Investigation of autism and GABA receptor subunit genes in multiple ethnic groups

Autism is a neurodevelopmental disorder of complex genetics, characterized by impairment in social interaction and communication, as well as repetitive behavior. Multiple lines of evidence, including alterations in levels of GABA and GABA receptors in autistic patients, indicate that the GABAergic system, which is responsible for synaptic inhibition in the adult brain, may be involved in autism. Previous studies in our lab indicated association of noncoding single nucleotide polymorphisms (SNPs) within a GABA receptor subunit gene on chromosome 4, GABRA4, and interaction between SNPs in GABRA4 and GABRB1 (also on chromosome 4), within Caucasian autism patients. Studies of genetic variation in African-American autism families are rare. Analysis of 557 Caucasian and an independent population of 54 African-American families with 35 SNPs within GABRB1 and GABRA4 strengthened the evidence for involvement of GABRA4 in autism risk in Caucasians (rs17599165, p=0.0015; rs1912960, p=0.0073; and rs17599416, p=0.0040) and gave evidence of significant association in African-Americans (rs2280073, p=0.0287 and rs16859788, p=0.0253). The GABRA4 and GABRB1 interaction was also confirmed in the Caucasian dataset (most significant pair, rs1912960 and rs2351299; p=0.004). Analysis of the subset of families with a positive history of seizure activity in at least one autism patient revealed no association to GABRA4; however, three SNPs within GABRB1 showed significant allelic association; rs2351299 (p=0.0163), rs4482737 (p=0.0339), and rs3832300 (p=0.0253). These results confirmed our earlier findings, indicating GABRA4 and GABRB1 as genes contributing to autism susceptibility, extending the effect to multiple ethnic groups and suggesting seizures as a stratifying phenotype.
Introduction
Autism is a neurodevelopmental disorder characterized by severe impairment in social interaction and communication, as well as repetitive behavior. Autism is part of a spectrum of disorders denoted autism spectrum disorders (ASD), which in addition to autism, include Asperger syndrome, childhood disintegrative disorder, and pervasive developmental disorder not otherwise specified (PDD-NOS). Onset of this disorder is typically before 3 years of age. The incidence of autism is estimated at approximately one in 1,000 individuals, with an increased incidence in males [1–3]. Incidences of approximately two to three in 1,000 are reported when a broader diagnosis includes the entire spectrum of pervasive developmental disorders [2, 4].
Evidence indicates that this disease has a strong genetic component. Twin studies have shown that monozygote twins have a higher recurrence rate than dizygotic twins. While the actual percentages have varied, recurrence rates have been measured as high as 95% in monozygote twins and 23% in dizygotic twins [5–7]. Despite this strong evidence for a genetic component, the inheritance appears to be complex, with estimates that more than 15 genes may be involved in its inheritance [8, 9], with different families carrying different combinations of contributing genes. The genes may act independently or interactively, adding to the complexity of determining the genetic contribution to this disorder.
There are two approaches to identifying genetic contributors to disease. The first is a genome wide search in which linkage or association analysis is used to identify regions of the genome that may contain autism susceptibility genes. The second is the candidate gene approach, which investigates a specific gene or genes for involvement in autism risk. In the candidate gene approach, genes are chosen for study based on either what is known about the gene’s function, its location (for example in a recognized linkage peak), or a combination of both. Several candidates are hypothesized to be involved in autism; however, no single candidate gene has consistently emerged as involved in autism risk.
One candidate pathway that is hypothesized to be involved in autism is the GABAergic system. Hussman [10] suggested that autism is the result of an imbalance of the excitatory glutamatergic and inhibitory GABAergic pathways, resulting in overstimulation in the brain and inability to filter out excess stimuli from environmental and intrinsic sources. This theory is supported by multiple lines of evidence. First, histological, biochemical, and molecular approaches have demonstrated altered levels and distribution of GABA and GABA receptors in peripheral blood and plasma, as well as in the brain, including decreased GABA-A receptors and benzodiazepine binding sites in the hippocampal formation [11–13]. There are also reported alterations in GABAergic neurons, as demonstrated by the increased packing density of GABAergic interneurons in the CA3 and CA1 subfields, and by the decreased numbers and reduced size of cerebellar GABAergic Purkinje cells [14, 15]. In addition, duplications and isodicentric chromosomes in the region containing the three clustered GABA receptor subunits GABRB3, GABRA5, and GABRG3 on chromosome 15q have been associated with autism [16, 17]. As well, evidence for both linkage and allelic association have been reported for this same GABA gene cluster, although the findings have not been consistent across datasets [18–22]. Investigation of association of GABA receptor subunits outside of the chromosome 15 region has been limited [23]. Lastly, mutations have been reported in multiple GABA receptor genes in families with epilepsy [24]. Given the high comorbidity of autism with epilepsy and seizures, these data suggest that a similar molecular etiology could exist between the disorders.
Signaling in the GABAergic system is mediated by receptors for the neurotransmitter GABA. There are 19 known GABA receptor subunits arranged in clusters throughout the genome. Functional pentamers formed by various combinations of these subunits result in receptors of varying properties and sensitivities. The amounts and functional capabilities of individual receptor subunits that form a specific pentamer can affect the amount and quality of signaling in different parts of the brain.
Previously published work from our laboratory analyzed 14 autosomal GABA receptor genes on four different chromosomes, using 70 SNPs in a Caucasian dataset of 470 families. This analysis revealed allelic association to rs1912960 (p=0.01) within gamma-aminobutyric acid (GABA) A receptor, alpha-4 (GABRA4) on chromosome 4 [23] and a significant interaction with rs2351299 within the neighboring gene gamma-aminobutyric acid (GABA) A receptor, beta-1 (GABRB1).
Despite similar prevalence rates between Caucasian and African-Americans [25, 26], autism studies in African-Americans are rare. Risk alleles may be different between ethnic groups or the same risk alleles may have differential effects in each ethnic group, warranting studies in multiple groups. Evidence that phenotypic factors, including indicators of language development, may be more severe in African-Americans, compared to non-Hispanic Caucasians, [27] is consistent with these possibilities and underscores the need to investigate autism in different ethnic groups. In this study, we present a validation of our previous report in an independent dataset of 54 African-American families, as well as confirmation of our previous results in an expanded Caucasian sample of 557 autism families.
Materials and methods
Samples
All families were drawn from a large multisite study of autism genetics conducted in the southeastern United States. These families are recruited through the Center for Human Genetics (CHG) at Duke University Medical Center (DUMC), the University of South Carolina, and the Center for Human Genetic Research at Vanderbilt (N=54 African-American and 557 Non-Hispanic Caucasian families) through support groups, advertisements, and clinical and educational settings. All sites recruited, enrolled, and sampled individuals with autism and family members, per study protocols approved by their respective institutional review boards (IRBs). Written informed consent was obtained from parents and from children who were able to give informed consent. Families were enrolled based on probands meeting the following core inclusion criteria of: (1) probands ranging from three to 21 years of age; (2) a presumptive clinical diagnosis of autism; and (3) an expert clinical diagnosis of autism using DSM-IV criteria [28], supported by the Autism Diagnostic Interview-Revised (ADI-R) [29] and in some cases, the Autism Diagnostic Observation Schedule (ADOS) [30]. Our original ascertainment protocol relied on clinical expertise and the ADI-R. The ADOS was subsequently added as a required diagnostic method, and families that were missing ADOS assessments were updated as available. To assure valid ADI-R results, all participants who met current diagnostic criteria for autism were included only if they had a minimal developmental level of 18 months, as extrapolated from the Vineland Adaptive Behavior Scale score [31], or had an IQ equivalent greater than 35 based on scores from a standardized measure of cognitive ability such as an the Wechsler Scale of Intelligence for Children—4th edition [32], Differential Abilities Scale [33], Mullen Scales of Early Learning [34] or Leiter Intelligence Scale-Revised [35]. IQ data was derived from medical records or direct assessment. Exclusion criteria for participation in the larger genetics study included: severe sensory problems (e.g., visual impairment or hearing loss), significant motor impairments (e.g., failure to sit by 12 months, or walk by 24 months), or identified metabolic, genetic, or progressive neurological disorders, based on screening by clinical staff. Additional samples are from the Autism Genetic Research Exchange (AGRE). These individuals were qualified using similar methods including ADI-R, ADOS, VABS, and a standardized measure of IQ.
Thirty-nine African-American families were used in an initial GABA receptor screen. Follow-up analysis of significant findings was performed in 54 African-American families. Analysis of the extended Caucasian dataset included 557 non-Hispanic Caucasian families. One-hundred and five new non-Hispanic Caucasian families were added to the analysis (18 families previously analyzed by Ma et al. [23] were newly identified as Hispanic, and were omitted from the current study in an effort decrease heterogeneity in the Caucasian dataset).
