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Objectives To confirm linkage to microsatellite markers on chromosome 8q, 12q, 13q and 22q in families with nocturnal enuresis/incontinence segregating with an autosomal dominant pattern, and to determine if there is an association between the clinical subtype and these linked loci.
Patients and methods Families with at least three members with nocturnal enuresis in two generations were included in the study. The index patient was 7 years old and had evidence of bladder dysfunction; all other family members were 5 years old. Bladder dysfunction in the index patients was documented by video-urodynamics when indicated. A nycthemeral rhythm of diuresis was documented in all index patients. The clinical diagnosis of all family members was based on a questionnaire on voiding problems and micturition habits, uroflowmetry, measurement of functional bladder capacity and nocturnal diuresis. Linkage was analysed using an autosomal dominant model with a gene frequency equal to 0.05 and a penetrance of 0.9.
Results Thirty-two families with nocturnal enuresis/incontinence (one with four, 25 with three and six with two generations) were included. The mean number of persons included per family was 10 and on average five members were symptomatic. Linkage of nocturnal enuresis to a region on chromosome 22q11 was found in nine families, to 13q13–14 in six and to 12q in four. There was no convincing evidence for linkage to chromosome 8q. Clinical findings in the proband and their family members with possible linkage to a given locus were heterogeneous, and hence no clear genotype/phenotype correlation could be postulated.
Conclusion These findings support the hypothesis of the genetic and phenotypic heterogeneity of nocturnal enuresis/incontinence. Putative linkage was confirmed to the same chromosomal loci as in previous studies of ‘monosymptomatic’ enuresis and different phenotypes were linked to the same loci.
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Nocturnal enuresis (NE) and incontinence affect 10–15% of 7-year-old children , making them one of the most important health problems in children worldwide. Until recently, NE was often considered as a self-limiting and therefore benign process. However, epidemiological studies have shown that 1% of adults also have these problems . Thus, if bedwetting is still a problem at the age of 7 years, the child has a 5–10% risk of having this problem in adulthood.
According to the International Children's Continence Society , enuresis is defined as normal voiding occurring at a socially inappropriate time and place, in children aged > 5 years. NE is voiding in bed without awakening; monosymptomatic NE is defined as enuresis with no daytime symptoms. In reality, enuresis/incontinence comprises a heterogeneous group of diseases, resulting from a combination of many exogenous and endogenous causes. However, the identification of clinically homogeneous subtypes seems to be difficult .
Despite the high incidence, a few studies have addressed the pathophysiology of NE [5,6]. Several pathophysiological mechanisms have been proposed , e.g. a small bladder volume , bladder dysfunction [9,10], abnormal nycthemeral vasopressin levels , nocturnal polyuria, abnormal sleep patterns and arousability . Monosymptomatic NE with no clear night-time polyuria or daytime bladder dysfunction is considered as the most frequent subgroup [13,14].
Familial and twin studies have suggested a genetic background for NE. Bakwin et al. showed that the incidence of NE is highest in families in which both parents have been enuretic (77%). A recent Finnish study in twins reported a concordance rate of 0.43 for monozygotic twins, against 0.19 for dizygotic twins in childhood, whereas it was 0.25 vs 0, respectively, in adulthood . Segregation studies have shown that in some families NE is inherited as a dominant trait with high penetrance . Linkage studies found evidence for linkage of primary monosymptomatic NE to regions on chromosomes 8q , 12q , 13q , and 22q . In 1995, Eiberg et al. found linkage to 13q13–14 in five of 11 Danish families with primary NE. Linkage to the same chromosomal region was confirmed in three of 16 Swedish families, whereas six of 16 families showed linkage to a region on 12q13–21 . In a subset of German families, linkage with those two loci was confirmed . These findings suggested a high genetic risk in all types of enuresis and not just in primary monosymptomatic NE . Subsequently, a third putative region on chromosome 8 was identified . Finally, in a single large family, linkage to a new region on chromosome 22q11 was identified, whereas this family initially had been linked to a region on 13q . Linkage to this region was confirmed in another set of families by one study .
