Background The rate of total edentulism is said to be increasing in developing countries and this had been attributed mainly to the high prevalence of periodontal diseases and caries. Several reports have shown that non-disease factors such as attitude, behavior, dental attendance, characteristics of health care systems and socio-demographic factors play important roles in the aetiopathogenesis of edentulism. The aim of this study was to assess the relationship between socio-demographic factors and edentulism.
Background
The rate of total edentulism is said to be increasing in developing countries and this had been attributed mainly to the high prevalence of periodontal diseases and caries. Several reports have shown that non-disease factors such as attitude, behavior, dental attendance, characteristics of health care systems and socio-demographic factors play important roles in the aetiopathogenesis of edentulism. The aim of this study was to assess the relationship between socio-demographic factors and edentulism.Methods A total of 152 patients made up of 80 (52.6%) males and 72 (47.4%) females who presented in two prosthetic clinics located in an urban and a rural area were included in the study. The relationship between gender, age, socio-economic status and edentulism in this study population was established.
Methods
A total of 152 patients made up of 80 (52.6%) males and 72 (47.4%) females who presented in two prosthetic clinics located in an urban and a rural area were included in the study. The relationship between gender, age, socio-economic status and edentulism in this study population was established.Results No significant relationship between gender and denture demand was noted in the study. The demand for complete dentures increased with age while the demand for removable partial dentures also increased with age until the 3 rd  decade and then started to decline. A significant relationship was found between denture demand and the level of education with a higher demand in lower educational groups (p < 0.001). In addition, the lower socio-economic group had a higher demand more for prostheses than the higher group.
Results
No significant relationship between gender and denture demand was noted in the study. The demand for complete dentures increased with age while the demand for removable partial dentures also increased with age until the 3 rd  decade and then started to decline. A significant relationship was found between denture demand and the level of education with a higher demand in lower educational groups (p < 0.001). In addition, the lower socio-economic group had a higher demand more for prostheses than the higher group.Conclusions The findings in this study revealed a significant relationship between socio-demographic variables and edentulism with age, educational level and socio-economic status playing vital roles in edentulism and denture demand.
Conclusions
The findings in this study revealed a significant relationship between socio-demographic variables and edentulism with age, educational level and socio-economic status playing vital roles in edentulism and denture demand.

Background Edentulism (partial or total) is an indicator of the oral health of a population [ 1 ]. It may also be a reflection of the success or otherwise of various preventive and treatment modalities put in place by the health care delivery system [ 2 ]. Many patients also regard edentulism as self-mutilating and may be a strong incentive to seek dental treatment [ 3 ]. While the rate of total edentulism is decreasing in developed countries, the reverse is the case with developing countries and this had been attributed mainly to the high prevalence of periodontal diseases and caries [ 5 - 7 ]. Previous studies have also shown that several non-disease factors such as attitude, behavior, dental attendance, characteristics of health care system and socio-demographic factors play important roles in the aetiopathogenesis of edentulism [ 3 ]. Some studies reported that the incidence of edentulism correlated with educational levels and income status, with those in the lower levels exhibiting higher risks of becoming totally edentulous [ 8 , 9 ]. In addition, a study done in a rural area of Eastern Guatemala showed that social and environmental influence such as poverty, lack of proper education and inadequate diet contributed to widespread premature and heavy losses of permanent teeth [ 10 ]. Although, Hoover and McDermount [ 11 ] reported a higher prevalence of edentulism in males than females, Marcus et al observed that the prevalence of edentulism had no relationship with gender [ 12 ]. They also observed that there was an inverse relationship between the level of education, income and edentulism. Studies among Nigerians have linked some of these socio-demographic factors with the prevalence, pattern and rate of dental diseases [ 13 , 14 ] but there has been no report on the influence of these on edentulism. The aim of this study therefore was to assess the relationship between socio-demographic variables with types of edentulism.
Background
Edentulism (partial or total) is an indicator of the oral health of a population [ 1 ]. It may also be a reflection of the success or otherwise of various preventive and treatment modalities put in place by the health care delivery system [ 2 ]. Many patients also regard edentulism as self-mutilating and may be a strong incentive to seek dental treatment [ 3 ].While the rate of total edentulism is decreasing in developed countries, the reverse is the case with developing countries and this had been attributed mainly to the high prevalence of periodontal diseases and caries [ 5 - 7 ].Previous studies have also shown that several non-disease factors such as attitude, behavior, dental attendance, characteristics of health care system and socio-demographic factors play important roles in the aetiopathogenesis of edentulism [ 3 ].Some studies reported that the incidence of edentulism correlated with educational levels and income status, with those in the lower levels exhibiting higher risks of becoming totally edentulous [ 8 , 9 ]. In addition, a study done in a rural area of Eastern Guatemala showed that social and environmental influence such as poverty, lack of proper education and inadequate diet contributed to widespread premature and heavy losses of permanent teeth [ 10 ].Although, Hoover and McDermount [ 11 ] reported a higher prevalence of edentulism in males than females, Marcus et al observed that the prevalence of edentulism had no relationship with gender [ 12 ]. They also observed that there was an inverse relationship between the level of education, income and edentulism.Studies among Nigerians have linked some of these socio-demographic factors with the prevalence, pattern and rate of dental diseases [ 13 , 14 ] but there has been no report on the influence of these on edentulism.The aim of this study therefore was to assess the relationship between socio-demographic variables with types of edentulism.Methods All patients that attended and were treated in the removable prosthetic units of Obafemi Awolowo University Teaching Hospitals Complex (OAUTHC), Ile-Ife (a rural area located in the south west of Nigeria) between the months of March and May year 2002 and Lagos University Teaching Hospitals (LUTH), Lagos (an urban area also located in south west Nigeria) between December 2002 and March 2003 were included in the study. Information such as age, gender, occupation and level of education attained were documented. The types of partial denture received following treatment at the clinics were also documented There has not been a consensus on various socio-economic classifications in Nigeria because of the unstructured nature of the society. Therefore, for the purpose of this study, a standard occupational classification system designed by Office of population Census and Surveys, London (OPCS 1991) [ 15 ] modified based on local reality was used and patients were classified into three socio-economic groups: Class 1 = Skilled worker e.g professionals and managerial officers and retirees of this cadre. Class 2 = Unskilled workers e.g. Artisans and traders Class 3 = Dependants. e.g. Retirees of class 2, those not on pensions, house wives of class 2 cadre, students whose parents are unskilled workers Data was analysed using SPSS for Windows version 10.0, (SPSS Inc Chicago Illinois, USA). Analysis included frequency, cross tabulations, calculation of means. Association between discrete variables was tested by Chi-Square and the level of significance was set at 5%.
Methods
All patients that attended and were treated in the removable prosthetic units of Obafemi Awolowo University Teaching Hospitals Complex (OAUTHC), Ile-Ife (a rural area located in the south west of Nigeria) between the months of March and May year 2002 and Lagos University Teaching Hospitals (LUTH), Lagos (an urban area also located in south west Nigeria) between December 2002 and March 2003 were included in the study. Information such as age, gender, occupation and level of education attained were documented. The types of partial denture received following treatment at the clinics were also documentedThere has not been a consensus on various socio-economic classifications in Nigeria because of the unstructured nature of the society. Therefore, for the purpose of this study, a standard occupational classification system designed by Office of population Census and Surveys, London (OPCS 1991) [ 15 ] modified based on local reality was used and patients were classified into three socio-economic groups:Class 1 = Skilled worker e.g professionals and managerial officers and retirees of this cadre.Class 2 = Unskilled workers e.g. Artisans and tradersClass 3 = Dependants. e.g. Retirees of class 2, those not on pensions, house wives of class 2 cadre, students whose parents are unskilled workersData was analysed using SPSS for Windows version 10.0, (SPSS Inc Chicago Illinois, USA). Analysis included frequency, cross tabulations, calculation of means. Association between discrete variables was tested by Chi-Square and the level of significance was set at 5%.Results One hundred and fifty two patients attended the prosthetic clinics during the study period. Eighty (52.6%) were males while 72 (47.4%) were females (Table  1 ). Their ages ranged from 8 to 84 years. The median age was 22.00 years, while the mean age was 41.8 (±19.5) years. The mean age for Ile-Ife study population was 41.3 (±20.46) years, while that of Lagos was 39.9 (±17.56) years. Table 1 Distribution by gender. Gender Ile-Ife Lagos Total No % No % No % Male 48 48.0 32 61.5 80 52.6 Female 52 52.0 20 38.5 72 47.4 Total 100 100.0 52 100.0 152 100.0 χ 2  = 2.515, df = 1, P = 0.113. There were no statistically significant age (p = 0.312) and gender (p = 0.113) differences between the populations from the two centers. (Tables  1  and  2 ). Table 2 Denture demand by age and center/clinic Age group Ile-Ife Lagos Total N % N % N % ≤20 18 18.0 5 9.6 23 15.13 21–40 35 35.0 22 42.3 57 37.5 41–60 25 25.0 17 32.7 42 27.63 ≥61 22 22.0 8 15.4 30 19.74 Total 100 100.0 52 100.0 152 100.00 χ 2  = 3.568, df = 3, P = 0.312 There was a highly significant difference in the educational status of patients seen at LUTH and OAUTHC with patients seen at LUTH being of higher educational levels than patients seen at OAUTHC. (χ 2  = 7.50 df = 3, P < 0.001) (Figure  1 ). Figure 1 Distribution of patients according to educational level. In terms of socioeconomic status, 28 (18.42%) patients belonged to class I; 43(28.29%) patients belonged to class II while 81(53.29%) belonged to class III. There was no statistically significant difference in the socio-economic status of patients from the two centers ((χ 2  = 5.70, df = 2, p = 0.057). (Figure  2 ) Figure 2 Socio-economic status distribution. In both centers, 134 patients (88.2%) received removable partial dentures, 13 patients (8.6%) received complete dentures while 5 patients (3.3%) received either upper or lower complete dentures. There was no significant difference in the demand for different types of dentures between the study locations. (P = 0.315). (Table  3 ). Table 3 Demand for various types of denture by study location. Types of dentures Ile-Ife Lagos Total N % N % N % Complete 11 11.0 2 3.8 13 8.6 Lower or upper complete denture 3 3.0 2 3.8 5 3.3 Removable partial denture 86 86.0 48 92.3 134 88.2 Total 100 100.0 52 100.0 152 100.0 χ 2  = 3.568, df = 3, P = 0.312 For the purpose of analysis, complete and lower/upper complete denture columns were merged. However, there was a significantly higher demand for removable partial dentures than any other type of prostheses. (P < 0.01). (Table  3 ) It was observed that as the age increased, the proportions demanding for complete dentures also increased. In addition, those in age group 21–40 years demanded more for removable partial denture than any other age groups. While those above 61 years asked more for removable complete dentures than removable partial dentures. (Table  4 ). Table 4 Types of denture demand within each age group. Age group Denture demanded TOTAL Complete Partial no % no % no ≤20 1 4.3 22 95.7 23 21–40 2 3.5 55 96.5 57 41–60 5 11.9 37 88.1 42 ≥61 10 33.3 20 66.7 30 Total 18 11.8 134 88.2 152 Likelihood-ratio χ 2  = 16.579, df = 3, P = 0.001 No significant relationship between gender and pattern of denture demand (p = 0.812) was noted and no statistically significant difference was noted in the pattern of denture demand between the two centers (p = 0.277). (Table  5  and Figure  3 ). Table 5 Demand for various prostheses in relation to gender. Type of Denture male Female Total Complete denture 6 7 13 Complete upper or Lower denture 3 2 5 Partial denture 71 63 134 Total 80 72 152 χ 2  = 0.57, df = 1, p = 0.812 For the purpose of analysis, complete and lower/upper complete denture columns were merged. Figure 3 Types of prostheses demanded by centers The lower educational groups demanded more for complete dentures among those asking for complete denture, while those with higher level of education asked more for removable partial denture. (P < 0.001). (Table  6 ). More over, those with tertiary level of education constituted the majority of the study population. (Table  6 ) Table 6 Demand for dentures according to educational level Educational Level Complete Denture Partial Denture Total No % No % No % Nil 11 61.1 12 8.9 23 15.1 Primary 3 16.7 8 5.9 11 7.2 Secondary 0 0.0 51 38.1 51 33.6 Tertiary 4 22.2 63 47.0 67 44.1 TOTAL 18 100.0 134 100.0 152 100.0 Fishers exact test P < 0.001 For the purpose of analysis, secondary and tertiary educational levels' rows were merged. Also complete denture and either upper or lower complete denture column were merged Among the patients that were completely edentulous, there was no significant difference in the demand for complete dentures between those with lower educational status and those with higher educational status. P = 0.276 (Table  7 ) Table 7 Relationship between age group, educational level and completely edentulous state Age group Educational Level Nil Primary Secondary/Tertiary ≤20 1(9.1%) - - 21–40 2(18.2%) - - 41–60 4(33.4%) 1(33.3%) >60 4(33.4%) 2(66.7%) 4(100%) Likelihood-ratio χ 2  = 7.515, df = 6 P = 0.276 It was noted that the lower the socio-economic status the higher the demand for dentures. This picture was independent of rural or urban dwelling. (Tables  8  and  9 ). However, 28.3 % of those in Class II who need dentures asked for complete as opposed to 3.6% in Class I and 8.6% of those in Class III. Table 8 Relationship between edentulous state and socio-economic status. Socio-economic status Edentulous state Total Partial complete No % No % No % Class I 27 96.4 1 3.6 28 18.4 Class II 33 76.7 10 23.3 43 28.3 Class III 74 91.4 7 8.6 81 53.3 Total 134 88.2 18 11.8 152 100 χ 2  = 7.992, df = 2, P = 0.018 Table 9 Socio-economic status distribution by centers Socio-economic Class Lagos center Ife center TOTAL NO % NO % No % Class I 8 15.4 20 20.0 28 18.4 Class II 21 40.4 22 22.0 43 28.3 Class III 23 44.2 58 58.0 81 53.3 TOTAL 52 100.0 100 100.0 152 100.0 χ 2  = 5.70, df = 2, p = 0.057
Results
One hundred and fifty two patients attended the prosthetic clinics during the study period. Eighty (52.6%) were males while 72 (47.4%) were females (Table  1 ). Their ages ranged from 8 to 84 years. The median age was 22.00 years, while the mean age was 41.8 (±19.5) years. The mean age for Ile-Ife study population was 41.3 (±20.46) years, while that of Lagos was 39.9 (±17.56) years.Distribution by gender.χ 2  = 2.515, df = 1, P = 0.113.There were no statistically significant age (p = 0.312) and gender (p = 0.113) differences between the populations from the two centers. (Tables  1  and  2 ).Denture demand by age and center/clinicAge groupIle-IfeLagosTotalTotal100100.052100.0152100.00χ 2  = 3.568, df = 3, P = 0.312There was a highly significant difference in the educational status of patients seen at LUTH and OAUTHC with patients seen at LUTH being of higher educational levels than patients seen at OAUTHC. (χ 2  = 7.50 df = 3, P < 0.001) (Figure  1 ).In terms of socioeconomic status, 28 (18.42%) patients belonged to class I; 43(28.29%) patients belonged to class II while 81(53.29%) belonged to class III. There was no statistically significant difference in the socio-economic status of patients from the two centers ((χ 2  = 5.70, df = 2, p = 0.057). (Figure  2 )In both centers, 134 patients (88.2%) received removable partial dentures, 13 patients (8.6%) received complete dentures while 5 patients (3.3%) received either upper or lower complete dentures. There was no significant difference in the demand for different types of dentures between the study locations. (P = 0.315). (Table  3 ).Demand for various types of denture by study location.Types of denturesIle-IfeLagosTotalTotal100100.052100.0152100.0χ 2  = 3.568, df = 3, P = 0.312For the purpose of analysis, complete and lower/upper complete denture columns were merged.However, there was a significantly higher demand for removable partial dentures than any other type of prostheses. (P < 0.01). (Table  3 )It was observed that as the age increased, the proportions demanding for complete dentures also increased. In addition, those in age group 21–40 years demanded more for removable partial denture than any other age groups. While those above 61 years asked more for removable complete dentures than removable partial dentures. (Table  4 ).Types of denture demand within each age group.Age groupDenture demandedTOTALCompletePartialno%no%noTotal1811.813488.2152Likelihood-ratio χ 2  = 16.579, df = 3, P = 0.001No significant relationship between gender and pattern of denture demand (p = 0.812) was noted and no statistically significant difference was noted in the pattern of denture demand between the two centers (p = 0.277). (Table  5  and Figure  3 ).Demand for various prostheses in relation to gender.Type of DenturemaleFemaleTotalTotal8072152χ 2  = 0.57, df = 1, p = 0.812For the purpose of analysis, complete and lower/upper complete denture columns were merged.The lower educational groups demanded more for complete dentures among those asking for complete denture, while those with higher level of education asked more for removable partial denture. (P < 0.001). (Table  6 ). More over, those with tertiary level of education constituted the majority of the study population. (Table  6 )Demand for dentures according to educational levelEducational LevelComplete DenturePartial DentureTotalNo%No%No%NilPrimarySecondaryTertiaryTOTAL18100.0134100.0152100.0Fishers exact test P < 0.001For the purpose of analysis, secondary and tertiary educational levels' rows were merged. Also complete denture and either upper or lower complete denture column were mergedAmong the patients that were completely edentulous, there was no significant difference in the demand for complete dentures between those with lower educational status and those with higher educational status. P = 0.276 (Table  7 )Relationship between age group, educational level and completely edentulous stateAge groupEducational LevelNilPrimarySecondary/TertiaryLikelihood-ratio χ 2  = 7.515, df = 6 P = 0.276It was noted that the lower the socio-economic status the higher the demand for dentures. This picture was independent of rural or urban dwelling. (Tables  8  and  9 ). However, 28.3 % of those in Class II who need dentures asked for complete as opposed to 3.6% in Class I and 8.6% of those in Class III.Relationship between edentulous state and socio-economic status.χ 2  = 7.992, df = 2, P = 0.018Socio-economic status distribution by centersSocio-economic ClassLagos centerIfe centerTOTALNO%NO%No%TOTALχ 2  = 5.70, df = 2, p = 0.057Discussion Tooth loss could occur as a result of caries, periodontal diseases, trauma, tooth impaction, orthodontic reasons, hypoplasia, over eruption, supernumerary teeth, attrition, neoplastic and cystic lesions [ 5 - 7 ]. Many studies have consistently shown the role of specific diseases like dental caries and periodontal disease as a major cause of tooth loss [ 7 , 13 , 14 ]. This same picture was noted in similar Nigerian studies [ 5 , 6 ]. Okoisor further established that the disease factors responsible for tooth loss was age related; with caries and periodontal diseases being the major causes of tooth mortality in children and adult respectively [ 5 ]. However, none of the studies done in Nigeria evaluated the role of other factors such as education, socio-economic status, gender, location of patients, dental attitude and behavior in the etiology of edentulism. The older age groups in this study required more of removable complete dentures than the younger age groups while the younger age groups required more of removable partial dentures. This is in agreement with the study done by Marcus et al [ 12 ]. Although there was an over representation of age groups >61 and 21–40 in our study population, the percentages of these age groups in Nigerian population are 4% and 30% respectively in both urban and rural areas [ 16 ]. Hence, these age groups have risk factors that might be responsible for their needing dentures. This age related changes may not be unconnected with the deteriorative physiological changes noticed after adolescence and which gets worse with increase in age, a situation which is changing rapidly in the developed countries due to improved social infrastructure and functional health system [ 17 , 18 ]. Most studies have also shown significant gender difference in edentulism with more males becoming edentulous than females [ 11 , 19 ]. This has been attributed to the fact that males are more active than females and do not pay much attention to oral care. A significant gender difference was not seen in this study although variation in site presentation was observed. In Lagos, an urban area, more males actually demanded for prostheses. However, in Ile-Ife, a rural area more females demanded for prostheses. This is in agreement with the studies done by Eklund and Burt [ 8 ] and Marcus et al [ 12 ]. Although no statistically significant difference was noted in the rural-urban gender presentation, a larger qualitative study alongside a quantitative study may be able to adduce possible reasons for this interesting observation. Majority of our study population belonged to the higher education status. This is because those with higher level of education are more informed about their health needs and may seek dental treatments earlier and more often than those of lower educational status who may only seek dental treatment when there is apparent morbidity. In addition, those of higher educational status are likely to be richer than those of lower educational status. Hence, they are able to afford the cost of dental treatments from time to time. Our study showed that the need for complete dentures decreased with increasing level of education (p < 0.001), hence the likelihood of retaining teeth in the