Classification of history of seizure activity in autism patients was based on question 92 from the ADI-R, which queries for both current and lifetime presence of convulsions, seizures, and epilepsy. Caregiver responses to question 92 are coded to indicate no seizure activity, seizure activity with no definitive diagnosis of epilepsy, and seizures with a definite diagnosis of epilepsy. Using lifetime ratings, two groups of families were defined: those in which no seizure activity was reported, and those in which seizure activity was present in at least one autism patient. In addition, question 92 allows for coding of febrile seizures. Families with only febrile seizures were classified as negative for seizure activity and not included in the seizure subset analysis. Both families with positive and negative history of seizure activity were included in our overall dataset. This resulted in a dataset of 41 Caucasian families with a positive history for seizures.
Molecular analyses and genotyping
The analysis of 14 GABA receptor subunit genes was performed in 39 African-American families as previously described [23]. Briefly, between three and seven intronic, UTR and synonymous coding SNPs within each gene were identified from Applied Biosystems (ABI, Foster City, CA, USA) Assay on Demand (AoD) products, resulting in 70 SNPs within 14 GABA receptor genes on four autosomes. Genes analyzed were: GABRA1, GABRA6, GABRB2, GABRG2, and GABRP from chromosome 5; GABRA2, GABRA4, GABRB1, and GABRG1 from chromosome 4; GABRB3, GABRA5, and GABRG3 from chromosome 15; and GABRR1 and GABRR2 from chromosome 6.
Additional SNPs within GABRA4 and GABRB1 were analyzed in the extended African-American (N=54) and Caucasian (N=557) datasets to expand the coverage of variation across this region. Thirty-five SNPs, representative of different linkage disequilibrium (LD) blocks across the two genes (20 in GABRA4 and 15 in GABRB1), were genotyped (Fig. 1). SNP’s for genotyping were selected from online databases (University of California Santa Cruz http://genome.ucsc.edu and NCBI dbSNP http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=snp) and from resequencing of exons and surrounding areas of both GABRB1 and GABRA4 genes.
Fig. 1Approximate locations of SNPs for extended analysis within GABRA4 and GABRB1. Horizontal arrows represent transcriptional direction. Vertical arrows represent SNPs, as numbered and identified below the diagram. Pter indicates the P-terminal end of the chromosome and cen indicates the centromere
SNP genotyping was performed using Taqman allelic discrimination assays (Applied Biosystems). DNA was extracted from whole blood according to established protocols [36], and 3 ng of genomic DNA was used per reaction. Amplification was performed on GeneAmp PCR Systems 9700 thermocyclers, with cycling conditions as recommended by Applied Biosystems. Fluorescence was measured using Applied Biosystem’s 7900. Genotype discrimination was conducted using ABI Prism SDS 2.1 software. Quality control, to ensure accurate genotyping, involved two different CEPH DNAs in quadruplicate on each 384 well plate, as well as the presence of samples which were replicated elsewhere in the sample list. Additionally, ≥95% genotyping efficiency is required.
Statistical analysis
To ensure genotyping quality, Pedcheck was run for detection of Mendelian inheritance inconsistency. One affected and one unaffected individual from each family were selected randomly for tests of Hardy–Weinberg equilibrium (HWE), which was assessed using exact tests from the Genetic Data Analysis program [37]. Pairwise LD between markers was calculated using graphical overview of linkage disequilibrium (GOLD) [38] in the parents of autism cases for both the African-American and Caucasian samples. LD was evaluated in parents to increase the available sample size for analysis and comparison between the two ethnic groups. The pedigree disequilibrium test (PDT) and its extension the genotypic pedigree disequilibrium test (genoPDT) [39, 40] were used to test for association to autism susceptibility.
The EMDR [23, 41], an extension of the MDR [42, 43], was used to test for potential gene–gene interaction, to identify specific locus combinations of interest for further investigation and validation of previous results. EMDR analysis was performed using seven SNPs, the four in GABRA4 found to show significant allelic or genotypic association in the Caucasian sample-set, and the three in GABRB1 found to be significant in the seizure subgroup. One-, two-, and three-way analysis was performed on the Caucasian dataset. For case-control pairs used in EMDR, the proband (or most completely genotyped affected child) from each multiplex and triad family was selected (n=470 total) as a case. Controls were generated using the untransmitted alleles from parental genotypes. In this study, a cross-validation option was not utilized. The sample size of the African-American dataset is too small to provide power for EMDR analysis, not allowing us to test for validation of the interaction seen in the paper of Ma et al. within the African-American dataset.
The haplotype family-based association test (HBAT; [44]) was used for haplotype association analysis using the significant SNPs in GABRA4.
Results
Allelic association studies of 70 SNPs across the 14 GABA receptor subunit genes in the 39 African-American screen set of families, revealed association in rs2280073 (GABRA4; p=0.0053) and hcv2119841 (GABRB1; p=0.0343), the same two genes identified through allelic association and interaction analysis in the Caucasian dataset [23]. Genotypic association analysis revealed the same GABRA4 SNP, rs2280073 (p=0.0262), and marginal significance within GABRP, rs1862242 (p=0.0471). The remaining SNPs showed no significant association (data not shown).
Analysis of the screening SNPs and newly identified SNPs within GABRA4 and GABRB1 in the Caucasian population (N=557), and within the extended African-American population (N=54) (Table 1), revealed new SNPs with significant association. In the Caucasian dataset, rs1912960, also significant in the study of Ma et al. (p=0.012), showed significant allelic association (p=0.0073). Additional significant SNPs were identified in GABRA4 as well, rs17599165 (p=0.0015) and rs17599416 (p=0.0040). Genotypic association was also seen in these SNPs (p=0.0046, 0.0009, and 0.0043, respectively), as well as in a fourth SNP, also in GABRA4 (rs7660336, p=0.0368). In the African-American dataset, rs2280073 (p=0.0287), identified in the smaller African-American dataset above, and rs16859788 (p=0.0253), were found to be associated with the allele based test. Genotypic association was also identified in rs16859788 (p=0.0412). No SNPs within GABRB1 were found to be associated with autism in either ethnic group.
Table 1Analysis of GABRA4 and GABRB1 in extended Caucasian and African-American datasetsGABRA4GABRB1SNPCaucasianAfrican-AmericanSNPCaucasianAfrican-AmericanPDTaGeno PDTaPDTaGeno PDTaPDTGeno PDTPDTGeno PDTRS76783380.93500.99230.22300.4190RS18669890.30710.38600.55530.7892RS64475170.88260.70340.5050.3930RS23512990.45290.08220.57750.8614RS175991020.80550.89130.75180.8805RS100163880.15850.26600.23670.4259RS76603360.08330.0368(G/G)0.51640.7410RS13724960.23620.37400.24820.3715RS15121360.90520.98690.28880.3575RS31140840.09340.19420.20590.3281RS175991650.0015(T)0.0009(T/T)0.65470.4304RS44827370.14950.25041.00001.0000HCV15925450.77980.94270.22300.5313HCV113535240.19590.31171.00001.0000RS76855531.00000.84190.49130.6044RS37755340.18930.18310.49130.4913RS19129600.0073(C)0.0046(C/C)0.41110.5110HCV21198410.33520.08380.22780.4111RS20559430.96710.94340.49270.7607RS62870.40450.35710.61710.6984RS22800730.14040.09550.0287(G)0.1100RS62890.93490.55540.86580.9220RS168597880.31730.31730.0253(A)0.0412(A/A)RS62900.42850.19730.31730.4594RS175994160.0040(A)0.0043(A/A)0.80840.80844P04131.00001.00000.73890.7389RS37922081.00000.49800.17970.1797RS100289450.32720.45841.00000.8179RS105171740.94840.08940.71500.8903RS38323000.40940.63520.65470.6547RS76940350.43370.62660.36570.3657RS37922110.90570.83820.65470.2895RS22299400.78730.93750.54850.7866RS131517590.75290.93260.23670.5759RS131517690.47400.74360.18240.4768Bold indicates significant valuesaAssociated allele/genotype shown in parenthesis
The majority of pairwise r2 values between the significant SNPs were less than 0.3, in both ethnic groups (Table 2). However, a few SNPs have values between 0.3 and 0.35. SNPs, rs17599165, and rs17599416 have r2 values of 0.709 in African-Americans and 0.853 in Caucasians, and rs7660336 and rs2280073 have a pairwise r2 of 0.907 in Caucasians and 0.905 in African-Americans. Allele frequencies are similar, yet not identical between the two groups. One SNP, however, did have large differences in minor allele frequencies (MAFs) between the two groups, with a MAF of 0.24 in African-Americans but only 0.001 in Caucasians (Table 2). Haplotype analysis, using the four SNPs with significant allelic or genotypic association in the Caucasian families, revealed a significant global test (p=0.014) in the Caucasian population, further supporting the involvement of these SNPs or another variant on the haplotype background. The haplotype composed of all risk alleles trends towards significance with autism as a risk haplotype (p=0.0763), while the haplotype of all protective alleles resulted in being highly significantly protective (p=0.0006; Table 3)
Table 2Minor allele frequencies and linkage disequilibrium in Caucasian and African-American datasetsBold indicates significant valuesaMAF minor allele frequencyTable 3Haplotype frequency and association in Caucasian populationRS7660336RS17599165RS1912960RS17599416Haplotype frequencyP-valueGTCA0.5030.0763CTCA0.2770.9214CTGA0.1240.6094CAGG0.0820.0006Bold indicates significant values
Subsetting of the GABRA4 and GABRB1 data to analyze all families with positive history for seizures revealed no association to GABRA4. However, three SNPs within GABRB1 were found to be both allelically and genotypically associated with autism: rs2351299 (p=0.0163 and p=0.0189 for PDT and genoPDT, respectively), rs4482737 (p=0.0339 and p=0.0339), and rs3832300 (p=0.0253 and p=0.0357). These three SNPs all had pairwise r2 values less than 0.1 (data not shown).