The present study is the first genetic study to attempt to define the clinical phenotype of NE more accurately. This attempt arose from two major concerns, i.e. about the definition of monosymptomatic NE and about the characterization of polyuria.
The classical definition of monosymptomatic NE by the International Children's Continence Society  has several shortcomings. If daytime symptoms are present, it is evident that there is an underlying bladder dysfunction. However, many children show no evidence of daytime symptoms on an initial questionnaire, but daytime symptoms are detected after completing a second or third questionnaire, or during registration in a diary. In other children daytime symptoms become apparent when fluid intake is increased during bladder volume training, or when frequent voiding is avoided. In these children, daytime symptoms are masked by a low fluid intake and/or high voiding frequency. Other children present with a bladder volume that is too small for age, and lower than their (normal) nocturnal diuresis volume. In most reports these patients are categorized as having monosymptomatic NE, because bladder volume is not considered. However, they undoubtedly have bladder dysfunction, if their volume is significantly too low. There are also children with enuresis with abnormal uroflowmetry and/or residual urine even in absence of daytime symptoms. All these patients can be considered as not having a normal bladder, and therefore do not fulfil the criteria for ‘true’ monosymptomatic NE.
Nocturnal diuresis may be influenced by several secondary defence mechanisms; children with small bladders tend to decrease their fluid intake during daytime to cope with their bladder dysfunction. Therefore, their total fluid intake is often so low that they have no nycthemeral rhythm of diuresis, but have to use their maximum concentration capacity during both day and night to achieve fluid homeostasis. The low fluid intake may also result in inappropriate low daytime osmotic excretion, and therefore higher renal osmotic load overnight, leading to a higher diuresis rate. Most of these children have a tendency to have their fluid intake after school hours; because of the high serum vasopressin level at that time, their urinary excretion will be postponed until the early night hours. These considerations make it difficult to define the presence or absence of nocturnal polyuria only from the initial screening data.
Thus classical subtyping, distinguishing monosymptomatic and other enuresis, is under discussion  and we therefore describe the characteristics of the patients as accurately as possible. The objective of the present study was to confirm or to refute possible linkage to known chromosomal markers on chromosome 8q, 12q, 13q and 22q of NE/incontinence in patients with evidence for underlying bladder dysfunction and/or polyuria. Furthermore, correlations between the linkage data and the different clinical phenotypes determined by the presence of nocturnal polyuria and the type of bladder dysfunction were sought.
Patients and methods
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- Patients and methods
Index patients were subtyped according to two main variables, i.e. the presence or absence of nocturnal polyuria, initially or subsequently (as defined below), and the type of bladder dysfunction. The inclusion criteria for the study were evidence for enuresis/incontinence based on these variables, respectively, in index patients aged 7 years, and aged 5 years for first- and second-degree family members. Patients with neurogenic bladder dysfunction and psychiatric problems were excluded. Only families with at least three affected first- and second-degree family members over two generations were included. The local ethical committee (project 98/80) approved this study, and all participants gave written informed consent.
The clinical diagnosis in the index patient was based on a careful history, including an extensive questionnaire about voiding problems and micturition habits. In addition, a micturition chart was completed over 14 days, collecting data on the voiding pattern and frequency, defecation pattern, NE, daytime incontinence and fluid intake.
The bladder volume was measured during forced diuresis; for 1 day the patient was asked to drink as much as possible and to restrain from voiding for as long as possible. The largest bladder volume was considered the maximum volume. The mean bladder volume was calculated as the mean of all representative volumes measured during 1 day . A 24-h urinary concentration profile was obtained at intake, to study the nycthemeral rhythm of diuresis and urinary osmolarity. Daytime and night-time were each divided in six similar periods for urine collection, and the urinary volume and osmolality measured for each period. Values were compared with control values from the same patient . Nocturnal polyuria was defined as a nocturnal diuresis higher than the normal bladder volume for age according to the Koff formula, with urinary osmolality < 800 mosmol/L in at least one overnight sample. If polyuria was obvious at the first screening, it was defined as initial polyuria. Nocturnal polyuria was re-evaluated later because an abnormal nycthemeral diuresis rhythm may be missed initially in children with underlying bladder dysfunction because of their secondary low fluid intake. During bladder volume training with a high fluid intake, this abnormal nycthemeral rhythm of diuresis can be disclosed. If polyuria is present after optimizing fluid intake, the polyuria is termed ‘subsequent’.