mouth becomes higher as the educational level increases. Although the educational status of the patients from the two centers differ, independent analysis of these centers still showed the same significant effect of educational status on the pattern of denture demand. This is in agreement with the findings of Brodeur et al, where the proportion of completely edentulous adults decreased from 26% in 1980 to 20 % in 1993 due to improved income and educational status [ 1 ]. The association between edentulism and educational status may be as a result of improved dental health awareness, increased utilization of oral health facilities, proper oral hygiene habits acquired during learning process and peer group influence. Interestingly, about 23.3 % of those in class II who need dentures asked for complete dentures as opposed to 3.6% in class I and 8.6% in class III. The reason for this may be as a result of the fact that they may not be able to afford the exorbitant cost of restorative procedures hence they wait until they have lost their set of teeth to have a complete removable denture which is cheaper. The present study showed that people with low socio-economic status demanded for more dentures than the high socio-economic group. Studies have long established a gradient relationship between socio-economic status and health [ 20 ]. In so many intricate ways, socio-economic status tends to affect health behaviors, the environment and social influences an individual is exposed to. Hunter and Arbona found that environmental influences such as land hunger, family poverty, and inadequate diet are of paramount importance in the cause of tooth loss [ 10 ]. They concluded, "Periodontal disease drives the poorest of the poor to spend disproportionately large sums on pain killers and destructive traditional medicine". The importance of socio-economic status is further reflected in the urban-rural variance noted in this study population's demand for denture. The Ile-Ife study group, a rural population with a lower socio-economic status, demanded for more dentures than the Lagos study group. However, this study population was an all-inclusive hospital based sample; the result may not be representative of the population at large. Hence, its use can only be limited to the study population. A randomized population based survey may be able to present a better picture among Nigerians. This study observed that edentulism is due to a combination of various factors. Poor education, a risk factor for poverty, has been identified as a major factor in edentulism. So also is the socio-economic status of the patient. These two factors, which are non-disease factors, affect the mortality of teeth arising from disease factors. There is therefore a need for oral health policy formulators to focus on improving the educational and socio-economic status of its citizens (a down stream approach) rather than the present emphasis on disease control (an up stream approach) in oral health care delivery. On the other hand, with increasing level of literacy, and positive social changes in Nigeria, Prosthodontists should brace up to face the challenges that may arise from increased removable partial denture demand and decreased demand for complete dentures. This is because, with increase in level of education and socio-economic status of patients, the demand for removable partial dentures is likely to increase while dentists may be confronted with a significant increase in the number of difficult edentulous mouths requiring treatment. In addition to addressing the non-disease factors, dental education should be targeted at the uneducated populace, the rural dwellers and low-income groups to reduce the rate of total edentulism.
Discussion
Tooth loss could occur as a result of caries, periodontal diseases, trauma, tooth impaction, orthodontic reasons, hypoplasia, over eruption, supernumerary teeth, attrition, neoplastic and cystic lesions [ 5 - 7 ].Many studies have consistently shown the role of specific diseases like dental caries and periodontal disease as a major cause of tooth loss [ 7 , 13 , 14 ]. This same picture was noted in similar Nigerian studies [ 5 , 6 ].Okoisor further established that the disease factors responsible for tooth loss was age related; with caries and periodontal diseases being the major causes of tooth mortality in children and adult respectively [ 5 ].However, none of the studies done in Nigeria evaluated the role of other factors such as education, socio-economic status, gender, location of patients, dental attitude and behavior in the etiology of edentulism.The older age groups in this study required more of removable complete dentures than the younger age groups while the younger age groups required more of removable partial dentures. This is in agreement with the study done by Marcus et al [ 12 ]. Although there was an over representation of age groups >61 and 21–40 in our study population, the percentages of these age groups in Nigerian population are 4% and 30% respectively in both urban and rural areas [ 16 ]. Hence, these age groups have risk factors that might be responsible for their needing dentures.This age related changes may not be unconnected with the deteriorative physiological changes noticed after adolescence and which gets worse with increase in age, a situation which is changing rapidly in the developed countries due to improved social infrastructure and functional health system [ 17 , 18 ].Most studies have also shown significant gender difference in edentulism with more males becoming edentulous than females [ 11 , 19 ]. This has been attributed to the fact that males are more active than females and do not pay much attention to oral care. A significant gender difference was not seen in this study although variation in site presentation was observed. In Lagos, an urban area, more males actually demanded for prostheses. However, in Ile-Ife, a rural area more females demanded for prostheses. This is in agreement with the studies done by Eklund and Burt [ 8 ] and Marcus et al [ 12 ]. Although no statistically significant difference was noted in the rural-urban gender presentation, a larger qualitative study alongside a quantitative study may be able to adduce possible reasons for this interesting observation.Majority of our study population belonged to the higher education status. This is because those with higher level of education are more informed about their health needs and may seek dental treatments earlier and more often than those of lower educational status who may only seek dental treatment when there is apparent morbidity. In addition, those of higher educational status are likely to be richer than those of lower educational status. Hence, they are able to afford the cost of dental treatments from time to time.Our study showed that the need for complete dentures decreased with increasing level of education (p < 0.001), hence the likelihood of retaining teeth in the mouth becomes higher as the educational level increases. Although the educational status of the patients from the two centers differ, independent analysis of these centers still showed the same significant effect of educational status on the pattern of denture demand. This is in agreement with the findings of Brodeur et al, where the proportion of completely edentulous adults decreased from 26% in 1980 to 20 % in 1993 due to improved income and educational status [ 1 ].The association between edentulism and educational status may be as a result of improved dental health awareness, increased utilization of oral health facilities, proper oral hygiene habits acquired during learning process and peer group influence.Interestingly, about 23.3 % of those in class II who need dentures asked for complete dentures as opposed to 3.6% in class I and 8.6% in class III. The reason for this may be as a result of the fact that they may not be able to afford the exorbitant cost of restorative procedures hence they wait until they have lost their set of teeth to have a complete removable denture which is cheaper.The present study showed that people with low socio-economic status demanded for more dentures than the high socio-economic group. Studies have long established a gradient relationship between socio-economic status and health [ 20 ]. In so many intricate ways, socio-economic status tends to affect health behaviors, the environment and social influences an individual is exposed to. Hunter and Arbona found that environmental influences such as land hunger, family poverty, and inadequate diet are of paramount importance in the cause of tooth loss [ 10 ]. They concluded, "Periodontal disease drives the poorest of the poor to spend disproportionately large sums on pain killers and destructive traditional medicine".The importance of socio-economic status is further reflected in the urban-rural variance noted in this study population's demand for denture. The Ile-Ife study group, a rural population with a lower socio-economic status, demanded for more dentures than the Lagos study group.However, this study population was an all-inclusive hospital based sample; the result may not be representative of the population at large. Hence, its use can only be limited to the study population. A randomized population based survey may be able to present a better picture among Nigerians.This study observed that edentulism is due to a combination of various factors. Poor education, a risk factor for poverty, has been identified as a major factor in edentulism. So also is the socio-economic status of the patient. These two factors, which are non-disease factors, affect the mortality of teeth arising from disease factors. There is therefore a need for oral health policy formulators to focus on improving the educational and socio-economic status of its citizens (a down stream approach) rather than the present emphasis on disease control (an up stream approach) in oral health care delivery.On the other hand, with increasing level of literacy, and positive social changes in Nigeria, Prosthodontists should brace up to face the challenges that may arise from increased removable partial denture demand and decreased demand for complete dentures. This is because, with increase in level of education and socio-economic status of patients, the demand for removable partial dentures is likely to increase while dentists may be confronted with a significant increase in the number of difficult edentulous mouths requiring treatment.In addition to addressing the non-disease factors, dental education should be targeted at the uneducated populace, the rural dwellers and low-income groups to reduce the rate of total edentulism.Conclusion No gender relationship with denture demand was noted this study. In addition, the demand for complete dentures increased with age. There was a statistically significant inverse relationship between educational levels and demand for dentures. There was more demand for prostheses among the lower socio economic groups.
Conclusion
No gender relationship with denture demand was noted this study. In addition, the demand for complete dentures increased with age. There was a statistically significant inverse relationship between educational levels and demand for dentures. There was more demand for prostheses among the lower socio economic groups.Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here:


Background To contribute further to the classification of three CFTR amino acid changes (p.I148T, p.R74W and p.D1270N) either as CF or CBAVD-causing mutations or as neutral variations.
Background
To contribute further to the classification of three CFTR amino acid changes (p.I148T, p.R74W and p.D1270N) either as CF or CBAVD-causing mutations or as neutral variations.Methods The CFTR genes from individuals who carried at least one of these changes were extensively scanned by a well established DGGE assay followed by direct sequencing and familial segregation analysis of mutations and polymorphisms.
