry mediator (HVEM) act as efficient receptors for both serotypes of HSV and are sufficient for disease development mediated by HSV-2 in mice. How HSV-1 enters skin and whether both nectin-1 and HVEM are involved are not known. We addressed the impact of nectin-1 during entry of HSV-1 into murine epidermis and investigated the putative contribution of HVEM. Using ex vivo infection of murine epidermis, we showed that HSV-1 entered the basal keratinocytes of the epidermis very efficiently. In nectin-1-deficient epidermis, entry was strongly reduced. Almost no entry was observed, however, in nectin-1-deficient keratinocytes grown in culture. This observation correlated with the presence of HVEM on the keratinocyte surface in epidermis and with the lack of HVEM expression in nectin-1-deficient primary keratinocytes. Our results suggest that nectin-1 is the primary receptor in epidermis, while HVEM has a more limited role. For primary murine keratinocytes, on which nectin-1 acts as a single receptor, electron microscopy suggested that HSV-1 can enter both by direct fusion with the plasma membrane and via endocytic vesicles. Thus, we concluded that nectin-1 directs internalization into keratinocytes via alternative pathways. In summary, HSV-1 entry into epidermis was shown to strongly depend on the presence of nectin-1, but the restricted presence of HVEM can potentially replace nectin-1 as a receptor, illustrating the flexibility employed by HSV-1 to efficiently invade tissue in vivo. IMPORTANCE: Herpes simplex virus (HSV) can cause a range of diseases in humans, from uncomplicated mucocutaneous lesions to life-threatening infections. The skin is one target tissue of HSV, and the question of how the virus overcomes the protective skin barrier and penetrates into the tissue to reach its receptors is still open. Previous studies analyzing entry into cells grown in vitro revealed nectin-1 and HVEM as HSV receptors. To explore the contributions of nectin-1 and HVEM to entry into a natural target tissue, we established an ex vivo infection model. Using nectin-1- or HVEM-deficient mice, we demonstrated the distinct involvement of nectin-1 and HVEM for HSV-1 entry into epidermis and characterized the internalization pathways. Such advances in understanding the involvement of receptors in tissue are essential preconditions for unraveling HSV invasion of skin, which in turn will allow the development of antiviral reagents.

The Accuracy of Small Area Sampling of Wireless Telephone Numbers.

The value of telephone surveys for assessing effects at geographic areas is impacted by increasing wireless telephone use. Highly accurate landline samples may be drawn for national, state, county, or even smaller areas; however, wireless samples have less geographic precision. This requires additional data collection effort and screening costs in order to ensure the appropriate geographic area is surveyed. In this paper, we examine the accuracy of wireless sample frame from the 2010-2011 National Flu Surveys. We illustrate differences and variations in wireless sampling accuracy for different geographic areas, focusing on variability by area in placement of wire centers related to residences. Our results suggest that the accuracy of wireless sampling may be dependent on differences in geographic areas with the accuracy of wireless sampling decreasing as the level of geographic aggregation gets more specific; landline accuracy remains relatively stable regardless of geographic specificity. To explain this phenomenon, we examine patterns of geographic dispersion of wireless telephone numbers related to telephone switch centers and geographic area. Based on the evidence from these surveys we present several options to estimate the geographic specificity of an area prior to sampling.

Endoplasmic reticulum stress in amelogenesis imperfecta and phenotypic rescue using 4-phenylbutyrate.

Inherited diseases caused by genetic mutations can arise due to loss of protein function. Alternatively, mutated proteins may mis-fold, impairing endoplasmic reticulum (ER) trafficking, causing ER stress and triggering the unfolded protein response (UPR). The UPR attempts to restore proteostasis but if unsuccessful drives affected cells towards apoptosis. Previously, we reported that in mice, the p.Tyr64His mutation in the enamel extracellular matrix (EEM) protein amelogenin disrupts the secretory pathway in the enamel-forming ameloblasts, resulting in eruption of malformed tooth enamel that phenocopies human amelogenesis imperfecta (AI). Defective amelogenin post-secretory self-assembly and processing within the developing EEM has been suggested to underlie the pathogenesis of X chromosome-linked AI. Here, we challenge this concept by showing that AI pathogenesis associated with the p.Tyr64His amelogenin mutation involves ameloblast apoptosis induced by ER stress. Furthermore, we show that 4-phenylbutyrate can rescue the enamel phenotype in affected female mice by promoting cell survival over apoptosis such that they are able to complete enamel formation despite the presence of the mutation, offering a potential therapeutic option for patients with this form of AI and emphasizing the importance of ER stress in the pathogenesis of this inherited conformational disease.