The uroflow was measured at least three times at a bladder volume of more than two-thirds of maximum bladder capacity. The interpretation of the curve included its shape and the presence or absence of residual urine. Ultrasonography of the kidneys was used to determine structural variables, and of the bladder to measure the thickness of the bladder wall and residual volume after micturition.
Video-urodynamics were undertaken if bladder dysfunction was suspected, based on the presence of diurnal incontinence, UTIs, a bladder volume too small for age, repeated abnormal uroflowmetry and residual urine. The type of bladder dysfunction was described from the characteristics of the filling phase (compliance and instability of detrusor) and/or the emptying phase (dysfunctional voiding, dyssynergia, organic abnormalities of the urethra). The methods and measurements of video-urodynamics conformed with ICCS standards . Patients were divided in four categories according to their urodynamic diagnosis, i.e. normal, urge syndrome, dysfunctional voiding and lazy bladder .
The family history was repeated twice independently to complete the pedigree information. After written informed consent was obtained, a questionnaire about micturition habits was completed and all participating family members were asked to register their maximum bladder volumes during one day and night of urinary production over two nights. Family members were considered affected if any of these variables was abnormal.
For the molecular studies, lymphocyte DNA was prepared from venous blood samples using an extraction kit (Qiagen Inc., USA). Microsatellites from the regions on chromosome 13q13–14 (D13S218, D13S1253, D13S263, D13S1297, D13S291, D13S155), 22q11 (D22S420, D22S425, D22S446, D22S257, D22S421, D22S689), 12q13–21 (D12S368, D12S90, D12S335, D12S43, D12S80, D12S82) and 8q (D8S1763, D8S1842) were amplified and subsequently the fragments analysed on an ABI-377 sequencer (Perkin-Elmer Biosystems, USA). After haplotyping all included individuals, two point lodscores were calculated with MLINK under an autosomal dominant model with a penetrance of 0.9 and a gene frequency of 0.05. Allele frequencies were calculated from the alleles present in the inter-married family members.
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The index patients in this study consisted of 48 children from 32 families (male : female ratio 2.2 : 1). One four-generation family, 25 three-generation and six two-generation families were included. In four families (nos 9, 63, 75, 78) the familial history showed that either the parents or their first-degree relatives were enuretic. In all, 325 persons from these 32 families were included, of whom 170 were considered to be affected. A mean (range) of 10 (4–24) individuals per family were assessed, from whom 5 (3–9) were affected. The mean age of the index patients was 11 years.
Complete clinical screening, as described, was undertaken in the 48 children from the 32 families. Except for the index patients of family 7 and 64, and index patient 48 : 2, there was evidence for bladder dysfunction, confirmed by video-urodynamics, in all index patients. In 11 families more than one index patient underwent video-urodynamics. The type of bladder dysfunction was concordant in only three of 11 families. Eleven children had polyuria at the initial screening and nine other children were confirmed to be polyuric after optimizing fluid intake. The clinical data from the 48 index children are summarized in Table 1.