Methods
The CFTR genes from individuals who carried at least one of these changes were extensively scanned by a well established DGGE assay followed by direct sequencing and familial segregation analysis of mutations and polymorphisms.Results Four CF patients (out of 1238) originally identified as carrying the p.I148T mutation in trans with a CF mutation had a second mutation (c.3199del6 or a novel mutation c.3395insA) on the p.I148T allele. We demonstrate here that the deletion c.3199del6 can also be associated with CF without p.I148T. Three CBAVD patients originally identified with the complex allele p.R74W-p.D1270N were also carrying p.V201M on this allele, by contrast with non CF or asymptomatic individuals including the mother of a CF child, who were carrying p.R74W-p.D1270N alone.
Results
Four CF patients (out of 1238) originally identified as carrying the p.I148T mutation in trans with a CF mutation had a second mutation (c.3199del6 or a novel mutation c.3395insA) on the p.I148T allele. We demonstrate here that the deletion c.3199del6 can also be associated with CF without p.I148T. Three CBAVD patients originally identified with the complex allele p.R74W-p.D1270N were also carrying p.V201M on this allele, by contrast with non CF or asymptomatic individuals including the mother of a CF child, who were carrying p.R74W-p.D1270N alone.Conclusion These findings question p.I148T or p.R74W-p.D1270N as causing by themselves CF or CBAVD and emphazises the necessity to perform a complete scanning of CFTR genes and to assign the parental alleles when novel missense mutations are identified.
Conclusion
These findings question p.I148T or p.R74W-p.D1270N as causing by themselves CF or CBAVD and emphazises the necessity to perform a complete scanning of CFTR genes and to assign the parental alleles when novel missense mutations are identified.

Background Cystic fibrosis (CF) is a common, often fatal disease with a well-defined genetic cause, so that it is now recommended in many countries in Europe and the United States to offer genetic screening for CF mutations to identify carriers among adults with a positive family history of CF, partners of individuals with CF, couples planning a pregnancy, couples seeking prenatal care and, recently, neonatal screening. Because of the mutational heterogeneity and the rarity of many mutations, most clinical DNA laboratories offer tests that aim to detect 75–95 % of CF alleles depending on the ethnic and geographic backgrounds of the population, using available commercial kits motsly including 20 to 31 mutations selected on the basis of their frequency as CF-causing mutations. A few laboratories (most often national reference laboratories) have developed the scanning of coding/flanking CFTR sequences to detect unknown mutations. As of February 2004, about 1200 disease-causing mutations have been identified in the cystic fibrosis transmembrane conductance regulator (CFTR) gene   Frameshift, splice-site, nonsense, and in-frame but nonfunctional deletions (such as p.F508del) are disease-causing mutations. By contrast, the status of some missense mutations is extremely difficult to assess and functional studies are not available to diagnostic laboratories. Missense mutations may represent, depending on the populations, up to 45% of mutations responsible for CF or CBAVD (congenital bilateral absence of vas deferens). Moreover, due to improved scanning strategies, a growing number of complex alleles (several sequence changes on the same gene) are thought to affect the expression of the disease phenotype by modulating the effect of a mutation [ 1 - 5 ]. The most striking exemple is the length of the intron 8 polythymidine tract (7, 9, or 5 thymidines) on exon 9 splicing as a genetic modifier of the severity of the p.R117H mutation [ 1 ]. Another exemple is the revertant mutation p.R553Q which, when carried on the same gene as p.F508del, is associated with a CF phenotype with normal chloride concentration in sweat test [ 6 ] and which, when expressed in heterologous cells, can partially correct the processing and Cl- channel gating defects caused by the p.F508del mutation [ 7 ]. With the start of population screening for CF carriers, new data on the prevalence of some missense mutations have been provided, questioning their involvement as disease-causing mutations. In North American populations, missense mutations p.I148T and p.D1270N were found >100 times and >200 times, respectively, more frequently in carrier screening than in CF patients [ 8 , 9 ]. Moreover, we and others have found that individuals affected with CF or CBAVD carry p.D1270N associated with p.R74W on the same allele [p.R74W;p.D1270N] [ 10 , 11 , 5 ]. Similarly, p.I148T has been shown to be associated with a CF phenotype only as a complex allele, i.e. when associated with mutation c.3199del6 on the same gene [ 8 ]. A completely asymptomatic male individual who is a compound heterozygote for p.D1270N and p.I148T has been recently identified [ 9 ]. These findings provided evidence that these missense changes may not be the true mutations and prompted us to reanalyze all the patients in our CF or CBAVD cohort who had been originally diagnosed as compound heterozygotes for either p.I148T or [p.R74W;p.D1270N] and another mutation on the other allele. The result of full scanning of CFTR sequences showed that a second mutation (c.3199del6 or the novel mutation c.3395insA) was associated in  cis  with p.I148T in all individuals with a CF phenotype, and that a third missense mutation (p.V201M) was associated in  cis  with complex allele [p.D1270N;p.R74W] in patients with a CBAVD phenotype in this series.
Background
Cystic fibrosis (CF) is a common, often fatal disease with a well-defined genetic cause, so that it is now recommended in many countries in Europe and the United States to offer genetic screening for CF mutations to identify carriers among adults with a positive family history of CF, partners of individuals with CF, couples planning a pregnancy, couples seeking prenatal care and, recently, neonatal screening. Because of the mutational heterogeneity and the rarity of many mutations, most clinical DNA laboratories offer tests that aim to detect 75–95 % of CF alleles depending on the ethnic and geographic backgrounds of the population, using available commercial kits motsly including 20 to 31 mutations selected on the basis of their frequency as CF-causing mutations. A few laboratories (most often national reference laboratories) have developed the scanning of coding/flanking CFTR sequences to detect unknown mutations. As of February 2004, about 1200 disease-causing mutations have been identified in the cystic fibrosis transmembrane conductance regulator (CFTR) gene   Frameshift, splice-site, nonsense, and in-frame but nonfunctional deletions (such as p.F508del) are disease-causing mutations. By contrast, the status of some missense mutations is extremely difficult to assess and functional studies are not available to diagnostic laboratories. Missense mutations may represent, depending on the populations, up to 45% of mutations responsible for CF or CBAVD (congenital bilateral absence of vas deferens). Moreover, due to improved scanning strategies, a growing number of complex alleles (several sequence changes on the same gene) are thought to affect the expression of the disease phenotype by modulating the effect of a mutation [ 1 - 5 ]. The most striking exemple is the length of the intron 8 polythymidine tract (7, 9, or 5 thymidines) on exon 9 splicing as a genetic modifier of the severity of the p.R117H mutation [ 1 ]. Another exemple is the revertant mutation p.R553Q which, when carried on the same gene as p.F508del, is associated with a CF phenotype with normal chloride concentration in sweat test [ 6 ] and which, when expressed in heterologous cells, can partially correct the processing and Cl- channel gating defects caused by the p.F508del mutation [ 7 ].With the start of population screening for CF carriers, new data on the prevalence of some missense mutations have been provided, questioning their involvement as disease-causing mutations. In North American populations, missense mutations p.I148T and p.D1270N were found >100 times and >200 times, respectively, more frequently in carrier screening than in CF patients [ 8 , 9 ]. Moreover, we and others have found that individuals affected with CF or CBAVD carry p.D1270N associated with p.R74W on the same allele [p.R74W;p.D1270N] [ 10 , 11 , 5 ]. Similarly, p.I148T has been shown to be associated with a CF phenotype only as a complex allele, i.e. when associated with mutation c.3199del6 on the same gene [ 8 ]. A completely asymptomatic male individual who is a compound heterozygote for p.D1270N and p.I148T has been recently identified [ 9 ]. These findings provided evidence that these missense changes may not be the true mutations and prompted us to reanalyze all the patients in our CF or CBAVD cohort who had been originally diagnosed as compound heterozygotes for either p.I148T or [p.R74W;p.D1270N] and another mutation on the other allele. The result of full scanning of CFTR sequences showed that a second mutation (c.3199del6 or the novel mutation c.3395insA) was associated in  cis  with p.I148T in all individuals with a CF phenotype, and that a third missense mutation (p.V201M) was associated in  cis  with complex allele [p.D1270N;p.R74W] in patients with a CBAVD phenotype in this series.ciscisMethods CFTR scanning for individuals with p.I148T or [p.D1270N;p.R74W] From 1990 to 2003, we have analysed for CFTR mutations genomic DNA from 437 families with CF and 170 with isolated azoospermia caused by CBAVD, using a combination of mutation screening for known and scanning for unknown mutations. The first step was the search of 31 CF mutations detected by the ABI oligonucleotide ligation assay and 3 common intronic mutations by using restriction analysis. The second step was the scanning of coding/flanking sequences by DGGE (Denaturing Gradient Gel Electrophoresis) using 32 GC-clamped amplimers (which in our experience detected 98% of CFTR mutants), followed by sequencing to resolve abnormal PCR products (BigDye terminator cycle sequencing on ABI 310 automate sequencer). Whenever possible, family members were assayed for the mutations and associated polymorphisms. We detected 160 different mutations in the CF group accounting for 97 % of CF alleles, and 64 different mutations in the CBAVD group accounting for 85% of CBAVD alleles, which represents one of the highest allelic heterogeneity reported so far. Usually, mutation scanning is stopped when two mutations are found to be in  trans . In this study, we analyzed by DGGE the entire coding and flanking regions of the CFTR gene of individuals who had been previously found to carry p.I148T or the complex allele [p.R74W;p.D1270N] and assayed their relatives for the additional sequence changes identified. We also re-analyzed the CFTR gene of a CF patient who had been originally described with c.394delTT in  trans  of c.3195del6 [ 12 ], now renamed c.3199del6 (see the Results). Studies to determine the frequency of each sequence alteration described in this report were performed on 600 chromosomes from our general population (Southern France). In addition, we also reanalyzed two additional CF patients previously found to carry p.I148T in the Lyon genetic center. The study was approved by the institutional ethical committees and informed consent was obtained from families. Nomenclature Gene variants and mutants are described using DNA and protein designation: intronic changes, deletions, insertions and frameshifts are reported at the cDNA level (c.) and amino acid changes at the protein level (p.), as recommended in the Human Genome Variation Society web page  .