Mutations in C4orf26, encoding a peptide with in vitro hydroxyapatite crystal nucleation and growth activity, cause amelogenesis imperfecta.

Autozygosity mapping and clonal sequencing of an Omani family identified mutations in the uncharacterized gene, C4orf26, as a cause of recessive hypomineralized amelogenesis imperfecta (AI), a disease in which the formation of tooth enamel fails. Screening of a panel of 57 autosomal-recessive AI-affected families identified eight further families with loss-of-function mutations in C4orf26. C4orf26 encodes a putative extracellular matrix acidic phosphoprotein expressed in the enamel organ. A mineral nucleation assay showed that the protein's phosphorylated C terminus has the capacity to promote nucleation of hydroxyapatite, suggesting a possible function in enamel mineralization during amelogenesis.

Phenotype-genotype correlations in mouse models of amelogenesis imperfecta caused by Amelx and Enam mutations.

Mutations in human and in mouse orthologous genes Amelx and Enam result in a diverse range of enamel defects. In this study we aimed to investigate the phenotype-genotype correlation between the mutants and the wild-type controls in mouse models of amelogenesis imperfecta using novel measurement approaches. Ten hemi-mandibles and incisors were dissected from each group of Amelx(WT), Amelx(X/Y64H), Amelx(Y/Y64H), Amelx(Y64H/Y64H), and Enam(WT), Enam(Rgsc395) heterozygous and Enam(Rgsc395) homozygous mice. Their macro-morphology, colour and micro-topography were assessed using bespoke 2D and 3D image analysis systems and customized colour and whiteness algorithms. The novel methods identified significant differences (p ≤ 0.05) between the Amelx groups for mandible and incisor size and enamel colour and between the Enam groups for incisor size and enamel colour. The Amelx(WT) mice had the largest mandibles and incisors, followed in descending order of size by the Amelx(X/Y64H), Amelx(Y/Y64H) and Amelx(Y64H/Y64H) mice. Within the Enam groups the Enam(WT) incisors were largest and the Enam(Rgsc395) heterozygous mice were smallest. The effect on tooth morphology was also reflected by the severity of the enamel defects in the colour and whiteness assessment. Amelogenin affected mandible morphology and incisor enamel formation, while enamelin only affected incisors, supporting the multifunctional role of amelogenin. The enamelin mutation was associated with earlier forming enamel defects. The study supported the critical involvement of amelogenin and enamelin in enamel mineralization.

Differences in the accumulation of mitochondrial defects with age in mice and humans.

Mitochondrial DNA mutations and associated defects in cytochrome c oxidase (COX) are proposed to play an important role in human ageing; however there have been limited studies on the frequency of these defects in normal mouse ageing. Here we compare COX-deficiency in two epithelial tissues; the colon and the ciliary epithelium, from human and mouse. The pattern of accumulation of COX-deficiency is similar in both tissues in the two species; however the frequency of colonic crypts with COX-deficiency in aged humans is significantly higher than in aged mice, whereas the levels of COX-deficiency in the ciliary epithelium are higher in the mouse than in humans. This suggests the impact of mitochondrial defects on normal ageing may differ significantly between species.

Whole-Exome sequencing identifies FAM20A mutations as a cause of amelogenesis imperfecta and gingival hyperplasia syndrome.

Amelogenesis imperfecta (AI) describes a clinically and genetically heterogeneous group of disorders of biomineralization resulting from failure of normal enamel formation. AI is found as an isolated entity or as part of a syndrome, and an autosomal-recessive syndrome associating AI and gingival hyperplasia was recently reported. Using whole-exome sequencing, we identified a homozygous nonsense mutation in exon 2 of FAM20A that was not present in the Single Nucleotide Polymorphism database (dbSNP), the 1000 Genomes database, or the Centre d'Etude du Polymorphisme Humain (CEPH) Diversity Panel. Expression analyses indicated that Fam20a is expressed in ameloblasts and gingivae, providing biological plausibility for mutations in FAM20A underlying the pathogenesis of this syndrome.

Quantifying bias in a health survey: modeling total survey error in the national immunization survey.