In the Caucasian population, EMDR verified the single locus effect identified through PDT analysis in rs1912960 (p=0.024), and identified two different significant two-locus gene–gene effects between GABRA4 and GABRB1, rs1912960 with rs2351299 (p=0.004), and rs17599416 with rs2351299 (p=0.014). Several three locus effects were also significant [rs7660336, rs1912960, and rs2351299 (p=0.012); rs17599165, rs1912960, and rs2351299 (p=0.012); rs1912960, rs17599416, and rs2351299 (p=0.038); and rs7660336, rs17599416, and rs2351299 (p=0.047)] (Table 4).
Table 4EMDR results in Caucasian dataset between GABRA4 and GABRB1Input SNPsSignificant interactionsGeneSNP numberSNPSNPsP-valuesGABRA41RS7660336One-way30.0242RS17599165Two-way3, 50.0043RS19129604, 50.0144RS17599416Three-way1, 3, 50.012GABRB15RS23512992, 3, 50.0126RS44827373, 4, 50.0387RS38323001, 4, 50.047
Discussion
We have confirmed the involvement of GABRA4 in autism through identification of significantly associated SNPs within an independent African-American population. Furthermore, we have strengthened our original findings, including identification of additional associated SNPs and a significant interaction between GABRA4 and GABRB1 and in an extended dataset (N=557) of Caucasian autism families. The AA dataset contains only 54 families, and in general power can be a problem in small sample sets if an effect is not seen. However, for the GABRA4 gene, we did find significant results in the AA dataset. Furthermore, studies in small datasets such as the AA dataset can also be less robust than in larger datasets. However, the fact that we find the same gene, GABRA4, significant in two different ethnic groups is supportive of the role of GABRA4 in autism risk.
The identification of two different two-way interactions between GABRA4 and GABRB1 provides additional evidence of the complex interaction of these two genes in autism. The rs1912960 (GABRA4) with rs2351299 (GABRB1) interaction is between the same two SNPs previously reported in the study of Ma et al., and is still significant in our larger dataset. A second significant two-way interaction was found, also including rs2351299 in GABRB1 as well as rs17599416 in GABRA4, further supporting that interactions between these two genes are involved in autism risk. Although rs2351299 does not have a significant PDT result, it is found in both significant interaction pairs, and in each of the significant three-way interactions. The MDR approach used in these analyses is specifically designed to detect interaction effects both in the presence and absence of main effects.
It is possible that both of the two-way interactions are being identified due to LD between the two GABRA4 SNPs (rs1912960 and rs17599416). There is significant correlation between the two SNPs, although the r2 value (Table 2) between them is not large (0.32). Given that these do not appear to be causative variants, it is likely that the true variant, yet to be identified, is in LD with these GABRA4 SNPs. Examination of interaction in the independent dataset of African-American families was not possible due to the limited sample size.
We also identified variants within GABRB1 as associated within the autistic population with seizures. One of these variants, rs2351299, also shows a significant interaction with SNPs in GABRA4 (Table 4). While no effect was seen in GABRA4 for the seizure subset, the sample size may be too small to conclusively determine its role in seizure status in autism. However, the enhanced findings in GABRB1 in the seizure subset implicate GABRB1 as a contributor to genetic risk in these patients.
Despite the identification of GABRA4 in both ethnic groups, different SNPs were found to be associated. The identification of distinct SNPs within these populations may indicate differences in allele frequency and linkage disequilibrium within the two racial groups, differences in the haplotypic background in which identical causative variations originated, or differences in the causative variation. SNP rs16859788 for example, which is significant in the African-American group, shows little variation in the Caucasian dataset, therefore, providing no power for detection of an effect in this group. Other SNPs, however, show similar allele frequencies between the two populations.
Some differences in LD do exist between the two ethnic groups as well; however, the majority of the differences are small. The largest differences in LD are in pairwise values with rs16859788, which appear to mostly be due to the fact that the SNP is practically monoallelic in the Caucasian population. The Caucasian dataset suggests that there is a significant association of SNP haplotypes with autism risk, while the African-American set does not. However, this difference may be due to the small size of the African-American dataset. Therefore, while it appears that minor allele frequency differences can account for the lack of association of rs16859788 in the Caucasian dataset, additional studies are needed to determine whether the other differences in results between the two ethnic groups are due to sample size differences, differential LD with the causative variation in the two populations, or are population-specific risk alleles.
While we have identified several associated SNPs, we do not predict any of the ones in GABRA4 to have functional consequences; therefore, it is unlikely that these are primary variants leading to the autism susceptibility. One of the SNPs identified in GABRB1 in the seizure subset, however, is in the 3′ untranslated region (UTR). Given that multiple GABA receptor subunits combine in varying combinations to form a functional GABA receptor, even minor changes in levels of a particular subunit may alter the makeup of receptors within a particular cell type, and alter the GABAergic signaling. Therefore, variations within potential regulatory regions, such as untranslated regions and promoters, could play an important role. It will be important to look at potential changes that may result from this and other potential GABRB1 UTR variations, as well as sequence coding and potential regulatory regions to identify the primary variation, or variations leading to altered autism susceptibility.
In summary, these data show that the GABA receptors are implicated in the etiology of autism in two different ethnic populations and suggest seizures as a stratifying phenotype. Furthermore, these results support our earlier findings, indicating GABRA4 and GABRB1 as genes contributing to autism susceptibility, independently, in the case of GABRA4, and through complex interactions with each other.

autism
gaba receptors
ethnicity
association
snps


Management and prognostic factors of recurrent pleomorphic adenoma of the parotid gland: personal experience and review of the literature

The aim of this study was to investigate the management and prognostic determinants of recurrent pleomorphic adenoma (RPA). A retrospective analysis was performed to examine the clinical features, the prevalence of surgical complications, and new recurrences of RPA. Tumor recurrence rate was estimated by the Kaplan–Meier method, and the prognostic value of some of the variables was tested by univariate analysis using the log rank test. The study focused on 33 patients, 18 female (54.5%) and 15 male (45.5%), aged 12–71 years (median 41). A total or extended total parotidectomy was performed in 16 cases (48.5%), a superficial parotidectomy in 10 cases (30.3%), and a local excision in 7 cases (21.2%). In ten patients (30.3%), a branch or the trunk of the facial nerve was deliberately sacrificed. Major complications included one unexpected definitive paralysis of the marginal mandibular branch of the facial nerve and 14 cases of Frey syndrome. Follow-up varied from 2 to 25 years (median 10.5 years), and there were 11 new recurrences (33.3%) within a period varying from 1 to 16 years (median 6 years). The estimated tumor recurrence rates were 14.1 ± 6.6% at 5 years, 31.4 ± 9.4% at 10 years, 43.0 ± 10.8% at 15 years, and 57.2 ± 14.8% at 20 years. Presence of a multinodular lesion and the type of intervention performed were significantly associated with a higher probability of recurrence. RPAs are prone to new recurrences, especially when multinodular and treated with a local excision. Surgical treatment should include facial nerve resection in selected cases. Follow-up for the patient’s lifetime is warranted.