Methods
CFTR scanning for individuals with p.I148T or [p.D1270N;p.R74W] From 1990 to 2003, we have analysed for CFTR mutations genomic DNA from 437 families with CF and 170 with isolated azoospermia caused by CBAVD, using a combination of mutation screening for known and scanning for unknown mutations. The first step was the search of 31 CF mutations detected by the ABI oligonucleotide ligation assay and 3 common intronic mutations by using restriction analysis. The second step was the scanning of coding/flanking sequences by DGGE (Denaturing Gradient Gel Electrophoresis) using 32 GC-clamped amplimers (which in our experience detected 98% of CFTR mutants), followed by sequencing to resolve abnormal PCR products (BigDye terminator cycle sequencing on ABI 310 automate sequencer). Whenever possible, family members were assayed for the mutations and associated polymorphisms. We detected 160 different mutations in the CF group accounting for 97 % of CF alleles, and 64 different mutations in the CBAVD group accounting for 85% of CBAVD alleles, which represents one of the highest allelic heterogeneity reported so far. Usually, mutation scanning is stopped when two mutations are found to be in  trans . In this study, we analyzed by DGGE the entire coding and flanking regions of the CFTR gene of individuals who had been previously found to carry p.I148T or the complex allele [p.R74W;p.D1270N] and assayed their relatives for the additional sequence changes identified. We also re-analyzed the CFTR gene of a CF patient who had been originally described with c.394delTT in  trans  of c.3195del6 [ 12 ], now renamed c.3199del6 (see the Results). Studies to determine the frequency of each sequence alteration described in this report were performed on 600 chromosomes from our general population (Southern France). In addition, we also reanalyzed two additional CF patients previously found to carry p.I148T in the Lyon genetic center. The study was approved by the institutional ethical committees and informed consent was obtained from families.
CFTR scanning for individuals with p.I148T or [p.D1270N;p.R74W]
From 1990 to 2003, we have analysed for CFTR mutations genomic DNA from 437 families with CF and 170 with isolated azoospermia caused by CBAVD, using a combination of mutation screening for known and scanning for unknown mutations. The first step was the search of 31 CF mutations detected by the ABI oligonucleotide ligation assay and 3 common intronic mutations by using restriction analysis. The second step was the scanning of coding/flanking sequences by DGGE (Denaturing Gradient Gel Electrophoresis) using 32 GC-clamped amplimers (which in our experience detected 98% of CFTR mutants), followed by sequencing to resolve abnormal PCR products (BigDye terminator cycle sequencing on ABI 310 automate sequencer). Whenever possible, family members were assayed for the mutations and associated polymorphisms. We detected 160 different mutations in the CF group accounting for 97 % of CF alleles, and 64 different mutations in the CBAVD group accounting for 85% of CBAVD alleles, which represents one of the highest allelic heterogeneity reported so far. Usually, mutation scanning is stopped when two mutations are found to be in  trans .transIn this study, we analyzed by DGGE the entire coding and flanking regions of the CFTR gene of individuals who had been previously found to carry p.I148T or the complex allele [p.R74W;p.D1270N] and assayed their relatives for the additional sequence changes identified. We also re-analyzed the CFTR gene of a CF patient who had been originally described with c.394delTT in  trans  of c.3195del6 [ 12 ], now renamed c.3199del6 (see the Results). Studies to determine the frequency of each sequence alteration described in this report were performed on 600 chromosomes from our general population (Southern France). In addition, we also reanalyzed two additional CF patients previously found to carry p.I148T in the Lyon genetic center. The study was approved by the institutional ethical committees and informed consent was obtained from families.transNomenclature Gene variants and mutants are described using DNA and protein designation: intronic changes, deletions, insertions and frameshifts are reported at the cDNA level (c.) and amino acid changes at the protein level (p.), as recommended in the Human Genome Variation Society web page  .
Nomenclature
Gene variants and mutants are described using DNA and protein designation: intronic changes, deletions, insertions and frameshifts are reported at the cDNA level (c.) and amino acid changes at the protein level (p.), as recommended in the Human Genome Variation Society web page  .Results A CF mutation (c.3199del6 or c.3395insA) is associated in  cis  with p.I148T in CF patients Two out of 437 CF patients analyzed in Montpellier and two out of 801 CF patients analyzed in Lyon were found to carry p.I148T, which was initially thought to be one of the two mutations responsible for CF in these patients. However, thorough re-analysis of the entire CFTR sequence determined that a CF mutation (c.3395insA or c.3199del6) was present on the same gene in both cases (table  1 ). Table 1 CFTR haplotypes associated with mutations found in CF patients carrying p.I148T in  cis  with c.3395insA or c.3199del6 and in one CF patient carrying c.3199del6 alone Indiv No. Age at Diagnosis Phenotype CFTR Mutations CFTR haplotype IVS1 IVS8 IVS8 IVS8 470 IVS17B IVS17B EGH a CA CA TGm Tn TA CA CF1 7 yrs CF-PI c.394delTT 21 23 10 9 M 36 13 B c.3199del6 22 16 11 7 V 7 17 C CF2 10 yrs CF-PS [c.3395insA;p.I148T] 21 23 10 9 M 7 17 B p.R334W 22 17 11 7 V 46 13 A CF3 6 ms CF-PI [c.3199del6;p.I148T] 21 23 10 9 M 7 17 B p.F508del 21 23 10 9 M 31 13 B CF4 3 yrs CF-PI [c.3199del6;p.I148T] 22 23 nd 9 M 7 17 B p.F508del 22 17 nd 9 M 31 15 B CF5 6 ms CF-PI [c.3199del6;p.I148T] 22 23 nd 9 M 7 17 B p.F508del 22 23 nd 9 M 31 13 B a EGH, extragenic haplotype XV2c/ TaqI , KM19/ PstI ;  nd, not determined Patients CF1-3 were from the cohort of Montpellier (n = 437), patients CF4-5 were from the cohort of Lyon (n= 801). Mutation c.3199del6 can also occur alone as a CF-causing allele Mutation c.3199del6 was found to be carried without p.I148T in a young CF male with 394delTT on the other allele, diagnosed at the age of 7 years on the basis of typical pulmonary disease, pancreatic insufficiency, poor growth and positive sweat test [ 12 ]. Mutation c.3199del6 was initially described by us in 1994 in this patient as c.3195del6 [ 12 ], in accordance with the first draft of mutation nomenclature [ 13 ]. However, it occurred in the same palindromic sequence in exon 17a than mutation c.3199del6 reported in 1998 by Bozon  et al . [ 14 ]. Both mutations are expected to delete either amino acids Val1022 and Ile1023, or Ile1023 and Val1024 from the CFTR protein. As it is impossible to determine at the genomic level in which part of the palindrome each of them occurred, the most 3'copy of the repeat is arbitrarily assigned to have been mutated, according to the current rule [ 15 ]. Consequently, mutations c.3195del6 and c.3199del6 should be considered as identical and reported as c.3199_3204del. The familial segregation analysis of polymorphisms covering the CFTR gene showed that p.I148T, when present in individuals with a CF phenotype, occurred on a unique haplotype carrying IVS8-9T whatever the mutation in  cis , c.3395insA or c.3199del6 (table  1 ). By contrast, c.3199del6 without p.I148T occurred on a different haplotype carrying IVS8-7T. Mutations p.I148T, c.3199del6 and c.3395insA were not found on 600 chromosomes from our general population. Triple-mutant allele [p.R74W;p.V201M;p.D1270N] is found in males with CBAVD whereas double-mutant allele [p.R74W;p.D1270N] is found in asymptomatic individuals Re-analysis of the CFTR gene in families carrying [p.R74W;p.D1270N] identified a third mutation (p.V201M) on the same chromosome in three unrelated individuals with CBAVD (table  2 ). Only the double-mutant p.R74W-p.D1270N was present in the two unaffected individuals who were found with these changes in our sample. The first case was a young boy who had been initially suspected of having CF at age 4 years because of allergic rhinitis but for whom the diagnosis of CF was later ruled out; no other CFTR sequence alteration could be identified and the sweat tests were negative (chloride values <40 mM). The second individual was the mother of a CF girl who was compound heterozygous for p.F508del and p.P67L. This woman, who was carrying p.P67L on one CFTR gene and [p.R74W-p.D1270N] on the other (table  1 ), was completely asymptomatic at age 45 years and displayed three negative sweat tests (chloride values <20 mM). The triple and double mutant alleles seem to have occurred on the same haplotype TG11-T7-V470. Table 2 CFTR sequence changes found in individuals carrying missense alterations p.R74W, p.D1270N, or p.V201M Mutations Haplotype IVS1 IVS8 IVS8 IVS8 470 IVS17B IVS17B CA CA TGm Tn TA CA CBAVD1 p.R1066C 22 16 11 7 V 30 13 [p.R74W;p.V201M;p.D1270N] 22 16 11 7 V 31 13 CBAVD2 p.M952I 26 17 10 7 M 7 17 [p.R74W;p.V201M;p.D1270N] 22 16 11 7 V 31 13 CBAVD3 [p.R74W;p.V201M;p.D1270N] 22 16 11 7 V 31 13 [p.R74W;p.V201M;p.D1270N] 22 16 11 7 V 31 13 Individual non affected with CF No mutation  21 nd 10 7 M 7 17 [p.R74W;p.D1270N] 22 nd 11 7 V 30 13 Asymptomatic mother of a CF affected girl p.P67L 23 16 10 7 M 7 17 [p.R74;p.D1270N] 22 16 11 7 V 31 13
Results
A CF mutation (c.3199del6 or c.3395insA) is associated in  cis  with p.I148T in CF patients Two out of 437 CF patients analyzed in Montpellier and two out of 801 CF patients analyzed in Lyon were found to carry p.I148T, which was initially thought to be one of the two mutations responsible for CF in these patients. However, thorough re-analysis of the entire CFTR sequence determined that a CF mutation (c.3395insA or c.3199del6) was present on the same gene in both cases (table  1 ). Table 1 CFTR haplotypes associated with mutations found in CF patients carrying p.I148T in  cis  with c.3395insA or c.3199del6 and in one CF patient carrying c.3199del6 alone Indiv No. Age at Diagnosis Phenotype CFTR Mutations CFTR haplotype IVS1 IVS8 IVS8 IVS8 470 IVS17B IVS17B EGH a CA CA TGm Tn TA CA CF1 7 yrs CF-PI c.394delTT 21 23 10 9 M 36 13 B c.3199del6 22 16 11 7 V 7 17 C CF2 10 yrs CF-PS [c.3395insA;p.I148T] 21 23 10 9 M 7 17 B p.R334W 22 17 11 7 V 46 13 A CF3 6 ms CF-PI [c.3199del6;p.I148T] 21 23 10 9 M 7 17 B p.F508del 21 23 10 9 M 31 13 B CF4 3 yrs CF-PI [c.3199del6;p.I148T] 22 23 nd 9 M 7 17 B p.F508del 22 17 nd 9 M 31 15 B CF5 6 ms CF-PI [c.3199del6;p.I148T] 22 23 nd 9 M 7 17 B p.F508del 22 23 nd 9 M 31 13 B a EGH, extragenic haplotype XV2c/ TaqI , KM19/ PstI ;  nd, not determined Patients CF1-3 were from the cohort of Montpellier (n = 437), patients CF4-5 were from the cohort of Lyon (n= 801).