Random-digit-dial telephone surveys are experiencing both declining response rates and increasing under-coverage due to the prevalence of households that substitute a wireless telephone for their residential landline telephone. These changes increase the potential for bias in survey estimates and heighten the need for survey researchers to evaluate the sources and magnitudes of potential bias. We apply a Monte Carlo simulation-based approach to assess bias in the NIS, a land-line telephone survey of 19-35 month-old children used to obtain national vaccination coverage estimates. We develop a model describing the survey stages at which component nonsampling error may be introduced due to nonresponse and under-coverage. We use that model and components of error estimated in special studies to quantify the extent to which noncoverage and nonresponse may bias the vaccination coverage estimates obtained from the NIS and present a distribution of the total survey error. Results indicated that the total error followed a normal distribution with mean of 1.72 per cent(95 per cent CI: 1.71, 1.74 per cent) and final adjusted survey weights corrected for this error. Although small, the largest contributor to error in terms of magnitude was nonresponse of immunization providers. The total error was most sensitive to declines in coverage due to cell phone only households. These results indicate that, while response rates and coverage may be declining, total survey error is quite small. Since response rates have historically been used to proxy for total survey error, the finding that these rates do not accurately reflect bias is important for evaluation of survey data. Published in 2011 by John Wiley & Sons, Ltd.

Is the 32-kDa fragment the functional enamelin unit in all species?

Enamelin is an extracellular enamel matrix protein essential for normal amelogenesis. After secretion, porcine enamelin is processed to generate several enamelin-degradation products. The cumulative 32-kDa enamelin is the most abundant enamelin present, and various roles for this molecule have been suggested. However, the proteolytic cleavage sites in porcine enamelin that generate the 32-kDa enamelin are not conserved across species, and the 32-kDa enamelin analogue may not be present in all species. To explore this we studied rat enamelin biochemistry using western blotting with anti-peptide IgGs to porcine 32-kDa enamelin and to the putative rat 32-kDa enamelin analogue. The dominant enamelins in secretory-stage rat enamel migrated at around 60-70 kDa. In contrast, the dominant enamelins in secretory-stage porcine enamel migrated at around 32 kDa. In contrast, secretory-stage porcine-enamel enamelins were dominated by the 32-kDa enamelin. Rat enamelin was completely removed from maturation-stage enamel without any accumulation of 32-kDa enamelin. We suggest that a discrete 32-kDa enamelin is not essential for normal amelogenesis in all species, and in pig it may be a processing product of a larger functional enamelin molecule. The pig may be an atypical model in terms of enamelin biochemistry and function, and caution should be exercised when assigning functional roles to the 32-kDa enamelin as a discrete enamel matrix entity.

Mitochondrial DNA defects and selective extraocular muscle involvement in CPEO.

PURPOSE. Chronic progressive external ophthalmoplegia (CPEO) is a prominent, and often the only, presentation among patients with mitochondrial diseases. The mechanisms underlying the preferential involvement of extraocular muscles (EOMs) in CPEO were explored in a comprehensive histologic and molecular genetic study, to define the extent of mitochondrial dysfunction in EOMs compared with that in skeletal muscle from the same patient. METHODS. A well-characterized cohort of 13 CPEO patients harboring a variety of primary and secondary mitochondrial (mt)DNA defects was studied. Mitochondrial enzyme function was determined in EOM and quadriceps muscle sections with cytochrome c oxidase (COX)/succinate dehydrogenase (SDH) histochemistry, and the mutation load in single muscle fibers was quantified by real-time PCR and PCR-RFLP assays. RESULTS. CPEO patients with mtDNA deletions had more COX-deficient fibers in EOM (41.6%) than in skeletal muscle (13.7%, P > 0.0001), and single-fiber analysis revealed a lower mutational threshold for COX deficiency in EOM. Patients with mtDNA point mutations had a less severe ocular phenotype, and there was no significant difference in the absolute level of COX deficiency or mutational threshold between these two muscle groups. CONCLUSIONS. The more pronounced mitochondrial biochemical defect and lower mutational threshold in EOM compared with skeletal muscle fibers provide an explanation of the selective muscle involvement in CPEO. The data also suggest that tissue-specific mechanisms are involved in the clonal expansion and expression of secondary mtDNA deletions in CPEO patients with nuclear genetic defects.

Defects in multiple complexes of the respiratory chain are present in ageing human colonic crypts.