Table 1. The clinical phenotype in 48 patients from 32 families
|Family no.||Generations||No./family||No. affected||Chromosome||Patient||Polyuria||Bladder dysfunction|
|54||3||8||4|| 0|| X|| 0||0||54–1 54–2 54–3||Yes Yes No||Dysfunctional voiding Urge syndrome Urge syndrome|
|39||3||15||9|| 0|| 0|| 0||0||39–1 39–2 39–3||No No No||Urge syndrome Urge syndrome Urge syndrome|
|78|| 3†||10||5|| 0|| 0|| 0||0||78–1||No||Dysfunctional voiding|
|80||3||6||4|| X|| 0|| 0||0||80–1 80–2||No No||Urge syndrome Urge syndrome|
|13||3||10||7|| X|| 0|| 0||0||13–1||No||Urge syndrome|
|89||3||18||7|| 0|| 0|| 0||0||89–1||Yes||Urge syndrome|
|92||3||15||7|| 0|| X|| 0||0||92–1||Yes*||Urge syndrome|
|28||3||12||8|| 0|| X|| X||0||28–1||No||Lazy bladder|
|48||2||4||3|| X|| X|| 0||0||48–1 48–2||No Yes||Urge syndrome Normal|
|23||3||15||6|| 0|| 0|| 0||0||23–1||No||Urge syndrome|
|53||3||21||6|| 0|| 0|| 0||0||53–1 53–2||No No||Dysfunctional voiding Urge syndrome|
| 4||4||11||6|| 0|| 0|| 0||0|| 4–1||No||Urge syndrome|
| 6||2||5||3|| X|| 0|| 0||0|| 6–1 6–2||Yes* Yes*||Dysfunctional voiding Urge syndrome|
| 3||3||11||8|| 0|| 0|| 0||X|| 3–1||No||Dysfunctional voiding|
|60||3||8||4|| X|| 0|| 0||0||60–1 60–2||No No||Urge syndrome Urge syndrome|
|51||2||20||9|| 0|| 0|| 0||0||51–1 51–2||Yes* Yes*||Dysfunctional voiding Urge syndrome|
|50||3||12||8|| 0|| 0|| 0||0||50–1||No||Urge syndrome|
| 7||2||5||3|| 0|| 0|| 0||0|| 7–1 7–2||Yes Yes||No examination No examination|
|64||3||9||3|| 0|| X|| X||0||64–1 64–2||Yes Yes||No examination No examination|
|46||3||8||3|| X|| 0|| X||0||46–1||No||Urge syndrome|
|74||3||8||4|| X|| 0|| 0||0||74–1||Yes||Dysfunctional voiding|
|32||3||10||5|| 0|| 0|| 0||0||32–1||No||Dysfunctional voiding|
|75|| 3†||8||5|| X|| 0|| 0||0||75–1||Yes||Urge syndrome|
|19||3||10||7|| 0|| 0|| 0||0||19–1||Yes*||Urge syndrome|
|47||3||7||3|| 0|| 0|| 0||0||47–1||No||Dysfunctional voiding|
|63|| 3†||8||4|| X|| 0|| 0||0||63–1||Yes*||Urge syndrome|
|88||3||7||4|| 0|| 0|| X||0||88–1||No||Dysfunctional voiding|
|18||3||10||6|| 0|| 0|| 0||0||18–1||Yes*||Urge syndrome|
|49||3||10||5|| 0|| 0|| 0||0||49–1||Yes*||Urge syndrome|
|55||2||15||8|| 0|| 0|| 0||0||55–1 55–2||No No||Dysfunctional voiding Urge syndrome|
| 9|| 3†||4||3|| 0|| X|| 0||X|| 9–1 9–2 9–3||No No No||Dysfunctional voiding Urge syndrome Urge syndrome|
|70||2||4||3|| 0|| 0|| 0||0||70–1 70–2||Yes Yes||Urge syndrome Urge syndrome|
In 15 of the 32 families there was a possible assignment of NE to at least one marker. In five families (nos 9, 28, 46, 48 and 64) possible linkage was found with more than one marker. In nine of 32 families (nos 6, 13, 46, 48, 60, 63, 74, 75 and 80) two-point lodscores were positive for marker D22S446. The cumulative lodscore was 3.63 (θ =0.1) for this marker, similar to the results obtained in previous studies [20,23]. None of the families was large enough to obtain a significant lodscore alone (Table 2), but the results confirm the possible involvement of a gene at 22q11 in NE.
Table 2. Two-point lodscores between markers and phenotype for θ=0, 0.1 and 0.2
|A B C Family no.||D13S218 D22S420 D12S368||D13S1253 D22S425 D12S90||D13S263 D22S446 D12S335||D13S1297 D22S257 D12S43||D13S291 D22S421 D12S80||D13S155 D22S689 D12S82|
|54|| −0.02||0.68||0.63|| −0.02||0.68||0.63|| −0.02||0.68||0.63|| −0.02||0.68||0.63|| −0.02||0.68||0.63|| −0.41||0.29||0.36|
|28||0||0||0||0.13||0.25||0.26||0.13||0.25||0.26|| −0.43|| −0.22|| −0.1||0.13||0.25||0.26||0.13||0.25||0.26|
|48||0||0||0||0.28||0.2||0.12||0.28||0.2||0.12||0.28||0.2||0.12||0.28||0.2||0.12|| −0.94|| −0.36|| −0.17|
|92||0.28||0.51||0.52|| −0.05||0.24||0.3|| −0.77||0.04||0.33|| −0.77||0.04||0.33|| −0.54|| −0.16||0.08|| −2.29|| −0.65|| −0.13|
|64||0||0||0|| −0.1|| −0.03||0||0.29||0.21||0.12||0.29||0.21||0.12||0||0||0|| −0.1|| −0.03||0|
|Zmax||0.54||1.39||1.27||0.51||1.53||1.43|| −0.09||1.38||1.46|| −0.38||1.10||1.22||0.12||1.16||1.21|| −3.34|| −0.31||0.44|
|80|| −0.19|| −0.12|| −0.06|| −1.05|| −0.24|| −0.09|| −1.21|| −0.26|| −0.1||0.84||0.63||0.41||0.84||0.63||0.42||0.36||0.32||0.24|
|13|| −1.92|| −0.42|| −0.07|| −0.52||0.48||0.5|| −0.52||0.48||0.5|| −0.52||0.05||0.26|| −0.8|| −0.19|| −0.07|| −1.35|| −0.67|| −0.32|
|46|| −0.21|| −0.09|| −0.04||0.26||0.18||0.11||0.94||0.69||0.44||0.29||0.21||0.12||0.82||0.6||0.38|| −1.09|| −0.03||0.07|
|74|| −1.12|| −0.23|| −0.07||0.55||0.38||0.22||0.85||0.63||0.4||0||0||0||0.85||0.63||0.4||0.85||0.63||0.4|
|75|| −2.62|| −0.82|| −0.45||0.45||0.31||0.19||0.95||0.73||0.5||0.42||0.29||0.18||0.52||0.45||0.33||1.24||0.94||0.63|
|63||0.42||0.43||0.32||0.43||0.3||0.18||0.33||0.35||0.26|| −0.41|| −0.06||0.01|| −0.71|| −0.33|| −0.15|| −2.67|| −0.61|| −0.29|
|Zmax|| −4.57|| −0.43||0.17||1.19||2.23||1.65||2.68||3.63||2.65||2.28||2.44||1.92||2.04||2.19||1.57|| −1.28||1.67||1.51|
|64|| −0.92|| −0.34|| −0.12||0.07||0.15||0.12||0.07||0.15||0.12||0.55||0.39||0.23||0.29||0.21||0.12||0.08||0.15||0.12|
|46||0.48||0.34||0.21||0.48||0.34||0.21||0.3||0.21||0.13|| −1.89|| −0.1||0.02||0.3||0.21||0.13||0.48||0.36||0.24|
In six families (nos 9, 28, 48, 54, 64 and 92) there was a weak indication for linkage with markers on chromosome 13q. The maximal cumulative lodscore was for marker D13S1253 (θ =0.1) was 1.53.
In four families (28, 46, 64, 88) the data suggested weak linkage with marker D12S80, resulting in a cumulative lodscore of 1.95 (θ =0). Finally in two families (3 and 9) linkage with a marker on chromosome 8q could not be excluded. The most informative families are shown in Fig. 1.
Figure 1. Pedigrees from family 54, 28 and 74 with haplotypes for, respectively, regions on chromosome 13q, 12q and 22q. The haplotype associated with enuresis/incontinence is indicated in red.
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From the nine families with suggestive linkage to chromosome 22q, five of 13 index patients presented with nocturnal polyuria, whereas two others became subsequently polyuric. Most (nine of 13) of the index patients from these families presented with urge syndrome. In four of six families with suggestive linkage to chromosome 13q polyuria was present in at least one child. The bladder function in the index patients from the 13q-linked families varied from normal, to urge syndrome and dysfunctional voiding to a lazy bladder. The clinical data in the families with possible linkage to 12q were highly variable.
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In previous genetic studies patient selection was biased by the use of (retrospective) questionnaires and the investigators only sought primary NE [17,19]. Except for the study by von Gontard et al., previous linkage studies of enuresis/incontinence did not distinguish between subtypes of enuresis. Von Gontard et al. distinguished monosymptomatic from ‘not monosymptomatic’ enuresis based on the presence of daytime incontinence. None of the present studies of the genetics of enuresis considered the presence of polyuria. The present genetic study attempted for the first time to determine correlations between chromosomal loci and the presence of bladder dysfunction and/or polyuria (initial and/or subsequent). Thus we assessed the index patients for both bladder dysfunction and/or polyuria as accurately as possible. Primary or secondary enuresis was not considered, as there seems sufficient evidence to accept that this is an arbitrary classification with no regard to the pathophysiology . Daytime incontinence and NE (with confirmed bladder dysfunction) might both result from the same cause. This is supported by the frequent association of both problems, easily detected from a meticulous family history.
The present results confirm a possible genetic linkage to three of the four loci already reported. Different studies have suggested linkage to these loci in patients with monosymptomatic NE. Most index patients in the present study did not fulfil this criterion because they had evidence of bladder dysfunction. In contrast to previous data, the clinical characteristics of the index patients are accurately described in the present study and the bladder characteristics of the other patients documented. In several families, putative linkage with more than one of the four chromosome regions was detected. This may be explained by a possible polygenic inheritance; the presence of two or more genes may cause the phenotype. It also excludes the presence of a monogenic cause for a subtype of enuresis, but confirms the role of genetic predisposition in enuresis. Despite the present study focusing on patients with evidence of bladder dysfunction, the results further support genetic and clinical heterogeneity, as described by von Gontard et al.. In contrast to those results, almost all patients in the present study underwent video-urodynamics to define bladder dysfunction. Despite this, there was no correlation between bladder dysfunction and different chromosomal loci. Linkage was associated with different types of enuresis and incontinence, with no specific trend. Both polyuria and bladder dysfunction-associated enuresis were linked to the same chromosomal loci. That families with urge syndrome and dysfunctional voiding were linked to the same chromosomal regions might indicate a common genetic background for these two subtypes.
The patients selected in the present study could arguably represent collection bias, as there were so many with bladder dysfunction. This might be a valid criticism, because the patients were recruited from a population referred to a university hospital and selected because they were difficult to treat by the referring paediatrician or urologist. However, as the present families with bladder dysfunction had linkages to the same chromosomal regions as had families reported previously (with so-called monosymptomatic enuresis) this might support the hypothesis that the latter entity is not real. Another explanation may be the presence of mild bladder dysfunction in patients with so-called monosymptomatic enuresis.
One of the limits of studying the genetics of enuresis is the definition of the enuresis/incontinence phenotype in adults. Often there is a stigma or emotional suffering associated with being or having been enuretic. Adult patients may have forgotten about their previous enuresis. Thus it is essential to repeat the clinical history and ask each adult about possible problems like urge, micturition frequency, night-time voiding, etc. A detailed history showed that family members who were not enuretic also had urge symptoms, repeated bladder infections, night-time awakening and high micturition frequency. Moreover there was a wide range in mean voided volumes, night-time urine production and maximal voided volume. The variation in genetic expression between persons might be considerable, which makes it difficult in some to clarify the carrier status. Hence the power of linkage studies is reduced by the presence of these phenocopies.