A CF mutation (c.3199del6 or c.3395insA) is associated in  cis  with p.I148T in CF patients
cisTwo out of 437 CF patients analyzed in Montpellier and two out of 801 CF patients analyzed in Lyon were found to carry p.I148T, which was initially thought to be one of the two mutations responsible for CF in these patients. However, thorough re-analysis of the entire CFTR sequence determined that a CF mutation (c.3395insA or c.3199del6) was present on the same gene in both cases (table  1 ).CFTR haplotypes associated with mutations found in CF patients carrying p.I148T in  cis  with c.3395insA or c.3199del6 and in one CF patient carrying c.3199del6 alonecis[c.3395insA;p.I148T]2123109M717B[c.3199del6;p.I148T]2123109M717B[c.3199del6;p.I148T]2223nd9M717B[c.3199del6;p.I148T]2223nd9M717Ba EGH, extragenic haplotype XV2c/ TaqI , KM19/ PstI ;  nd, not determined Patients CF1-3 were from the cohort of Montpellier (n = 437), patients CF4-5 were from the cohort of Lyon (n= 801).TaqIPstI ;Mutation c.3199del6 can also occur alone as a CF-causing allele Mutation c.3199del6 was found to be carried without p.I148T in a young CF male with 394delTT on the other allele, diagnosed at the age of 7 years on the basis of typical pulmonary disease, pancreatic insufficiency, poor growth and positive sweat test [ 12 ]. Mutation c.3199del6 was initially described by us in 1994 in this patient as c.3195del6 [ 12 ], in accordance with the first draft of mutation nomenclature [ 13 ]. However, it occurred in the same palindromic sequence in exon 17a than mutation c.3199del6 reported in 1998 by Bozon  et al . [ 14 ]. Both mutations are expected to delete either amino acids Val1022 and Ile1023, or Ile1023 and Val1024 from the CFTR protein. As it is impossible to determine at the genomic level in which part of the palindrome each of them occurred, the most 3'copy of the repeat is arbitrarily assigned to have been mutated, according to the current rule [ 15 ]. Consequently, mutations c.3195del6 and c.3199del6 should be considered as identical and reported as c.3199_3204del. The familial segregation analysis of polymorphisms covering the CFTR gene showed that p.I148T, when present in individuals with a CF phenotype, occurred on a unique haplotype carrying IVS8-9T whatever the mutation in  cis , c.3395insA or c.3199del6 (table  1 ). By contrast, c.3199del6 without p.I148T occurred on a different haplotype carrying IVS8-7T. Mutations p.I148T, c.3199del6 and c.3395insA were not found on 600 chromosomes from our general population.
Mutation c.3199del6 can also occur alone as a CF-causing allele
Mutation c.3199del6 was found to be carried without p.I148T in a young CF male with 394delTT on the other allele, diagnosed at the age of 7 years on the basis of typical pulmonary disease, pancreatic insufficiency, poor growth and positive sweat test [ 12 ]. Mutation c.3199del6 was initially described by us in 1994 in this patient as c.3195del6 [ 12 ], in accordance with the first draft of mutation nomenclature [ 13 ]. However, it occurred in the same palindromic sequence in exon 17a than mutation c.3199del6 reported in 1998 by Bozon  et al . [ 14 ]. Both mutations are expected to delete either amino acids Val1022 and Ile1023, or Ile1023 and Val1024 from the CFTR protein. As it is impossible to determine at the genomic level in which part of the palindrome each of them occurred, the most 3'copy of the repeat is arbitrarily assigned to have been mutated, according to the current rule [ 15 ]. Consequently, mutations c.3195del6 and c.3199del6 should be considered as identical and reported as c.3199_3204del. The familial segregation analysis of polymorphisms covering the CFTR gene showed that p.I148T, when present in individuals with a CF phenotype, occurred on a unique haplotype carrying IVS8-9T whatever the mutation in  cis , c.3395insA or c.3199del6 (table  1 ). By contrast, c.3199del6 without p.I148T occurred on a different haplotype carrying IVS8-7T.et alcisMutations p.I148T, c.3199del6 and c.3395insA were not found on 600 chromosomes from our general population.Triple-mutant allele [p.R74W;p.V201M;p.D1270N] is found in males with CBAVD whereas double-mutant allele [p.R74W;p.D1270N] is found in asymptomatic individuals Re-analysis of the CFTR gene in families carrying [p.R74W;p.D1270N] identified a third mutation (p.V201M) on the same chromosome in three unrelated individuals with CBAVD (table  2 ). Only the double-mutant p.R74W-p.D1270N was present in the two unaffected individuals who were found with these changes in our sample. The first case was a young boy who had been initially suspected of having CF at age 4 years because of allergic rhinitis but for whom the diagnosis of CF was later ruled out; no other CFTR sequence alteration could be identified and the sweat tests were negative (chloride values <40 mM). The second individual was the mother of a CF girl who was compound heterozygous for p.F508del and p.P67L. This woman, who was carrying p.P67L on one CFTR gene and [p.R74W-p.D1270N] on the other (table  1 ), was completely asymptomatic at age 45 years and displayed three negative sweat tests (chloride values <20 mM). The triple and double mutant alleles seem to have occurred on the same haplotype TG11-T7-V470. Table 2 CFTR sequence changes found in individuals carrying missense alterations p.R74W, p.D1270N, or p.V201M Mutations Haplotype IVS1 IVS8 IVS8 IVS8 470 IVS17B IVS17B CA CA TGm Tn TA CA CBAVD1 p.R1066C 22 16 11 7 V 30 13 [p.R74W;p.V201M;p.D1270N] 22 16 11 7 V 31 13 CBAVD2 p.M952I 26 17 10 7 M 7 17 [p.R74W;p.V201M;p.D1270N] 22 16 11 7 V 31 13 CBAVD3 [p.R74W;p.V201M;p.D1270N] 22 16 11 7 V 31 13 [p.R74W;p.V201M;p.D1270N] 22 16 11 7 V 31 13 Individual non affected with CF No mutation  21 nd 10 7 M 7 17 [p.R74W;p.D1270N] 22 nd 11 7 V 30 13 Asymptomatic mother of a CF affected girl p.P67L 23 16 10 7 M 7 17 [p.R74;p.D1270N] 22 16 11 7 V 31 13
Triple-mutant allele [p.R74W;p.V201M;p.D1270N] is found in males with CBAVD whereas double-mutant allele [p.R74W;p.D1270N] is found in asymptomatic individuals
Re-analysis of the CFTR gene in families carrying [p.R74W;p.D1270N] identified a third mutation (p.V201M) on the same chromosome in three unrelated individuals with CBAVD (table  2 ). Only the double-mutant p.R74W-p.D1270N was present in the two unaffected individuals who were found with these changes in our sample. The first case was a young boy who had been initially suspected of having CF at age 4 years because of allergic rhinitis but for whom the diagnosis of CF was later ruled out; no other CFTR sequence alteration could be identified and the sweat tests were negative (chloride values <40 mM). The second individual was the mother of a CF girl who was compound heterozygous for p.F508del and p.P67L. This woman, who was carrying p.P67L on one CFTR gene and [p.R74W-p.D1270N] on the other (table  1 ), was completely asymptomatic at age 45 years and displayed three negative sweat tests (chloride values <20 mM). The triple and double mutant alleles seem to have occurred on the same haplotype TG11-T7-V470.CFTR sequence changes found in individuals carrying missense alterations p.R74W, p.D1270N, or p.V201M[p.R74W;p.V201M;p.D1270N]2216117V3113[p.R74W;p.V201M;p.D1270N]2216117V3113[p.R74W;p.V201M;p.D1270N]2216117V3113[p.R74W;p.V201M;p.D1270N]2216117V3113[p.R74W;p.D1270N]22117V3013[p.R74;p.D1270N]2216117V3113Discussion p.I148T is a low penetrance CF mutation or a neutral polymorphism Since its initial description in a CF Canadian patient with pancreatic insufficiency [ 16 ], the mutation p.I148T, which changes a conserved amino acid and occurs in the first cytoplasmic loop of the CFTR protein, has been considered as a severe CF allele in many countries. It was thought to be the second most common CF mutation in the French Canadian population, accounting for 9.1% of the French Canadian chromosomes [ 17 ], whereas in France, p.I148T accounted for only 0.11 % of the CF alleles in a sample of 3,710 patients affected with the disease [ 5 ]. p.I148T can now be detected by several commercially available kits developed for routine screening of CF carriers and for CF neonatal screening, and recently it has been included in the core panel of 25 CF mutations recommended by the American College of Medical Genetics (ACMG) [ 18 ]. Thousands of individuals are being screened for this mutation worldwide and it is possible that several prenatal diagnosis have been or will be performed. However there are now several lines of evidence that question the role of p.I148T by itself in causing disease. First, compound heterozygosity for p.I148T and a severe CF mutation was recently identified in several healthy individuals [ 9 , 19 ]. When affected and unaffected individuals carrying apparently the same mutational genotype were re-analyzed for additional changes that could explain the different phenotypes, p.I148T was found to be associated in  cis  with another mutation, c.3199del6, in patients with a classic CF phenotype, whereas healthy adults who were compound heterozygous for p.I148T and a severe CF mutation or homozygous for p.I148T did not carry the deletion [ 8 ]. In a recent study, the p.I148T mutation has been further documented to be linked with the 3199del6 mutation in all 24 CF patients of French Canadian descent originally identified as compound heterozygous for the p.I148T mutation and a second severe CFTR mutation [ 20 ]. Second, p.I148T was found to be over 100 times more common in two independent U.S. carrier screening programmes than in CF patients: it accounted for 6.4 to 7.7% of chromosomes detected in the screened populations versus 0.06 to 0.068% of CF chromosomes in CF patients [8. 