Mitochondrial DNA (mtDNA) mutations accumulate in a number of ageing tissues and are proposed to play a role in the ageing process. We have previously shown that colonic crypt stem cells accumulate somatic mtDNA point mutations during ageing. These mtDNA mutations result in the loss of the activity of complex IV (cytochrome c oxidase (COX)) of the respiratory chain in the stem cells and their progeny, producing colonic crypts which are entirely COX deficient. However it is not known whether the other complexes of the respiratory chain are similarly affected during ageing. Here we have used antibodies to individual subunits of complexes I-IV to investigate their expression in the colonic epithelium from human subjects aged 18-84. We show that in approximately 50% of crypts with any form of respiratory chain deficiency, decreased expression of subunits of multiple complexes is observed. Furthermore we have sequenced the entire mitochondrial genome of a number of cells with multiple complex defects and have found a wide variety of point mutations in these cells affecting a number of different protein encoding and RNA encoding genes. Finally we discuss the possible mechanisms by which multiple respiratory chain complex defects may occur in these cells.

A mutation in the mouse Amelx tri-tyrosyl domain results in impaired secretion of amelogenin and phenocopies human X-linked amelogenesis imperfecta.

Amelogenesis imperfecta (AI) describes a broad group of clinically and genetically heterogeneous inherited defects of dental enamel bio-mineralization. Despite identification of a number of genetic mutations underlying AI, the precise causal mechanisms have yet to be determined. Using a multi-disciplinary approach, we describe here a mis-sense mutation in the mouse Amelx gene resulting in a Y --> H substitution in the tri-tyrosyl domain of the enamel extracellular matrix protein amelogenin. The enamel in affected animals phenocopies human X-linked AI where similar mutations have been reported. Animals affected by the mutation have severe defects of enamel bio-mineralization associated with absence of full-length amelogenin protein in the developing enamel matrix, loss of ameloblast phenotype, increased ameloblast apoptosis and formation of multi-cellular masses. We present evidence to demonstrate that affected ameloblasts express but fail to secrete full-length amelogenin leading to engorgement of the endoplasmic reticulum/Golgi apparatus. Immunohistochemical analysis revealed accumulations of both amelogenin and ameloblastin in affected cells. Co-transfection of Ambn and mutant Amelx in a eukaryotic cell line also revealed intracellular abnormalities and increased cytotoxicity compared with cells singly transfected with wild-type Amelx, mutant Amelx or Ambn or co-transfected with both wild-type Amelx and Ambn. We hypothesize that intracellular protein-protein interactions mediated via the amelogenin tri-tyrosyl motif are a key mechanistic factor underpinning the molecular pathogenesis in this example of AI. This study therefore successfully links phenotype with underlying genetic lesion in a relevant murine model for human AI.

Hereditary dentine disorders: dentinogenesis imperfecta and dentine dysplasia.

The hereditary dentine disorders, dentinogenesis imperfecta (DGI) and dentine dysplasia (DD), comprise a group of autosomal dominant genetic conditions characterised by abnormal dentine structure affecting either the primary or both the primary and secondary dentitions. DGI is reported to have an incidence of 1 in 6,000 to 1 in 8,000, whereas that of DD type 1 is 1 in 100,000. Clinically, the teeth are discoloured and show structural defects such as bulbous crowns and small pulp chambers radiographically. The underlying defect of mineralisation often results in shearing of the overlying enamel leaving exposed weakened dentine which is prone to wear. Currently, three sub-types of DGI and two sub-types of DD are recognised but this categorisation may change when other causative mutations are found. DGI type I is inherited with osteogenesis imperfecta and recent genetic studies have shown that mutations in the genes encoding collagen type 1, COL1A1 and COL1A2, underlie this condition. All other forms of DGI and DD, except DD-1, appear to result from mutations in the gene encoding dentine sialophosphoprotein (DSPP), suggesting that these conditions are allelic. Diagnosis is based on family history, pedigree construction and detailed clinical examination, while genetic diagnosis may become useful in the future once sufficient disease-causing mutations have been discovered. Differential diagnoses include hypocalcified forms of amelogenesis imperfecta, congenital erythropoietic porphyria, conditions leading to early tooth loss (Kostmann's disease, cyclic neutropenia, Chediak-Hegashi syndrome, histiocytosis X, Papillon-Lefevre syndrome), permanent teeth discolouration due to tetracyclines, Vitamin D-dependent and vitamin D-resistant rickets. Treatment involves removal of sources of infection or pain, improvement of aesthetics and protection of the posterior teeth from wear. Beginning in infancy, treatment usually continues into adulthood with a number of options including the use of crowns, over-dentures and dental implants depending on the age of the patient and the condition of the dentition. Where diagnosis occurs early in life and treatment follows the outlined recommendations, good aesthetics and function can be obtained.