Introduction
Pleomorphic adenoma is the most common neoplasm of the parotid gland. It is a benign tumor composed of epithelial and myoepithelial cells arranged in various morphological patterns. Thinning or absence of the pseudocapsule and the presence of fingerlike projections of the tumor have been observed in all histologic subtypes of pleomorphic adenoma, in particular the myxoid type [1]. Even though many hypotheses for recurrences of parotid gland pleomorphic adenoma have been advanced, including cell biological and genetic factors [2, 3], obvious or underestimated tumor spillage, incomplete excision, and violation of the pseudocapsule of the tumor are considered the only proven reasons contributing to recurrent disease [4].
This is why the abandonment of enucleation techniques in favor of more extended surgical procedures, which require the tumor to be excised with the surrounding normal tissue and the facial nerve to be identified and preserved, have dramatically reduced the recurrence rate of pleomorphic adenomas of the parotid gland from 20–45% to less than 4% in the last decades [5–7].
However, pure en bloc excision in parotid benign tumor disease has been considered impossible by most surgeons when the pleomorphic adenoma is lying on the facial nerve, and so partial enucleation is often the only surgical option in those cases [5, 7], thus explaining the possibility of recurrence even after proper surgery.
Most of the recent literature on recurrent pleomorphic adenoma (RPA) of the parotid gland has attempted to analyze the causes of primary failure and discusses other possible risk factors for recurrence [8–12]; however, there are very few papers focused on the prognostic factors of RPA and retreatment policies [13–18].
The present study was undertaken to address these important issues based on a follow-up period of up to 20 years. Here, we present our findings and review some of the more recent literature.
Materials and methods
A retrospective analysis of the charts of patients treated for benign parotid neoplasm between 1983 and 2004 was performed to identify those patients operated on for RPA. We collected information on patient demographics and clinical history, clinical features of the lesions, diagnostic work-up, the type of surgery, operative findings, facial nerve management, and adjuvant treatment.
The study analyzed the prevalence of surgical complications and new recurrences. Statistical analysis was performed using the SPSS statistical package. Tumor recurrence rate was estimated by the Kaplan–Meier method: the entry point was the date of surgery for a recurrent tumor seen for the first time in our department, and the end point was the date of a new recurrence or the date of last consultation for censored observations.
The prognostic value of certain categorical variables (age, gender, number of previous operations, type of previous operation, lobe of origin, type of surgery performed for RPA, maximum diameter, facial nerve resection, presence of a pseudocapsule at the surgical margins, rupture of the pseudocapsule, number of neoplastic nodules, postoperative radiotherapy) was tested by univariate analysis using the log rank test.
Results
Among 524 patients treated for a benign parotid neoplasm between 1983 and 2004, 33 underwent surgical treatment for RPA. Eighteen (54.5%) patients were female and 15 (45.5%) male; their ages ranged from 12 to 71 years (median 41). None had been previously affected by a malignant neoplasm or submitted to radiation treatment. Seven patients were previously operated on in our department (with a prevalence of recurrence after first treatment of 1.3%) and 26 patients had previous surgical treatment(s) performed elsewhere. A summary of the patient’s previous clinical history is reported in Table 1. A swelling in the parotid region was the most significant complaint for 87.9% (29/33) of patients when they came to our attention for the recurrence; pain or facial nerve paresis was associated with the swelling in two cases. In four (12.1%) cases, the relapse was recognized by routine postoperative follow-up examinations. Other complaints, present from previous surgeries, were facial nerve paralysis in four patients and Frey syndrome in one.
Table 1Summary of clinical history of patients treated for the first time at the Department of Otolaryngology of the University of Brescia for RPA of the parotid glandPrevious recurrences  One63.6% (21)  Two 18.2% (6)  Three 12.1% (4)  Four 6.1% (2)Median time to recurrence  First recurrence (33) 6 years (range 1–23)  Second recurrence (12) 5 years (range 2–28)  Third recurrence (6) 5.5 years (range 3–19)  Fourth recurrence (2) 8 years (both patients)Last operation  Subtotal parotidectomy 33.3% (11)  Local excision 33.3% (11)  Superficial parotidectomy 21.2% (7)  Not reported 12.1% (4)Number of patients in parentheses
Ultrasonography was the most frequently used diagnostic tool (23 patients, 69.7%), followed by fine needle aspiration biopsy in 21 (63.6%) patients, magnetic resonance imaging in 14 (42.4%), computed tomography in 3 (9.1%), and sialography in 2 (6.1%). The different types of diagnostic procedures employed reflect the multi-decade period analyzed. At present, after ultrasonography, which is routinely adopted for follow-up, magnetic resonance imaging is used to delineate the extension of the recurrent lesions.
In 12 (36.4%) patients, the lesion involved the superficial lobe of the parotid gland, in 9 (27.3%) both lobes, and in 12 (36.4%) only the deep lobe was involved. In 5 (15.2%) patients the lesion also extended to the parapharyngeal space. Information on multinodularity of the lesion could be obtained for 31 patients: multiple lesions were observed in 24 (77.4%) of them.
Surgical treatment was tailored to the single patient in an attempt to obtain a margin of healthy tissue (Table 2). Postoperative complications are listed in Table 3. Nine (27.3%) patients were submitted to postoperative radiotherapy. There were no standard criteria for postoperative radiotherapy, but it was performed when the excision did not guarantee sufficient free margins of healthy tissue based on the judgment of the surgeon.
Table 2Surgical treatment for RPA of the parotid gland at the Department of Otolaryngology of the University of BresciaTotal or extended total parotidectomy 48.5% (16)Superficial parotidectomy 30.3% (10)Local excision 21.2% (7)Partial or total facial nerve resection30.3% (10)Number of patients in parenthesesTable 3Postoperative complications of patients treated for RPA of the parotid gland at the Department of Otolaryngology of the University of BresciaFrey syndrome 43.7% (14/32)Temporary paresis of a branch or of the entire facial nerve30.4% (7/23)Definitive paralysis of the marginal mandibular branch of the facial nerve4.3% (1/23)Salivary fistula3.0% (1/33)Keloid3.0% (1/33)
The patients were followed by yearly ultrasonography. Follow-up varied from 2 to 25 years (median 10.5 years). Three patients died from a metachronous malignant neoplasm, one of them after a new recurrence. The other patients are still alive. Overall, 11 (33.3%) new recurrences were observed. No patient with a single node had a recurrence. Four of the new recurrences had been operated on again and one had a further recurrence. Seven patients with small (less than 1 cm) and stable new recurrences are still on follow-up. The time for new recurrence varied from 1 to 16 years (median 6 years). There were 2 cases of focal atypical cells but no malignant transformation in the new recurrences. Distribution of the recurrences in relation to the different variables under analysis is shown in Table 4. The estimated tumor recurrence rates were 14.1 ± 6.6% at 5 years, 31.4 ± 9.4% at 10 years, 43.0 ± 10.8% at 15 years, and 57.2 ± 14.8% at 20 years (Fig. 1). Among the variables evaluated with the log-rank test, only the presence of a multinodular recurrence and the type of intervention performed were significantly associated with a higher probability of a new recurrence (Table 5). The high variation in tumor recurrence rates observed after 20 years of follow-up (Table 5) is probably related to the low number of patients with longer follow-up times.
Table 4Distribution of the new recurrences after treatment for RPA of the parotid gland at the Department of Otolaryngology of the University of Brescia (No. = 11; 33.3%)Variable%P-valueAge≤30 years (3/10)30.01>30 years (8/23)34.8GenderFemale (4/18)22.20.1Male (7/15)46.7Number of previous operations1 (7/21)33.31>1 (4/12)33.3Type of previous operationsEnucleation (2/11)18.20.2Parotidectomy (8/18)44.4Lobe of originSuperficial (5/21)23.80.1Deep (6/12)50.0Intervention performedParotidectomy (6/26)23.10.02Other (5/7)71.4Maximum diameter≤2 cm (7/16)43.80.3>2 cm (3/15)20.0Facial nerve resectionNo (7/23)30.40.7Yes (4/10)40.0Involvement of surgical marginsNo (8/23)34.80.5Yes (2/8)25.0Capsular ruptureNo (6/23)26.10.2Yes (4/8)50.0Multiple nodulesNo (0/7)00.04Yes (10/24)41.7Postoperative radiotherapyNo (9/24)37.50.3Yes (2/9)22.2Fig. 1Estimated recurrence rate after treatment for recurrent pleomorphic adenoma of the parotid gland at the Department of Otolaryngology of the University of Brescia (Kaplan–Meier method)Table 5Estimated tumor recurrence rates after treatment for RPA of the parotid gland at the Department of Otolaryngology of the University of Brescia (log-rank test)VariableEstimated tumor recurrence rates (%)P-value5-year10-year15-year20-yearAge≤30 years (10)12.5 ± 11.741.7 ± 18.6––0.8> 30 years (23)14.7 ± 7.926.9 ± 10.542.4 ± 12.856.8 ± 15.7GenderFemale (18)13.4 ± 9.030.7 ± 13.130.7 ± 13.130.7 ± 13.10.4Male (15)14.3 ± 9.430.7 ± 13.056.7 ± 15.171.1 ± 16.3Number of previous operations1 (21)11.8 ± 7.925.4 ± 11.145.6 ± 14.763.7 ± 17.80.9>1 (12)18.5 ± 11.941.8 ± 16.341.8 ± 16.341.8 ± 16.3Type of last previous operationEnucleation (11)9.1 ± 8.724.2 ± 15.624.2 ± 15.624.2 ± 15.60.2Parotidectomy (18)20.0 ± 10.345.1 ± 14.058.9 ± 15.9100Lobe of originSuperficial (21)013.8 ± 9.132.1 ± 13.654.8 ± 20.60.07Deep (12)35.8 ± 14.459.9 ± 16.459.9 ± 16.459.9 ± 16.4Intervention performedParotidectomy (26)5.3 ± 5.116.4 ± 8.730.5 ± 11.747.9 ± 17.40.001Other (7)42.9 ± 18.7–––Maximum diameter≤2 cm (16)19.6 ± 10.226.9 ± 11.644.3 ± 14.072.2 ± 20.90.4>2 cm (15)10.0 ± 9.532.5 ± 15.532.5 ± 15.532.5 ± 15.5Facial nerve resectionNo (23)9.4 ± 6.327.2 ± 10.635.3 ± 12.151.4 ± 16.70.4Yes (10)25.0 ± 15.340.0 ± 18.2––Involvement of surgical marginsNo (23)14.0 ± 7.524.7 ± 9.737.9 ± 11.753.4 ± 16.00.3Yes (8)25.0 ± 21.762.5 ± 28.6––Capsular ruptureNo (23)16.4 ± 8.838.7 ± 12.838.7 ± 12.838.7 ± 12.80.8Yes (8)12.5 ± 11.712.5 ± 11.740.0 ± 18.2100Multiple nodulesNo (7)00000.02Yes (24)19.6 ± 8.936.8 ± 11.253.2 ± 13.4100Postoperative radiotherapyNo (24)14.2 ± 7.734.3 ± 11.848.9 ± 12.965.9 ± 16.40.3Yes (9)12.5 ± 11.625.0 ± 15.325.0 ± 15.325.0 ± 15.3
Discussion
It is generally accepted that the standard treatment of pleomorphic adenoma of the parotid gland is a formal parotidectomy or at least the removal of the tumor with a surrounding cuff of normal parotid tissue after identification, dissection and preservation of the facial nerve [7]. Unfortunately, if the tumor is in contact with the facial nerve, even a total parotidectomy can not guarantee the presence of a cuff of normal tissue, because the surgeon must carry on the dissection between the tumor and the branches of the nerve with the risk of leaving microscopic disease behind or causing microscopic spillage of the tumor. This accounts for 1–4% probability of recurrence [5, 7]. These low percentages, together with the increasing number of institutions that treat parotid neoplasms, have reduced the number of cases of RPA being referred to a single institution; this, in turn, has contributed to a lack of experience in managing these tumors. Moreover, with the number of reported patients usually varying from less than 15 to about 50 [6, 8, 9, 11, 12, 14–17, 19–24], and only a few studies reporting about a hundred patients [10, 13, 18, 25], there has been relatively little discussion of the prognostic factors for RPA [13–18].
Follow-up of parotidectomies is currently performed by ultrasonography, but most patients do not avail themselves of regular follow-up and only 12.1% of our patients with an RPA were diagnosed by routine ultrasonography, while the majority came to our observation for a swelling in the parotid region. In 77.4% of our patients, there was a multinodular lesion (in Stennert et al. [4], 90% were multinodular at pathologic examination), reinforcing the pathogenetic hypothesis of incomplete excision, violation of the pseudocapsule of the tumor, or obvious or underestimated tumor spillage during the first surgery due to the impossibility of obtaining healthy free margins when the lesion is adjacent to the facial nerve.
Delineation of the extension of the RPA is better depicted by MRI, which better demonstrates the multinodular nature of the disease [24] and potential deep-lobe or parapharyngeal extension. Many patients that we treated came from other institutions without clear documentation of their previous treatment(s); in such cases, MRI can also help in determining the amount of parotid gland still present after any previous surgery. The signal and enhancement characteristics of RPA of the parotid gland are nonspecific [26]. The lesions are usually round; they are of low intensity on T1-weighted images and of high intensity on T2-weighted MR images. With contrast administration, the lesions show mild enhancement [26]. However, MRI can also be inadequate for identifying all nodules, and frequently the surgeon’s microscope or the pathologist’s microscope reveals many more nodules than suspected from clinical assessment [4].
Management options for RPA include observation only, localized resection, resection with facial nerve dissection, wide-field resection with facial nerve sacrifice, and radiotherapy [2, 4, 24]. Only observation has been suggested for the elderly or medically infirm patient [2, 4, 24]. When surgical excision is considered, it should be tailored to the individual patient [19, 23]. In general, it is recommended to resect the scars of any previous surgery. Localized resection of the tumor has been employed after multiple recurrences or after previous total parotidectomy when it is the only option to preserve the facial nerve [10, 14]. Superficial or total parotidectomy, depending on the location of the recurrence, has been suggested when the previous operation was a simple local excision [6, 8–15, 17, 18, 20, 23–25]. Radical and extended parotidectomies have been considered for patients with infiltration of branches or the main trunk of the facial nerve, and in cases of multinodular recurrences [9, 11–13, 15, 18, 23–25]. In particular, facial nerve resection has been suggested for patients with a history of multiple recurrences or failed radiotherapy [24]. In 30.3% of our patients, the resection of at least a branch of the facial nerve was performed (Table 2). Facial nerve dissection and preservation were performed in all cases with still healthy parotid tissue between the nerve and the tumor, and in the case of facial nerve encasement when a patient refused facial nerve resection. The observation of many cases with unexpected multinodularity persuaded Stennert et al. [4] to routinely perform a total parotidectomy for all RPAs. However, such an aggressive policy of treatment does not prevent leaving microscopic residuals; indeed, a recent study by the same group reported a 52% probability of new recurrences [18].
The role of radiotherapy remains questionable [9, 13, 16, 17, 24, 27]. Some authors suggest that radiotherapy should be reserved only for malignant tumors since it carries, especially in young people, a risk of malignant transformation that increases over time [17]. Nonetheless, its use is not infrequent [6, 9, 13–16, 19, 20, 27]. Jackson et al. [9] used postoperative radiotherapy in patients in whom histology showed that resection margins of the specimen were not free of tumor or in those with intraoperative spillage of tumor. Renehan et al. [13] recommended postoperative radiotherapy in multinodular recurrences. Douglas et al. [16] reported their experience of fast neutron radiotherapy and recommended radiotherapy for selected cases: when complete extirpation is not possible, when sacrifice of the facial nerve is necessary, or after multiple recurrences. Glas et al. [17] suggested postoperative radiotherapy for difficult cases in which further surgical treatment of recurrent disease is not recommended for technical reasons. Kamida et al. [27] suggested the use of stereotactic radiosurgery for difficult selected cases, such as the patient they treated for skull base invasion with no regrowth after 5 years. Our experience, including only nine patients, is too limited to draw any conclusion about indications for radiation therapy.
Complications of treatment of RPA are similar to those for primary surgery for parotid pleomorphic adenoma. The main difference is the higher risk of facial nerve damage in the former, with unexpected permanent facial nerve paralysis in more than 30% of cases [24].
Even though we did not have cases of malignant transformation, a high incidence is expected, especially after multiple recurrences, reaching up to 15% in the experience of Mercante et al. [22].
The main purpose of our study was to analyze risk factors for new recurrence after treatment for RPA. The percentage of new recurrences varies from 10 to 58% [8, 10, 12–15, 17–20, 23–25]. In two surveys of the literature with a 10-year follow-up, the prevalence of a new recurrence was 43 and 45%, respectively [12, 25]. Wittekindt et al. [18] reported a recurrence rate increasing from 42% at 5 years to 75% at 15 years. We observed a prevalence of 33.3% with an estimation of recurrence rate increasing from 14.1 ± 6.6% at 5 years to 31.4 ± 9.4% at 10 years, 43.0 ± 10.8% at 15 years, and 57.2 ± 14.8% at 20 years (Fig. 1).
Among the variables tested with the log-rank test against the estimated new recurrence rate (Table 5), just two were significantly associated with the likelihood of developing a new recurrence: local excision of the RPA instead of a formal parotidectomy and the presence of multinodular disease. To date, no new recurrences have been observed for uninodular recurrences. There was a trend toward increased risk of a new recurrence for deep-lobe localization, although it was not statistically significant.
Few papers have analyzed prognostic factors for RPA [13–18]. Renehan et al. [13] studied 114 patients with first-recurrence RPA and reported significantly better, new recurrence-free survival for patients treated with postoperative radiotherapy when the RPA was multinodular. Age, gender, time to first recurrence, and the type of prior treatment were all not significant predisposing factors for a new recurrence [13]. Yugueros et al. [14] followed 39 patients treated for RPA at their institution: they found no significant difference in development of another recurrence in relation to gender, time after the previous treatment, size of the tumor, type of resection, and postoperative radiotherapy. Carew et al. [15] analyzed 31 patients treated for RPA and concluded that the extent of the first parotid operation was the only factor with a statistically significant impact on tumor control after resection of the recurrence. Local control was achieved in all patients whose initial procedures involved local excision. Superficial lobe location and postoperative radiotherapy showed better results but were not significant [15]. Fast neutron radiotherapy of 16 patients with residuals of RPA stopped progression of the tumor in patients without gross residuals, without multifocal disease and with an interval from diagnosis of less than 25 years; however, the results were not statistically significant [16]. Glas et al. [17] analyzed 52 patients with RPA, treated after one or more recurrences: no significant influence was seen in the recurrence rate with respect to gender, age at initial treatment, time of first recurrence, and intraoperative tumor spillage. Wittekindt et al. [18], in a group of 108 patients, observed a significantly higher rate of new recurrences in female patients, in younger patients, and in patients treated with a simple enucleation.
Conclusion
The management of RPA is a major challenge for the clinician. RPAs, particularly multinodular tumors, are prone to new recurrences especially when treatment of the initial tumor is performed according to currently accepted standards. MRI is considered to be the best tool to delineate the extension of the lesion, although it can miss microscopic nodules. Options for management include pure observation, not only for the elderly or infirm, but also in cases of small lesions until they begin to grow. When surgery is elected, it should be tailored to the single patient, because even if on one side a limited local excision is considered acceptable, on the other side a total or extended parotidectomy may be inadequate to control an RPA adjacent to the nerve. In these cases, facial nerve resection and reconstruction must also be considered and discussed with the patient in the preoperative counseling. Postoperative radiotherapy is an option, particularly for older patients for whom the risk of inducing other malignancies is considered to be low. Follow-up for patients treated for RPA should be done with regular ultrasonography for the lifetime of the patient and with magnetic resonance in selected cases with deep lobe or parapharyngeal involvement. All patients should be informed about the possibility of the need for multiple treatment procedures.

pleomorphic adenoma
tumor recurrence
parotid gland neoplasms


Methodological considerations concerning the development of oral dental erosion indexes: literature survey, validity and reliability

Within the context of preventing non-communicable diseases, the World Health Report (2002) and the WHO Global Oral Health Program (2003) put forward a new strategy of disease prevention and health promotion. Greater emphasis is placed on developing global policies in oral health promotion and oral disease prevention. The Decayed, Missing, Filled Teeth (DMFT) index does not meet new challenges in the field of oral health. Dental erosion seems to be a growing problem, and in some countries, an increase in erosion of teeth is associated with an increase in the consumption of beverages containing acids. Therefore, within a revision of the WHO Oral Health Surveys Basic Methods, new oral disease patterns, e.g. dental erosion, have to be taken into account. Within the last 20 years, many studies on dental erosion have been carried out and published. There has been a rapid growth in the number of indexes quantifying dental erosion process in different age groups. However, these indexes are not comparable. This article discusses quality criteria which an index intended for assessing tooth erosion should possess.
Introduction
Within the context of preventing non-communicable diseases, the World Health Report (2002) and the WHO Global Oral Health Program (2003) put forward a new strategy of disease prevention and health promotion [52]. The main orientation of the WHO Oral Health Program (2003) is that oral health is integral, essential and interrelated to general health and a determinant factor for quality of life. Proper oral health care reduce the burden of disease as well as premature mortality. Risk factors for oral diseases are common with cardiovascular diseases, diabetes, cancer and chronic obstructive pulmonary diseases.
The actual WHO Global Oral Health Program focuses on priority action areas as: “Diet, nutrition and oral health”, “Oral health and fluorides”, “Tobacco and oral health”, “School children”, “Elderly people” and “Oral health services”. Nutrition affects oral health in many ways, e.g. dental erosion. To minimize the occurrence of dental erosion which is particularly related to acidic beverages, the WHO/FAO (Food and Agricultural Organization of the UN) recommends (within their recently published Global Strategy on Diet, Physical Activity and Health) a reduction in the amount and frequency of intake of soft drinks and juices. WHO recommends that the Ministries of Health should ensure that the mechanisms for intersectorial collaboration are strengthened. Strategies include taxation and pricing, food labeling, school lunch policies and nutrition programs. This WHO Programme is elaborated for all countries of the World. The Regional Offices of WHO play an important role in the improvement of oral health within risk factor approach in disease prevention and health promotion. Therefore, the Global Oral Health Programme will help to achieve greater equity in oral health. But to evaluate the Global Oral Health Programme, we need suitable and validated indicators and indexes [52]. There is currently only one global oral health index to measure oral health. It is the Decayed, Missing, Filled Teeth (DMFT) index. According to the WHO Oral Health Data Bank in the year 2000 from 184 countries, 68% had a DMFT index less than 3 [52]. Up to now, there is only the DMFT index for 12-year-old children in the Health for all 21 Database (www.euro.who.int: HFA-21 DB, downloaded June 13, 2007).
The European Commission launched from 2003 to 2005 the EU-Project: European Global Oral Health Indicators coordinated by the Université Claude Bernard de Lyon, to support European Member States in their efforts to reduce the toll of morbidity, disability related to oral health diseases and especially [18]:
To identify indicators of oral health, of critical oral health care, its quality of care and of essential health resourcesTo strengthen the ability at the local, national and regional levels to measure, compare and determine the effects of oral health services and use of resourcesTo identify indicators of oral health (problems, determinants and risk factors related to lifestyle) of critical oral health careTo identify the types of data generation and management problems within the Health Information SystemTo identify principles for guiding the selection and use of oral health indicatorsTo identify a set of core indicators for oral healthTo review the recent oral health-indicator selection efforts
In total, 66 indicators are proposed, but no indicator for dental erosion is considered [18]. Therefore up to now, the only international reference in the area of oral health is the DMFT index. The European Commission assesses the quality of oral health data as inadequate for planning, implementation, management and evaluation. The number of internationally recommended indicators complicates the national selection of indicators and the comparability of indicators and indexes [18]. The statements of the European Global Oral Health Indicators Report coincide with the experience in the field of development and using of different erosion indexes. Therefore, it would be necessary to strengthen the international alliance of dentists, epidemiologists, statisticians and other scientists and politicians, to develop an adequate oral health indicators set, i.e. as a subset or user-window of the ECHI-Database (European Community Health Indicators-Database) and within the Oral Databank of the WHO and to develop an internationally agreed and accepted erosion index.
Societies around the world today are increasingly witnessing significant changes in diets and lifestyles which have an impact on oral health. Dental erosion seems to be a growing problem, and in some countries, an increase in erosion of teeth is associated with a higher consumption of beverages containing acids [52]. Within a revision of the WHO Oral Health Surveys Basic Methods, new oral disease patterns, e.g. dental erosion, have to be taken into account [52]. The WHO urges the development of methodologies and approaches for evaluating the effectiveness of community oral health programs focusing on health promotion and disease prevention. Additionally, the formulation of new WHO oral health goals up to the year 2020 have been initiated [52].
Most indexes use different clinical examination standards for measuring tooth erosion especially in preschool- and schoolchildren [25]. Such examination standards could be:
–The full mouth or partial recording–The examination of primary and/or of only permanent teeth–The examination of all surfaces or partial recording of surfaces
On this basis, a lot of indexes for the clinical diagnosis of erosive tooth wear have been proposed, which are more or less modifications of combinations of the index published by Eccles [17] or Smith and Knight [48]. The most cited examples of Erosion Indexes developed during the last 20 years are [adopted from 20]:
–The Smith and Knight Tooth Wear Index (TWI) (1984) [48]–The Eccle’s Index (1979) [17]–UK National Survey of Children’s Dental Health Index (1999/2003) [35]–Erosion Index according to Lussi (1996) [33]–Modified scoring system of Linkosalo and Markkanen (1985) [19]–Aine Index 1993 [1]–The Larsen and Westergaard Index (2000) [30]–The O’Sullivan Index (2000) [39]
Recently, the question has arisen how reliable and valid current diagnostic criteria and data on erosion are. The indexes developed and used during the last 20 years are not comparable; a gold standard does not exist, and validation studies have not had the effect of identifying an index that could be used as a standard for assessing tooth erosion. The following questions should be answered to advance the definition and assessment of tooth erosion:
–Which erosion indexes are mostly used in the scientific literature?–Which quality criteria should indexes possess?–What differences exist between individual- and population-based erosion indexes?
Finally in the discussion, we will debate whether it is possible to involve erosion indexes into Health Indicator Sets and into International Classification of Diseases (ICD; e.g. ECHI, HFA21, country databases on oral Health, ICD-11).
Erosion index in the scientific literature
A literature analysis with regard to erosion indexes for the period 2000–2006 was carried out. For this purpose, a Medline research was done considering mesh terms and keywords “tooth erosion” and “dental erosion” in connection with “index”. All human studies published in English found by this search strategy were analyzed for the erosion indexes used. In the literature analyses, a total of 1,380 articles were found with regard to the mesh terms and keywords “tooth erosion” or “dental erosion”. Approximately 326,112 articles were found with regard to the mesh term and keyword “index”. The conjunction of both revealed a total of 91 articles. From these articles, 40 were excluded as they were published before the year 2000, three were excluded because they were not English-language publications, eight studies were not included as no humans were involved, and one review was excluded. In total, 40 publications were considered in the literature review. Of 22 publications, 14 cross sectional studies were found in children, and 18 publications/cross sectional studies were found in adults. The erosion indexes used are presented in Tables 1 and 2 for children and adults, respectively. In children, the UK children dental health survey index [35] was used in four studies, the O’Sullivan Index [39] in three, an index developed in 1993 by Aine et al. [1] was used in two studies, and the Smith and Knight TWI [48] in one study. However, in most studies (n = 4), differently designed erosion indexes were considered. In adults, the Smith and Knight TWI [48] was used in most studies (n = 10) followed by the Eccle’s Index [17] (n = 2), Lussi Index [33] (n = 1), UK adult dental health survey index (n = 1) and an index developed by Schweizer-Hirt et al. 1978 [47]. In one study, the method for estimating the tooth erosion was not mentioned.
Table 1Used dental erosion indexes in human cross-sectional studies for children found in Medline from the years 2000–2006Aim of the studynStudy populationReferred indexSourceErosive tooth wear463Kindergarten, 3- to 5-year-old childrenO’Sullivan IndexWiegand et al. [54]Dental erosion15311-year-oldsO’Sullivan IndexCaglar et al. [10]Prevalence of dental erosion499School setting; 12-year-old childrenO’Sullivan IndexPeres et al. [43]Prevalence of dental erosion1,949Preschool children; 3- to 5-year-old childrenUK Children dental health survey indexLuo et al. [32]Prevalence of dental erosion8326- and 12-year-old school childrenOwn erosion indexTruin et al. [50]SES and ethnicity and oral health1,753Random sample of 12- and 14-year-old childrenUK Children dental health survey indexDugmore and Rock [16]Accuracy and reproducibility of school dental screening570Primary-school childrenNew index was developedHetherington and White [24]Prevalence of and risk factors for dental erosion95Children and adultsOwn erosion indexJohansson et al. [28]Oral health and gastro-oesophageal reflux52Children with gastro-oesophageal reflux diseaseAine IndexLinnett et al. [31]Asthma and dental erosion418Random Sample of 14-year-old childrenSmith and Knight TWIAl-Dlaigan et al. [2]Prevalence of dental erosionOral health and gastro-oesophageal reflux37Children with gastro-oesophageal reflux diseaseAine IndexDahshan et al. [15]Prevalence of dental erosions987Preschool children, 2- to 5-year-old childrenUK Children dental health survey indexAl-Malik et al. [4]Oral health of children with clefts914-, 8-, and 12-year-old childrenUK Children dental health survey indexChapple and Nunn [11]Dental erosion and consumption of oranges1,01012-year-old childrenOwn erosion indexKünzel et al. [29]Table 2Used dental erosion indexes in human cross-sectional studies for adults found in Medline from the years 2000–2006Aim of the studynStudy populationRefered indexSourceTooth survey (surface?) check! loss155Patients attending a dental hospitalUK Adult dental health survey indexRafeek et al. [44]Tooth wear among psychiatric patients143Psychiatric patientsSmith and Knight TWIAl-Hiyasat et al. [3]Tooth wear in elderly690Local survey, elderlyEccles IndexTaiwo et al. [49]Tooth surface in winemakers36WinemakersSchweizer-Hirt et al. 1978Chikte et al. [12]Workplace and dental erosion20Silicon workersOwn erosion indexJohansson et al. [27]Oral and dental health34Inpatients in treatment of alcohol disordersNo information givenAraujo et al. [6]Dental erosion and gastro-oesophageal reflux?Patients with gastro-oesophageal reflux diseaseSmith and Knight TWIMoazzez et al. [36]Prevalence of dental erosion18,555Permanent dentitionSmith and Knight TWIBorcic et al. [9]Dental erosion and gastro-oesophageal reflux253Patients with gastro-oesophageal reflux diseaseEccles IndexMunoz et al. [37]Monitoring of tooth wear500Patients referred for a variety of restorative procecduresSmith and Knight TWIBartlett [7]Risk factors of tooth wear506Patients attending a dental hospitalSmith and Knight TWIChuajedong et al. [13]Sport drinks and dental erosion304AthletesLussi IndexMathew et al. [34]Prevalence of tooth wear126Patients attending a dental hospitalSmith and Knight TWIOginni and Olusile [41]Recreational drug and tooth surface loss13Undergraduate studentsSmith and Knight TWINixon et al. [38]Risk factors for dental erosion10Male military Saudi inducteesOwn erosion indexJohansson et al. [28]Oral health status of workers68Workers exposed to acid fumesSmith and Knight TWIAmin et al. [5]Methamphetamine and tooth wear43Methamphetamine usersSmith and Knight TWIRichards and Brofeldt [45]Dental erosion and gastro-oesophageal reflux20Patients with gastro-oesophageal reflux diseaseSmith and Knight TWIGregory-Head et al. [21]
Quality criteria of indexes for measuring tooth erosion
Instruments or indexes for measuring a construct of interest—quality of life or intelligence, mental health status or tooth erosion—should possess certain quality characteristics. Otherwise, the scientific value of the obtained results is questionable [8]. Important quality characteristics are validity and reliability as well as sensitivity and specificity, which will be explained in the following paragraphs.
The validity of an instrument indicates to what extent it measures what it is supposed to measure. There are different—partly overlapping—types of validity which emphasize different aspects [42]. The most important types are content, construct, and criterion validity which will be explained in the following paragraphs.
Content validity describes whether all aspects, which are relevant to grasp the construct of interest, have been considered adequately. For example, a test that is supposed to measure the ability to calculate should not be restricted only to addition and subtraction but include all basic arithmetic operations. In our context, e. g. the erosion of surfaces of all teeth has to be considered, not only the erosion of incisors. The extent to which an instrument possesses content validity cannot be determined numerically. It is solely based upon the subjective meaning of established experts [8, 23]. Therefore, a major step in developing a new instrument is a thorough discussion of which aspects should be included.
The optimal method to validate a newly developed instrument is the comparison with a so-called gold standard, which measures the same construct. A gold standard is “the method, procedure or measurement that is widely accepted as being the best available” [46]. The so-called criterion validity is high if the results of the new instrument and the gold standard are highly correlated. For example, a thermometer might serve as a gold standard for self-reported temperature [23].
However, because a gold standard is seldom available, construct validity is of major importance [8]. Construct validity is subdivided into convergent and discriminant validity. There is convergent validity if the results obtained by the new instrument are correlated with the results of an established instruments that measures similar aspects. If, for example, results of a new instrument for measuring physical health are correlated with the results of an established instrument which measures activity of daily living, convergent validity is indicated [8]. Or in our context: if the results of a tooth-wear index is correlated with tooth-erosion index, convergent validity is indicated. On the other hand, an instrument possesses discriminant validity if the results of this instrument are not too highly correlated with the results of an established instrument that measures a different construct. For example, results of an instrument for measuring mental health should not be too highly correlated with an instrument for measuring physical function [23]. With regard to teeth, a high correlation between tooth erosion and wedge-shaped defects might indicate insufficient discriminant validity. Both convergent and discriminant validity have to be given for a complete confirmation of construct validity.
The reliability of an instrument indicates how precise it is able to measure, independent of whether it really measures what it is supposed to (this is a question of validity) [42]. There are different aspects of reliability. For our purposes, inter-examiner reliability and intra-examiner reliability are important.
Inter-examiner reliability can be determined if two or more dentists (the “examiner”) assess dental erosion of a number of patients independently of each other [14]. Roughly spoken, the more often the examiners agree in their assessment, the higher the inter-examiner reliability. Intra-examiner reliability or test–retest reliability can be determined if each dentist rates dental erosion of a group of patients twice [23]. The period of time between the two assessments should be fairly long, otherwise the examiners may be overly consistent because they remember their former ratings. On the other hand, if the time interval between the two assessments is too long, changes in the erosion status might bias the reliability estimates. The more often the assessments at the two points in time concerning each examiner are identical, the higher the intra-examiner reliability. A widespread index to calculate the degree of agreement is Cohen’s Kappa [14], which “is now fairly well disseminated as one of the standard summary statistics used in the medical literature” [22]. The calculation of Kappa is based on the percentage of agreement which is adjusted for agreement expected by chance.
The sensitivity of an instrument indicates its ability to detect—in our case—dental erosion. In contrast, an instrument with high specificity is able to indicate no dental erosion if dental erosion is not present. Both assessment of sensitivity and specificity require the comparison with a gold standard. Comparing a new instrument that is supposed to detect dental erosion with an instrument that is known to be able to detect dental erosion, i.e. a gold-standard instrument, can yield one of the four following outcomes: (1) dental erosion is indicated by the new instrument if there is in fact dental erosion (correct positive), (2) dental erosion is indicated if there is no dental erosion (false positive), (3) dental erosion is not indicated if there is dental erosion (false negative) and (4) dental erosion is not indicated if there is no dental erosion (correct negative). The degree of sensitivity is calculated by dividing the frequency of correct positive results by the sum of correct positive and false negative results, while the degree of specificity is determined by dividing the frequency of correct negative results by the sum of correct negative and false positive results [46].
Individual versus population-based erosion indexes
An individual-based index should fulfill other criteria than a population-based index. The first should allow assessment of the full truth of the construct “tooth erosion”. Therefore, a very good individual-based erosion index should nearly be a gold-standard instrument. Using a population-based erosion index, the individual assessment should take less time to allow an assessment in big population samples. Therefore, for practical reasons, a population-based erosion index is often a short form of an individual-based index. However, a good population-based erosion index should possess very high validity as well as high reliability.
Discussion
There is no common sense in the usage of the above-mentioned erosion indexes. A wide range of different forms and indexes was used in the last 6 years. Therefore, comparability between different studies was not given, and meta-analyses were not possible. If anything, it can be stated that for adults, the Smith and Knight TWI [48] has been widely used in most Medline-cited cross-sectional studies. For children, there is no index that is most used. However, the literature analysis is limited. Only one search strategy was used. Some articles which do not use the mentioned key words or mesh headings might not be included in the analysis. However, the results presented help to get a first view on the different indexes on tooth erosion cited in the last 6 years.
In all these indexes, the criteria to record the grading for erosion differed [26]. Most of the indexes used are based on the clinical severity of erosion, focusing on accessible teeth but not all [29]. For example, the structure of the Smith and Knight TWI is shown in Table 3. However, this index is made for scoring tooth wear in general irrespective of its predominant aetiology. It is only a system for quantifying and grading the amount of tissue loss but does not include diagnostic criteria for erosion. The UK National Survey of Children’s Dental Health Index was a modified version of the Smith and Knight TWI assessing only buccal or palatal surfaces of maxillary incisors and added erosion criteria. However, while using the first index, the conclusion was drawn that the figures might not be completely accurate [40]. The next version focused on erosion of the palatal surface of at least one maxillary anterior tooth with the majority of surfaces having dentin involvement [29]. To date, literature assessing the prevalence of erosion has been published from different countries. Erosion was recorded using various indexes. In addition, selection criteria of study populations, sampling techniques and considered age groups differed. The prevalence of dental erosion of different studies is therefore difficult and nearly impossible to compare [29].
Table 3Smith and Knight Tooth Wear Index [48]ScoreSurfaceCriterion0B/L/O/I/CNo loss of surface characteristic, no loss of contour1B/L/O/I/CLoss of enamel surface characteristics, minimal loss of contour2B/L/OLoss of enamel exposing dentine for less than one third of the surfaceILoss of enamel just exposing dentineCDefect less then 1 mm deep3B/L/OLoss of enamel exposing dentine for more than one third of surfaceILoss of enamel and substantial loss of dentine not exposing secondary dentine or pulpCDefect 1–2 mm deep4B/L/OComplete loss of enamel, or pulp exposure, or exposure of secondary dentineIPulp exposure or exposure of secondary dentineCDefect more than 2 mm deep, or pulp exposure, or exposure of secondary dentineB Buccal or labial, L lingual or palatal, O occlusal, I incisal, C cervical
A good index is characterized by a conducted validation study. For those indexes for which a validation study has not been conducted so far, that should be made up to check whether they possess the quality criteria as described above. In a validation study, it is not only the validity of an index that is examined but also aspects of reliability as well. Intra-examiner and inter-examiner reliability should be checked. That is done quite easily as described in “Quality criteria of indexes for measuring tooth erosion”. At the beginning of the study, it has to be considered how many patients and how many examiners respectively have to be involved. This is because the number of patients and examiners should be as small as possible for practical and economic reasons. On the other hand, the number of patients and examiners has to be sufficiently high because otherwise a certain degree of reliability, which actually exists, might be overlooked. Therefore, solid sample-size calculations should be conducted with the help of an experienced statistician.
So far, there is no consensus concerning a gold-standard instrument. However, a gold standard is important for comparison. Otherwise, criterion validity cannot be checked. If one attempts to develop a gold-standard instrument, the first step would be a thorough discussion of which aspects of the construct “tooth erosion” have to be included. This should be conducted by established experts to ensure content validity. When the instrument is constructed, inter-examiner reliability and intra-examiner reliability can be examined as described above. However, this should be done not only in a national but in an international context.
A gold-standard instrument would explain the full truth of the construct “tooth erosion”. Nevertheless, the measurement procedure in general takes a long time. It is not possible to deal with such a comprehensive assessment instrument in a population-based study design. Therefore, more simple assessment instruments should be available. These assessment instruments should fulfill the following qualifications: (a) a validation study should be available where all quality criteria of the assessment instrument were tested and (b) the instrument should be used in other scientific studies, too. If the population-based short version of an assessment instrument and the gold-standard instrument are measuring the same construct, and when both conditions are fulfilled, calibration studies might be possible. Calibration refers to a process in which values from one method are quantitatively related to values from a superior, gold-standard method. The aim of such an analysis is to ensure that the measurement uncertainty is known and is consistent with the required measurement capability. In a so-called calibration study, known data on the observed relationship between an independent variable of the population-based short version of the assessment instrument and the dependent variable of the gold-standard instrument are used to make estimates of other “true” values of the independent variable from new observations of the gold standard.
One of the prospective targets ought to be the inclusion of indexes on dental health prevention especially of an internationally agreed “erosion index” into the existing Health Indicator Sets. These would be the Health for All Database of the WHO (HFA21), The European Community Health Indicator Set (ECHI), Country Databases on Oral Health and the inclusion of Dental Erosion into the International Classification of Diseases and Health-Related Problems.
In April 2007, the World Health Organization announced plans to revise the ICD-10, in other words, to prepare for ICD-11. Within the actual ICD-10, there exists two codes for Dental Diseases: “K02” for Caries and “K03” for “Other diseases of hard tissues of teeth” including with “K03.2—Erosion of teeth.” On the four-digit level, we find the following different diagnoses presented in Table 4.
Table 4Code description for K03.2—Erosion of teeth (ICD 10 code) [51]ICD 10 codeDescriptionK03.2Erosion of teethK03.20Occupational erosion of teethK03.21Erosion of teeth due to persistent regurgitating or vomitingK03.22Erosion of teeth due to dietK03.23Erosion of teeth due to drugs and medicamentsK03.24Idiopathic erosion of teethK03.28Other specified erosion of teethK03.29Erosion of teeth, unspecified
The Updating and Revision Committee proposes major changes like addition of new codes or deletion of codes and developed submission guidelines. Therefore, it would be possible to check critically if the codes for dentistry within the Application of the International Classification of Diseases to Dentistry and Stomatology are adequate to future developments or have to be replaced or completed [53].
Conclusion
Further efforts have to be made in the development of an internationally agreed index which is able to assess dental erosion with as much reliability and validity as possible. Current recommendations of the WHO and the European Union and health task forces within countries and Dental Associations should be used to develop and to discuss the concept of tooth erosion and the development of a unified erosion index.

index
validity
reliability
tooth erosion
gold standard