9]. This discrepancy suggests that p.I148T is either a poorly penetrant mutation or a neutral polymorphism. Third, when transiently expressed in epithelial cells, p.I148T mutant protein is normally processed and is able to mediate normal chloride transport with properties identical with those of wild-type cells [ 21 ]. As the mutant seems to suppress the ability of CFTR to support HCO3- transport, it has been hypothesized that p.I148T may contribute to disease through Cl- coupled HCO3- altered transport; however, the major CFTR functions are retained by the mutant [ 21 ]. Fourth, we show in this study for the first time that p.I148T can be associated with a frameshift mutation c.3395insA in exon 17b instead of in-frame deletion c.3199del6 in exon 17a. Insertion c.3395insA (designated as c.3395_3396insA) is a previously unreported mutation that is predictive of premature termination of translation at amino acid residue 1155. The truncated protein lacking the 325 last amino acids is believed to be not functional and be degraded rapidly, generating no detectable protein. A CFTR alteration producing a premature termination signal is a class I mutation, considered severe enough to cause CF by itself and exclude the contribution of any other sequence change on the same allele. Fifth, in contrast with other studies that stated that only the complex allele [p.I148T;9T;c.3199del6] appeared to be associated with a classic CF phenotype [ 8 ], we demonstrate that c.3199del6 is associated with a CF phenotype even if the deletion occurs on a chromosome that does not carry p.I148T, which adds further value to the consideration that p.I148T is not a true mutation but simply a polymorphism. Although no functional test was performed to prove its contribution to the severe phenotype, mutation c.3199del6 has been considered as a defective allele as it results in the loss of two amino acid residues in the TM10 domain of the CFTR protein and has not been detected in non-CF alleles. Our data fully support the recent recommendation that p.I148T should not be included in the mutation panel selected for prenatal screening strategy [ 22 ]. The complex allele [p.R74W;p.D1270N] may be not enough to cause disease We and others had initially described p.R74W [ 23 ] and p.D1270N [ 24 ] in isolation but they have since been found in association in many CBAVD or CF patients [ 10 , 11 ] and these two changes were thought to be deleterious, alone or in combination. A few complex alleles have been expressed in heterologous systems to evaluate the impact on CFTR processing and Cl- channel activity and better understand the contribution of each missense mutation on phenotype. When expressed in HeLa cells, mutant p.R74W, p.D1270N and [p.R74W;p.D1270N] did not affect CFTR processing, however a lower cAMP-responsive anion conductance was observed with the double mutant [p.R74W;p.D1270N] [ 3 ]. The assay suggested that p.R74W alone should be considered as a polymorphism, p.D1270N alone could generate a CBAVD phenotype while the complex allele could produce a more severe phenotype as p.R74W could enhance the effect of p.D1270N [ 3 ]. However these findings have not yet been confirmed by other studies. We have found here that a triple-mutant [p.R74W;p.V201M;p.D1270N] allele was carried in all three patients with CBAVD whereas only the double mutant [p.R74W;p.D1270N] allele was present in two asymptomatic individuals including an obligate carrier who was compound heterozygous for a CF mutation. Another mother carrying [p.R74W;p.D1270N]  in trans  of a CF mutation has been described previously; despite two positive sweat tests she was absolutely asymptomatic [ 25 ]. Missense p.V201M in exon 6a changes a valine for a methionine in the third transmembrane domain; it was initially reported alone in a French patient with CBAVD [ 26 ], then in Brazilian patients with CF [ 27 ]. Recent large scale screening for CF carrier showed that p.D1270N was present 205 times more commonly in the screened population than in the CF patients (frequency of 14% versus 0.068%); in addition, a completely asymptomatic adult compound heterozygote for p.D1270N and p.I148T has been identified [ 9 ]. Although it is not known whether these alleles are associated or not with the third change p.V201M, there are now enough evidence to question the role of the complex allele [p.R74W;p.D1270N] as being a CF or CBAVD mutation. Further experimental and genetic investigations will be necessary to demonstrate the role of p.V201M in causing disease.
Discussion
p.I148T is a low penetrance CF mutation or a neutral polymorphism Since its initial description in a CF Canadian patient with pancreatic insufficiency [ 16 ], the mutation p.I148T, which changes a conserved amino acid and occurs in the first cytoplasmic loop of the CFTR protein, has been considered as a severe CF allele in many countries. It was thought to be the second most common CF mutation in the French Canadian population, accounting for 9.1% of the French Canadian chromosomes [ 17 ], whereas in France, p.I148T accounted for only 0.11 % of the CF alleles in a sample of 3,710 patients affected with the disease [ 5 ]. p.I148T can now be detected by several commercially available kits developed for routine screening of CF carriers and for CF neonatal screening, and recently it has been included in the core panel of 25 CF mutations recommended by the American College of Medical Genetics (ACMG) [ 18 ]. Thousands of individuals are being screened for this mutation worldwide and it is possible that several prenatal diagnosis have been or will be performed. However there are now several lines of evidence that question the role of p.I148T by itself in causing disease. First, compound heterozygosity for p.I148T and a severe CF mutation was recently identified in several healthy individuals [ 9 , 19 ]. When affected and unaffected individuals carrying apparently the same mutational genotype were re-analyzed for additional changes that could explain the different phenotypes, p.I148T was found to be associated in  cis  with another mutation, c.3199del6, in patients with a classic CF phenotype, whereas healthy adults who were compound heterozygous for p.I148T and a severe CF mutation or homozygous for p.I148T did not carry the deletion [ 8 ]. In a recent study, the p.I148T mutation has been further documented to be linked with the 3199del6 mutation in all 24 CF patients of French Canadian descent originally identified as compound heterozygous for the p.I148T mutation and a second severe CFTR mutation [ 20 ]. Second, p.I148T was found to be over 100 times more common in two independent U.S. carrier screening programmes than in CF patients: it accounted for 6.4 to 7.7% of chromosomes detected in the screened populations versus 0.06 to 0.068% of CF chromosomes in CF patients [8. 9]. This discrepancy suggests that p.I148T is either a poorly penetrant mutation or a neutral polymorphism. Third, when transiently expressed in epithelial cells, p.I148T mutant protein is normally processed and is able to mediate normal chloride transport with properties identical with those of wild-type cells [ 21 ]. As the mutant seems to suppress the ability of CFTR to support HCO3- transport, it has been hypothesized that p.I148T may contribute to disease through Cl- coupled HCO3- altered transport; however, the major CFTR functions are retained by the mutant [ 21 ]. Fourth, we show in this study for the first time that p.I148T can be associated with a frameshift mutation c.3395insA in exon 17b instead of in-frame deletion c.3199del6 in exon 17a. Insertion c.3395insA (designated as c.3395_3396insA) is a previously unreported mutation that is predictive of premature termination of translation at amino acid residue 1155. The truncated protein lacking the 325 last amino acids is believed to be not functional and be degraded rapidly, generating no detectable protein. A CFTR alteration producing a premature termination signal is a class I mutation, considered severe enough to cause CF by itself and exclude the contribution of any other sequence change on the same allele. Fifth, in contrast with other studies that stated that only the complex allele [p.I148T;9T;c.3199del6] appeared to be associated with a classic CF phenotype [ 8 ], we demonstrate that c.3199del6 is associated with a CF phenotype even if the deletion occurs on a chromosome that does not carry p.I148T, which adds further value to the consideration that p.I148T is not a true mutation but simply a polymorphism. Although no functional test was performed to prove its contribution to the severe phenotype, mutation c.3199del6 has been considered as a defective allele as it results in the loss of two amino acid residues in the TM10 domain of the CFTR protein and has not been detected in non-CF alleles. Our data fully support the recent recommendation that p.I148T should not be included in the mutation panel selected for prenatal screening strategy [ 22 ].
p.I148T is a low penetrance CF mutation or a neutral polymorphism
Since its initial description in a CF Canadian patient with pancreatic insufficiency [ 16 ], the mutation p.I148T, which changes a conserved amino acid and occurs in the first cytoplasmic loop of the CFTR protein, has been considered as a severe CF allele in many countries. It was thought to be the second most common CF mutation in the French Canadian population, accounting for 9.1% of the French Canadian chromosomes [ 17 ], whereas in France, p.I148T accounted for only 0.11 % of the CF alleles in a sample of 3,710 patients affected with the disease [ 5 ]. p.I148T can now be detected by several commercially available kits developed for routine screening of CF carriers and for CF neonatal screening, and recently it has been included in the core panel of 25 CF mutations recommended by the American College of Medical Genetics (ACMG) [ 18 ]. Thousands of individuals are being screened for this mutation worldwide and it is possible that several prenatal diagnosis have been or will be performed. However there are now several lines of evidence that question the role of p.I148T by itself in causing disease. First, compound heterozygosity for p.I148T and a severe CF mutation was recently identified in several healthy individuals [ 9 , 19 ]. When affected and unaffected individuals carrying apparently the same mutational genotype were re-analyzed for additional changes that could explain the different phenotypes, p.I148T was found to be associated in  cis  with another mutation, c.3199del6, in patients with a classic CF phenotype, whereas healthy adults who were compound heterozygous for p.I148T and a severe CF mutation or homozygous for p.I148T did not carry the deletion [ 8 ]. In a recent study, the p.I148T mutation has been further documented to be linked with the 3199del6 mutation in all 24 CF patients of French Canadian descent originally identified as compound heterozygous for the p.I148T mutation and a second severe CFTR mutation [ 20 ]. Second, p.I148T was found to be over 100 times more common in two independent U.S. carrier screening programmes than in CF patients: it accounted for 6.4 to 7.7% of chromosomes detected in the screened populations versus 0.06 to 0.068% of CF chromosomes in CF patients [8. 9]. This discrepancy suggests that p.I148T is either a poorly penetrant mutation or a neutral polymorphism. Third, when transiently expressed in epithelial cells, p.I148T mutant protein is normally processed and is able to mediate normal chloride transport with properties identical with those of wild-type cells [ 21 ]. As the mutant seems to suppress the ability of CFTR to support HCO3- transport, it has been hypothesized that p.I148T may contribute to disease through Cl- coupled HCO3- altered transport; however, the major CFTR functions are retained by the mutant [ 21 ]. Fourth, we show in this study for the first time that p.I148T can be associated with a frameshift mutation c.3395insA in exon 17b instead of in-frame deletion c.3199del6 in exon 17a. Insertion c.3395insA (designated as c.3395_3396insA) is a previously unreported mutation that is predictive of premature termination of translation at amino acid residue 1155. The truncated protein lacking the 325 last amino acids is believed to be not functional and be degraded rapidly, generating no detectable protein. A CFTR alteration producing a premature termination signal is a class I mutation, considered severe enough to cause CF by itself and exclude the contribution of any other sequence change on the same allele. Fifth, in contrast with other studies that stated that only the complex allele [p.I148T;9T;c.3199del6] appeared to be associated with a classic CF phenotype [ 8 ], we demonstrate that c.3199del6 is associated with a CF phenotype even if the deletion occurs on a chromosome that does not carry p.I148T, which adds further value to the consideration that p.I148T is not a true mutation but simply a polymorphism. Although no functional test was performed to prove its contribution to the severe phenotype, mutation c.3199del6 has been considered as a defective allele as it results in the loss of two amino acid residues in the TM10 domain of the CFTR protein and has not been detected in non-CF alleles. Our data fully support the recent recommendation that p.I148T should not be included in the mutation panel selected for prenatal screening strategy [ 22 ].cisThe complex allele [p.R74W;p.D1270N] may be not enough to cause disease We and others had initially described p.R74W [ 23 ] and p.D1270N [ 24 ] in isolation but they have since been found in association in many CBAVD or CF patients [ 10 , 11 ] and these two changes were thought to be deleterious, alone or in combination. A few complex alleles have been expressed in heterologous systems to evaluate the impact on CFTR processing and Cl- channel activity and better understand the contribution of each missense mutation on phenotype. When expressed in HeLa cells, mutant p.R74W, p.D1270N and [p.R74W;p.D1270N] did not affect CFTR processing, however a lower cAMP-responsive anion conductance was observed with the double mutant [p.R74W;p.D1270N] [ 3 ]. The assay suggested that p.R74W alone should be considered as a polymorphism, p.D1270N alone could generate a CBAVD phenotype while the complex allele could produce a more severe phenotype as p.R74W could enhance the effect of p.D1270N [ 3 ]. However these findings have not yet been confirmed by other studies. We have found here that a triple-mutant [p.R74W;p.V201M;p.D1270N] allele was carried in all three patients with CBAVD whereas only the double mutant [p.R74W;p.D1270N] allele was present in two asymptomatic individuals including an obligate carrier who was compound heterozygous for a CF mutation. Another mother carrying [p.R74W;p.D1270N]  in trans  of a CF mutation has been described previously; despite two positive sweat tests she was absolutely asymptomatic [ 25 ]. Missense p.V201M in exon 6a changes a valine for a methionine in the third transmembrane domain; it was initially reported alone in a French patient with CBAVD [ 26 ], then in Brazilian patients with CF [ 27 ]. Recent large scale screening for CF carrier showed that p.D1270N was present 205 times more commonly in the screened population than in the CF patients (frequency of 14% versus 0.068%); in addition, a completely asymptomatic adult compound heterozygote for p.D1270N and p.I148T has been identified [ 9 ]. Although it is not known whether these alleles are associated or not with the third change p.V201M, there are now enough evidence to question the role of the complex allele [p.R74W;p.D1270N] as being a CF or CBAVD mutation. Further experimental and genetic investigations will be necessary to demonstrate the role of p.V201M in causing disease.
The complex allele [p.R74W;p.D1270N] may be not enough to cause disease
We and others had initially described p.R74W [ 23 ] and p.D1270N [ 24 ] in isolation but they have since been found in association in many CBAVD or CF patients [ 10 , 11 ] and these two changes were thought to be deleterious, alone or in combination. A few complex alleles have been expressed in heterologous systems to evaluate the impact on CFTR processing and Cl- channel activity and better understand the contribution of each missense mutation on phenotype. When expressed in HeLa cells, mutant p.R74W, p.D1270N and [p.R74W;p.D1270N] did not affect CFTR processing, however a lower cAMP-responsive anion conductance was observed with the double mutant [p.R74W;p.D1270N] [ 3 ]. The assay suggested that p.R74W alone should be considered as a polymorphism, p.D1270N alone could generate a CBAVD phenotype while the complex allele could produce a more severe phenotype as p.R74W could enhance the effect of p.D1270N [ 3 ]. However these findings have not yet been confirmed by other studies.We have found here that a triple-mutant [p.R74W;p.V201M;p.D1270N] allele was carried in all three patients with CBAVD whereas only the double mutant [p.R74W;p.D1270N] allele was present in two asymptomatic individuals including an obligate carrier who was compound heterozygous for a CF mutation. Another mother carrying [p.R74W;p.D1270N]  in trans  of a CF mutation has been described previously; despite two positive sweat tests she was absolutely asymptomatic [ 25 ]. Missense p.V201M in exon 6a changes a valine for a methionine in the third transmembrane domain; it was initially reported alone in a French patient with CBAVD [ 26 ], then in Brazilian patients with CF [ 27 ].in transRecent large scale screening for CF carrier showed that p.D1270N was present 205 times more commonly in the screened population than in the CF patients (frequency of 14% versus 0.068%); in addition, a completely asymptomatic adult compound heterozygote for p.D1270N and p.I148T has been identified [ 9 ]. Although it is not known whether these alleles are associated or not with the third change p.V201M, there are now enough evidence to question the role of the complex allele [p.R74W;p.D1270N] as being a CF or CBAVD mutation. Further experimental and genetic investigations will be necessary to demonstrate the role of p.V201M in causing disease.Conclusions This report further corroborates the recent hypothesis [ 9 ] that p.I148T and p.R74W-p.D1270N may not be true CF/CBAVD mutations. If these observations are further confirmed by a large multicentric study, they will have important implications for genetic counseling of patients and couples found to carry p.I148T or [p.R74W;p.D1270N]. They also pinpoint several important points in genetic testing for CF : first, the necessity of scanning the whole regions of the CFTR gene for diagnosis purposes, whatever the cost; second the necessity to better standardize mutation nomenclature, and third the usefulness of confirming inheritance of mutations from both parents whenever possible to avoid the risk for erroneously reporting changes in  trans  that are in fact complex alleles.
Conclusions
This report further corroborates the recent hypothesis [ 9 ] that p.I148T and p.R74W-p.D1270N may not be true CF/CBAVD mutations. If these observations are further confirmed by a large multicentric study, they will have important implications for genetic counseling of patients and couples found to carry p.I148T or [p.R74W;p.D1270N]. They also pinpoint several important points in genetic testing for CF : first, the necessity of scanning the whole regions of the CFTR gene for diagnosis purposes, whatever the cost; second the necessity to better standardize mutation nomenclature, and third the usefulness of confirming inheritance of mutations from both parents whenever possible to avoid the risk for erroneously reporting changes in  trans  that are in fact complex alleles.transCompeting interests None declared.
Competing interests
None declared.Authors'contributions JPA, CG, CT and FC carried out the molecular genetic studies. MDG coordonated the molecular analysis. MC conceived the study and drafted the manuscript. All authors read and approved the final manuscript.
Authors'contributions
JPA, CG, CT and FC carried out the molecular genetic studies. MDG coordonated the molecular analysis. MC conceived the study and drafted the manuscript. All authors read and approved the final manuscript.Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here: