From the Department of Molecular Medicine Karolinska Institutet, Stockholm, Sweden
GENETIC STUDIES OF HYPOSPADIAS
Louise Frisén
Stockholm 2002 Supervisors Agneta Nordenskjöld, Associate Professor Department of Molecular Medicine Karolinska Institutet, Stockholm, Sweden
Holger Luthman, Professor Department of Endocrinology Lund University, University Hospital MAS, Malmö, Sweden
Opponent Juha Kere, Professor Department of Biosciences at Novum Karolinska Institutet Huddinge University Hospital, Huddinge, Sweden
Thesis committee Göran Annerén, Professor Department of Clinical Genetics Uppsala University Hospital, Uppsala, Sweden
Jan Hillert, Professor Department of Neuroscience Karolinska Institutet Huddinge University Hospital, Huddinge, Sweden
Olle Söder, Professor Department of Woman and Child Health Karolinska Institutet, Stockholm, Sweden
All previously published papers were reproduced with permission from the publisher.
Published and printed by Karolinska University Press Box 200, SE-171 77 Stockholm, Sweden
Genetic studies of hypospadias © Louise Frisén, 2002 ISBN 91-7349-397-X In most cases the cause of hypospadias is unknown. In our case the family believes that the condition originated by imprinting, when the mother of patient IV-12, while being pregnant, stared at a camel (camels apparently urinate backwards).
Frydman et al, American Journal of Medical Genetics, 1995
CONTENTS
ABSTRACT I LIST OF PUBLICATIONS II ABBREVIATIONS III INTRODUCTION 1 CHARACTERISTICS OF HYPOSPADIAS 2 MALE SEX DIFFERENTIATION 4 EPIDEMIOLOGY IN HYPOSPADIAS 5 GENETICS AND HYPOSPADIAS 8 COMPLEX TRAITS 11 GENE MAPPING 13 Linkage analysis 13 Parametric versus non-parametric linkage analysis 14 Affective relative pair method 14 Power estimations 16 Significance levels 17 Association mapping 18 Success so far and alternative strategies 19 AIMS OF THE STUDY 21 MATERIALS 22 ETHICAL PERMISSIONS 26 METHODS 27 RESULTS AND DISCUSSION 32 GENERAL DISCUSSION 44 SUMMARY IN SWEDISH 46 ACKNOWLEDGEMENTS 48 REFERENCES 51 PAPER I-V ABSTRACT
Hypospadias is defined as an abnormal opening of the urethra on the underside of the penis. It is a frequently found malformation with an incidence of 3 per 1000 males. The aim of this thesis was to identify genetic and environmental factors in the pathogenesis of hypospadias. For this purpose, a variety of genetic methods were used in a nation-wide material corresponding to half of all registered cases of hypospadias in Sweden.
We identified 18 monozygotic twins discordant for hypospadias. In 16 of these, the twin with lowest birth weight was affected with hypospadias. This shows that low birth weight is important for hypospadias, regardless of the genetic constitution (paper I).
We investigated the familial rate and analyzed the association with low birth weight in 2138 families with at least one boy with hypospadias. There was a familial rate of 7% and a significant lower birth weight in cases with hypospadias compared with their respective brothers, used as controls (p=5x10-13). An increased frequency of dizygotic as well as monozygotic male-male twins was found, with a skewed distribution towards monozygotic twins. This paper (II) also includes a description of the ethnic background in the material and the distribution of phenotypes.
A complex segregation analysis was performed to define the mode of inheritance in a material consisting of 2005 pedigrees. We found a best fit for the multifactorial model and a heritability of 0.99. This is interpreted as monogenic effects acting in some of the families but a multifactorial cause in the majority (paper III).
A genome-wide linkage analysis based on a non-parametric affected relative pair method was used in 69 families. All available family members were genotyped with 360 polymorphic PCR based microsatellite markers with a mean interval of 9.5 cM. Five chromosomal regions reaching the level of suggestive significance were identified (paper IV). These need to be investigated further to identify hypospadias susceptibility genes.
Linkage analysis and subsequent mutation analysis in a family with autosomal dominant inheritance of hypospadias revealed a novel mutation in the HOXA13 gene (paper V). This suggests the diagnosis of hand-foot-genital syndrome although the phenotype in this family is atypical compared with previously reported families.
In this thesis, several lines of evidence suggesting genetic factors in the pathogenesis of hypospadias are presented, including the identification of five chromosomal regions in which genes for hypospadias are likely to be located and a novel mutation in the HOXA13 gene. It is also shown that low birth weight is an important risk factor for hypospadias.
Key words: hypospadias, genetic, complex trait, low birth weight, twin study, segregation analysis, genome-wide linkage analysis, mutation analysis, HOXA13 gene
I LIST OF PUBLICATIONS
This thesis is based on the following papers, which will be referred to by their Roman numerals
I Louise Fredell, Paul Lichtenstein, Nancy L Pedersen, Jan Svensson and Agneta Nordenskjöld Hypospadias is related to birth weight in discordant monozygotic twins The Journal of Urology 160, 2197-2199, 1998
II Louise Fredell, Ingrid Kockum, Einar Hansson, Staffan Holmner, Lars Lundquist, Göran Läckgren, Jörgen Pedersen, Arne Stenberg, Gunnar Westbacke and Agneta Nordenskjöld Heredity of hypospadias and the significance of low birth weight The Journal of Urology 167, 1423-1427, 2002
III Louise Fredell, Lennart Iselius, Andy Collins, Einar Hansson, Staffan Holmner, Lars Lundquist, Göran Läckgren, Jörgen Pedersen, Arne Stenberg, Gunnar Westbacke and Agneta Nordenskjöld Complex segregation analysis of hypospadias Human Genetics 111, 231-234, 2002
IV Louise Frisén, Cilla Söderhäll, Margareta Tapper-Persson, Holger Luthman, Ingrid Kockum and Agneta Nordenskjöld Genome-wide linkage analysis for hypospadias susceptibility genes In manuscript
V Louise Frisén, Kristina Lagerstedt, Margareta Tapper-Persson, Ingrid Kockum and Agneta Nordenskjöld A novel duplication in the HOXA13 gene in a family with atypical hand-foot-genital syndrome Submitted
II ABBREVIATIONS
AMH anti Müllerian hormone AR the androgen receptor gene bp base pairs cM centiMorgan ddNTP dideoxynucleotide DNA deoxyribonucleic acid FSH follicle stimulating hormone hCG human chorionic gonadotropin HFGS hand-foot-genital syndrome HOX homeobox gene IBD identical by descent IBS identical by state kb kilobase pairs LH luteinizing hormone LOD logarithm of the odds Mb megabase pairs PCR polymerase chain reaction RFLP restriction fragment length polymorphisms SNP single nucleotide polymorphism SRD5A2 the 5-alpha-reductase gene SRY the sex-determining region Y gene
III INTRODUCTION
The term hypospadias is derived from the Greek words hypo (under) and spad (something torn, from span to tear, pluck off) meaning that the urethral orifice is located on the underside of the penis (Merriam-Webster 2002).
Hypospadias was first described in the second century A.D. by Galen (c. 130-201 A.D.). At this time, as well as during the following 1000 years, the treatment of choice was amputation beyond the urethral orifice (Adams 1844-1847). The importance of chordee (curvature of the penis) was also recognized by Galen as “men inflicted with hypospadias find it impossible to beget children, the meatus being turned away from the extremity of the penis by the frenum, not because they lack fertile sperm, but because the curvature of the penis prevents its normal overflow from being conveyed forwards. This theory is confirmed by the ability to beget children if the frenum is divided” (Galen c. 130-201 A.D.). In the 16th century this was illustrated in the successful correction of the chordee of King Henry II of France, after which he fathered ten children in his marriage to Catherine de Medici (Smith 1997).
The husband to a Maltese woman, Mathia, did not manage as well during this era. The marriage was annulled by the Roman Catholic Church on request from Mathia, who described her husband’s hypospadias as “a defect in the configuration of his virile member on account of which he did not urinate in a natural way like other men”. The medical examination took place in court and resulted in the following report: “John’s male member was inept or incapable and also useless for deflorating or perforating because it was short and curved, this curvation tending to become more pronounced with rigidity of the penis” (Cassar 1974).
1 CHARACTERISTICS OF HYPOSPADIAS
“Eddings had no injuries except several old scars, mostly on his knee. But biology had dealt him an earlier blow called hypospadias, which meant his urethra opened on the underside of his penis instead of in the center. This moderate defect would have caused him a great deal of anxiety, especially as a boy. As a man he may have suffered sufficient shame that he was reluctant to have sex.”
Patricia Cornwell, Cause of Death, 1996
The main features of hypospadias are the abnormal opening of the urethral orifice and different degrees of curvature of the penis (chordee) (figure 1).
Figure 1
Hypospadias is classified into glandular, penile, penoscrotal, scrotal or perineal according to the localization of the urethral orifice. A cleaved prepuce is considered a mild variant of hypospadias. Cryptorchidism, bifid scrotum and micropenis are occasionally associated with the condition, increasing in frequency with the severity of hypospadias. In the most severe cases, there may be ambiguity regarding the sex of the newborn child, warranting further investigations in order to determine this.
Hypospadias is considered to be restricted to males but females with hypospadias-like manifestations have been reported (Knight et al. 1995; Ronzoni et al. 2001).
2 During normal male sex differentiation, the urethral folds close in fetal weeks 8-16 (figure 2). Hypospadias results from a failure in this fusion process. The severity of hypospadias is a continuum, and depends on when during the embryonic period the fusion fails.
Figure 2 The urethral folds gradually fuse during fetal weeks 8-16.
The development of the penis and the urethra is an androgen dependent process, relying on proper timing as well as quantity of the required hormones. This suggests that hypospadias may be due to impairment in the androgen pathway. During the first trimester, fetal androgen production is induced by maternal gonadotropins, preferably human chorionic gonadotropin (hCG) produced in the placenta, whereas later in gestation, this is provided by gonadotropins (i.e. LH, FSH) produced in the fetal pituitary.
Surgical correction of hypospadias is recommended before the age of school start. Preoperative treatment with hCG or testosterone result in decreased chordee and increased penile length, thus facilitating the surgical treatment (Davits et al. 1993; Koff and Jayanthi 1999). Severe hypospadias can be detected with prenatal ultrasonography (Sides et al. 1996; Meizner et al. 2002). However, in most cases, there is no indication for routinely carrying out prenatal diagnosis for hypospadias, since it is a mild malformation in which surgical correction is almost always successful.
As a group, boys with hypospadias undergo normal psychological and sexual development and exhibit no divergent gender identity (Sandberg et al. 1995; Sandberg et al. 2001). The sexual debut may be delayed but is in that case related to the individuals´ genital perception (Bracka 1999). Depending on the severity of hypospadias and on the underlying cause, the fertility is sometimes reduced. In the majority of cases with mild variants and satisfactory surgical correction, there is no evidence for reduced fertility.
3 MALE SEX DIFFERENTIATION
At fertilization, the fusion of an egg carrying 22 autosomal chromosomes and an X chromosome with a sperm with 22 autosomal chromosomes and either an X or a Y chromosome determines the sex (chromosomal sex). An XX individual develops into a female and an XY individual into a male (phenotypic sex). Until fetal week 6, the embryo is sexually undifferentiated. Two bipotential gonads will ultimately differentiate into testes or ovaries (gonadal sex). At this point, there are also two undifferentiated bilateral duct systems, the Müllerian and the Wolffian ducts, generating internal female or male genital organ, respectively.
chromosomal sex (XX or XY)
gonadal sex (testis or ovary)
phenotypic sex (male or female)
While female sex differentiation is the default pathway, male sex differentiation relies on a series of crucial events. First, the SRY gene on the Y chromosome is solely responsible for initiating the male differentiation of the XY embryo in fetal week 6. The SRY gene encodes a transcription factor, which induces the differentiation of the indifferent gonads into testes. Shortly after the onset of SRY expression, cells in the developing testis differentiate into Sertoli cells, producing anti Müllerian hormone (AMH) and Leydig cells, producing testosterone. AMH induces the regression of the Müllerian ducts whereas testosterone induces the differentiation of Wolffian ducts into the epididymis, vas deferens and seminal vesicles. In target tissues, testosterone is converted intracellularly into dihydrotestosterone through the 5-alpha-reductase enzyme. Dihydrotestosterone modulates differentiation of the prostate gland, penis and scrotum. Testosterone as well as dihydrotestosterone binds to its intracellular receptor (the androgen receptor). Ultimately this hormone-receptor complex binds to DNA to regulate transcription.
In fetal week 8, male external genitalia start forming. As the phallus elongates it pulls the urethral folds forward so that they form the lateral walls of the urethral groove. The epithelial lining of the groove is of endodermal origin and forms the urethral plate. In fetal week 12, the two urethral folds close over the urethral plate, thus forming the penile urethra. Later, the most distal part of the urethra is formed when ectodermal cells from the tip of glans penetrate inward, forming a short epithelial cord. The formation of external genitalia, including closure of the prepuce, is completed in fetal week 16 (Sadler 1990).
4 EPIDEMIOLOGY IN HYPOSPADIAS
Hypospadias is one of the most prevalent malformations in man and by far the most common urogenital malformation. There are large geographical differences in reported hypospadias rates, ranging from 0.3 per 1000 male births (Japan) to 7 per 1000 (The Netherlands) (Paulozzi 1999; Virtanen et al. 2001; Pierik et al. 2002). However, it is difficult to make comparisons between countries due to various inclusion criteria and incomplete ascertainment. There are about 5000 cases with hypospadias in the Swedish Malformation Registry. The incidence in Sweden has remained at a stable rate of 3 per 1000 male births since the beginning of the 1970s, when the registered incidence increased from 0.8 per 1000 infants in 1969 to 1.5 per 1000 infants in 1973 (Källén and Winberg 1982). The reason for this has not been explained. An overview of epidemiological studies of hypospadias is shown in table 1.
In some of these, an association for hypospadias with low birth weight is found (Chen and Woolley 1971; Monteleone Neto et al. 1981; Calzolari et al. 1986). Low weight at birth reflects a growth retardation throughout the gestation and has been shown to be correlated to sub-optimal first-trimester growth (Smith et al. 1998). Up to a 10-fold increase of hypospadias was found in infants small for gestational age (Akre et al. 1999; Weidner et al. 1999; Gatti et al. 2001; Hussain et al. 2002). The poor intrauterine growth was shown to be of early gestational cause (Hussain et al. 2002). Interestingly, the degree of growth retardation is not correlated to the severity of hypospadias, indicating that this is determined by genetic factors (Calzolari et al. 1986; Gatti et al. 2001; Hussain et al. 2002).
It is unclear whether the growth retardation in itself renders the fetus more susceptible for other predisposing factors (genetic or environmental), or if there is a common denominator in the genesis of the two conditions. A placentary malfunction is suggested by the association with a low weight of the placenta (Stoll et al. 1990) and severe preeclampsia (Akre et al. 1999). Abnormalities of the fetal-placental-maternal interaction may also explain the finding that women giving birth to boys with hypospadias had a higher rate of weak contractions during birth, induced deliveries and caesarean sections (Källén 1988).
5 Table 1 Epidemiological studies of hypospadias
Author Incidence Data Numbers Familial rate % of sibs Recurrence Heritability Twin rate Birth Phenotype (male births) source included affected risk for sib weight Sørensen 1953 3.3/1000 Hospital 103 28%** 9.6% (monozygotic:dizygotic 4.5:1) 75% glandular 12.5% penile 12.5% severe
Chen&Wolley 1971 Hospital 50 9.7% 10.4% 0.74 10% (=5 dizygotic twins) low*** 40% glandular 46% penile 14% severe
Roberts&Lloyd 1973 1.98/1000 Registry 93 6.5% (monozygotic:dizygotic 16:1) n.s. 85% glandular 14% penile 1% severe
Sweet 1974 8.2/1000 Hospital 113 8% of FDR 87% glandular 10% penile 3% severe
Czeizel 1979 4.4/1000 Hospital 294 4% of FDR** 0.65 2% (monozygotic:dizygotic 2:1) low
Bauer 1981 Hospital 307 21%** 14% 12% 32% glandular 56% penile 12% severe
Monteleone 1981 0.76/1000* Registry 324 6.1% 0.68 3.4% (male-male dominance) low*** 72% glandular 18.5% proximal 9.5% unknown
Calzolari 1986 Registry 168 9.1% 0.67 5.4% low*** 75% glandular 21.4% penile 3.6% severe
Källén 1986 1.53/1000* Registry 262 4.2% (male-male dominance) low 75% glandular 12.5% penile 12.5% severe
Stoll 1990 2.89/1000 Registry 176 17% 0.57 low 69% glandular 25% penile 6% severe *per live births **the more severe the malformation, the higher the recurrence risk in relatives ***p<0.001
6 An increased risk for hypospadias following in vitro fertilization and intracytoplasmic sperm injection has been found (Macnab and Zouves 1991; Silver et al. 1999b; Wennerholm et al. 2000; Ericson and Källén 2001). This may be explained by the administration of high doses of gestagens interfering with androgen production in the male fetus (Silver et al. 1999a). Furthermore, children born after in vitro fertilization have lower birth weight (Bergh et al. 1999), an established risk factor for hypospadias. Older mothers run a higher risk of giving birth to a boy with hypospadias but it is unclear if this is a direct consequence of the mothers age or of other factors, such as a higher frequency of in vitro fertilization in older women (Fisch et al. 2001). The increased frequency of hypospadias after intracytoplasmic sperm injection may be related to paternal subfertility (Wennerholm et al. 2000).
Hypospadias is not associated with maternal use of oral contraceptives in early pregnancy (Källén et al. 1991). An increased frequency of hypospadias in boys born to 2780 Swedish women with intake of loratadine (a non-prescription antihistamine) during pregnancy was recently reported in the daily press. However, the number of cases was small (15 observed boys with hypospadias to be compared with the expected 5-6) and a biological mechanism for this remains to be explained (Hellbom 2002).
The increasing incidences of hypospadias reported in some countries have raised speculations on the involvement of environmental endocrine disrupters, such as estrogenic and anti-androgenic chemicals, in the pathogenesis of hypospadias (Toppari et al. 1996; Paulozzi et al. 1997). Possibly relating to this is the finding that mothers with a vegetarian diet during pregnancy have a five-fold increased risk of giving birth to a boy with hypospadias (North and Golding 2000). This has been suggested to result from increased levels of phytoestrogens acting anti-androgenic in the developing male fetus. However, studies of boys born to women in gardening occupations, exposed to pesticides with presumed estrogenic or anti-androgenic properties, showed no increased frequency of hypospadias (Weidner et al. 1998).
7 GENETICS AND HYPOSPADIAS
Several observations in hypospadias suggest that genetic factors are involved in the pathogenesis. Familial clustering has been reported in 4% to 28% of cases (table 1). The more severe the malformation of the index patient, the higher is the recurrence risk for the next male sibling, ranging between 4% and 17% (table 1).
In some ethnic groups in which consanguinity is a common feature, hypospadias has a particularly high incidence (Frydman et al. 1985; Tsur et al. 1987). In these families recessive inheritance can be suspected. An autosomal dominant mode of inheritance has also been observed (Lowry and Kliman 1976; Page 1979).
Furthermore, hypospadias is a feature in more than one hundred genetically caused syndromes (McKusick 2002). In some of these, mutations have been identified in genes involved in sex differentiation, e.g. the X-linked partial androgen insensitivity syndrome caused by mutations in the androgen receptor gene (AR, Xq11-12) and the recessive 5- alpha-reductase deficiency due to mutations in the 5-alpha-reductase gene (SRD5A2, 2p23) (Imperato-McGinley et al. 1974; Wilson et al. 1993; Quigley et al. 1995). However, these syndromes are characterized by severe hypospadias in association with other genital malformations such as cryptorchidism, bifid scrotum and penoscrotal transposition. Infrequently, mutations in the androgen receptor gene (Hiort et al. 1994; Allera et al. 1995) and the 5-alpha-reductase gene (Silver and Russell 1999) have been identified in isolated hypospadias.
Although there is without doubt a familial aggregation in hypospadias that in some cases are caused by a mutation in a single gene, susceptibility genes in common forms of the malformation have not yet been identified. Before undertaking an investigation for disease-causing genes, it is important to estimate the contribution of genetic effects.
An overview of various strategies in identifying the genetic component of a disease is presented in figure 3.
8 Figure 3 Strategies in the identification of disease-causing genes
Definition of phenotype
Identification of genetic component
Relative risk Segregation analysis Pedigree analysis Twin study
Identification of mode of inheritance
Complex Monogenic
Non-parametric Parametric linkage analysis linkage analysis in many families, Mutation analysis in large families, few affected/family in candidate gene many affected
Association studies
9 The relative risk (λ) is used to estimate the contribution of genetic factors by dividing the λ λ recurrence risk in family members ( r), or in siblings ( s), with the risk in the general population (Risch 1990a). Because λ is a ratio, the prevalence in relatives as well as in the general population affects its size. Thus, a strong familial aggregation in a common disease results in a smaller λ than the same degree of aggregation in a rare disease. In general, λ>2 indicate a significant genetic component and λ>40 suggests a strong genetic component (Kruglyak and Lander 1995b). With an incidence of 0.3% and a familial λ recurrence risk of about 10%, the r for hypospadias is 33.
Segregation analysis is used to evaluate whether a major gene contributes to the phenotype, and if that is the case, to determine the mode of inheritance. Ideally, the segregation analysis results in correct estimation of the genetic model, the penetrance of the disease allele and its frequency. These parameters can then be used in a linkage analysis. A successful example of this is the segregation analysis in breast cancer (Iselius et al. 1991), eventually resulting in the identification of the BRCA1 and BRCA2 genes (Miki et al. 1994; Wooster et al. 1995). If no major genes are involved and the data instead suggest a polygenic model, the segregation analysis is useful to quantify the degree of genetic contribution, i.e. heritability (Khoury et al. 1993).
Heritability is defined as the proportion of the phenotypic variance due to the additive effects of many genes (the polygenic component) (Haines and Pericak-Vance 1998). Heritability for hypospadias has been estimated to be between 0.57 and 0.74 using simple multifactorial threshold models (Chen and Woolley 1971; Czeizel et al. 1979; Monteleone Neto et al. 1981; Calzolari et al. 1986; Stoll et al. 1990). In a complex segregation analysis of 103 families, the heritability was 0.99 (Harris and Beaty 1993).
One per 80 pregnancies results in twins. One third of twins are monozygotic and two thirds are dizygotic. Studies in twins are valuable in order to disentangle genetic and environmental factors. This is based on the fact that monozygotic twins are genetically identical whereas dizygotic twins, like siblings, share on average half of their genetic material. Higher concordance rates (i.e. both twins affected) in monozygotic twins than in dizygotic twins suggest that genetic factors are important in the pathogenesis of a disease.
Several studies have reported an increased frequency of hypospadias in twins, with a skewed distribution towards monozygotic twins (Sørensen 1953; Roberts and Lloyd 1973; Czeizel et al. 1979). The increase in frequency is related to the sex distribution in the twins, with a higher incidence of hypospadias in male-male twins (regardless of
10 zygosity), but lower among males in male-female twins (Källén et al. 1986; Ramos- Arroyo 1991).
Twins that are monozygotic and discordant constitute a powerful model for estimating the role of environmental factors. Monozygotic twins discordant for hypospadias were first described by Lamy (Lamy 1952). Sørensen identified five twin pairs discordant for hypospadias, in which the twin with lowest birth weight was affected. By outward resemblance and blood groups, zygosity could be established in four of these. Three of the twin couples were monozygotic and one was dizygotic (Sørensen 1953).
Although several lines of evidence suggest that genetic factors are involved in the pathogenesis of hypospadias, environmental factors are also important. Hypospadias is therefore considered a complex trait caused by the combined influence of genes and environment.
COMPLEX TRAITS
Monogenic diseases are consequences of mutations in a single gene, transmitted according to Mendelian models of inheritance (e.g. autosomal dominant, autosomal recessive or X-linked). In contrast, complex traits do not exhibit Mendelian inheritance attributable to a single gene locus but is rather believed to result from interactions of several genes (i.e. susceptibility genes). The more genes involved, the smaller the contribution of each of them, making them difficult to detect (Konig et al. 2001). Also, individuals with the same phenotype may have different genetic background (genetic heterogeneity) and susceptibility genes are believed to interact at a genetic level (epistasis) as well as with environmental factors. In some individuals, the phenotype results from environmental factors only (phenocopies), whereas others are unaffected despite carrying the susceptibility allele(s) (reduced penetrance).
Thus, many different genes acting together and in various combinations, with or without environmental factors, comprise the liability, or predisposition, to a complex trait. Although the phenotype is qualitative (i.e. affected or unaffected), liability to the disease is measured on a quantitative scale (figure 4). The proportion of affected relatives will be highest among severely affected persons, since their liability is further beyond the threshold than that for mildly affected persons. In line with this, the risk is higher for closely related family members and increases with the number of cases in the family.
11 Figure 4 A multifactorial threshold model describing the situation in which a genetically predisposed individual is affected when exceeding a threshold of genetic and/or environmental factors (Falconer 1965). The lower right-shifted curve illustrates an increased liability compared to the top curve.
12 GENE MAPPING
In about 1500 monogenic diseases, the mutated gene has been identified (Peltonen and McKusick 2001). For complex traits, the situation is completely different and few susceptibility genes have been found so far. Figure 3 illustrates the various strategies that can be used to identify the gene(s) involved in a disease. The candidate gene approach can be used directly in monogenic as well as complex traits. However, in some cases the prior identification of chromosomal regions is required. For this purpose, linkage analysis is used.
Linkage analysis The term linkage refers to the tendency for two loci on the same chromosome to be inherited together. During meiosis the two homologous chromosomes exchange genetic material, a process known as recombination. This occurs at least once per chromosome arm in each pair of homologous chromosomes. Loci close to each other are separated by recombination less often than loci far apart. A probability of 1% for recombination between two loci corresponds to a genetic distance of 1 cM. This is roughly equal to a physical distance of 1 Mb. The probability of recombination to occur is called the recombination fraction (θ). Recombination enables the construction of genetic maps with the use of genetic markers. Genetic markers can be any part of the DNA reflecting normal sequence variations. Genetic maps based on direct measurements of DNA sequence variation was first constructed using restriction fragment length polymorphisms (RFLP) (Botstein et al. 1980). The basis for the polymorphism in a RFLP marker is a single base pair change that introduces or abolishes a cleavage site for restriction enzymes. These variabilities are now known as single nucleotide polymorphisms (SNP) scattered throughout the genome (1 per 1250 base pair) and potentially useful for genome-wide association mapping (Lander et al. 2001; Venter et al. 2001).
A breakthrough for the mapping of genetic diseases was achieved in the 1990s by the accessibility of microsatellite markers (Sheffield et al. 1995). These repetitive segments of DNA (e.g. di, tri or tetranucleotiderepeats) have a high variability between individuals (i.e. heterozygosity), which makes them useful in linkage analysis. The microsatellite markers are spread all over the genome, approximately one every 2 kb (Lander et al. 2001) and are flanked by unique sequences that enable amplification with Polymerase Chain Reaction (PCR). This permits a high-throughput methodology, which is essential since many microsatellite markers are used in a genome-wide linkage analysis. A minimum of 300 microsatellite markers giving a mean distance of 10 cM is required to cover the whole genome, but more markers generally increase the power.
13 In linkage analysis, genetic markers are used to trace the gene(s) involved in a disease. A genetic marker close to the disease locus will be inherited together with the disease more often than expected by random segregation (i.e. it is linked). The probability of linkage is given as the logarithm of the odds (LOD). This is derived from the log10 of the ratio between the two likelihoods under the null and the alternative hypothesis. The null hypothesis is that the two loci are not linked and the alternative hypothesis that the two loci are linked (Morton 1955). A LOD score of 3 means that the odds that the loci are linked are 1000 times greater than the odds that they are not linked. This corresponds to a significance level of 5%, taking into consideration the inherent probability of linkage (≈1/50) (Ott 1991).
Linkage analysis can be performed in a single-point or a multipoint fashion. In single- point analysis, LOD score calculations are performed for each marker in relation to the disease locus, independent of the surrounding markers. Multipoint analysis refers to the simultaneous analysis of several markers with known location on a genetic map, which increases information in the region.
Parametric versus non-parametric linkage analysis Linkage analysis is used to identify loci in monogenic as well as in complex traits. In monogenic diseases, parametric linkage analysis is the method of choice. This involves the assumption of certain parameters such as the mode of inheritance, the penetrance of the disease-causing allele and its frequency in the population. The chance of success is dependent on the recruitment of families large enough to give significant evidence for linkage. However, if that is impossible, LOD scores can be added between different families with the same phenotype.
Although the power of parametric linkage analysis greatly exceeds that of non-parametric linkage analysis, the latter method is preferred in the mapping of susceptibility genes in common, genetically complex, traits. This is due to the fact that no assumptions about the essential parameters can be reliably made. Instead, a non-parametric linkage analysis including many affected relative pairs is preferred (Lander and Schork 1994).
Affective relative pair method The simplest form of an affected relative pair method is the affected sib pair analysis first proposed by Penrose in 1935 (Penrose 1935). This is based on the assumption that two affected siblings share the chromosomal segment carrying the disease locus more often than expected by chance alone. Since they will show excess allele sharing even in the presence of incomplete penetrance, phenocopies, genetic heterogeneity and high-
14 frequency disease alleles, this type of linkage analysis is more robust than classical parametric linkage analysis (Lander and Schork 1994). It is, however, less powerful than a correctly specified linkage model.
With the use of genetic markers spread all over the genome, shared regions can be identified (Kruglyak and Lander 1995a). The method relies on calculations based on deviations from allele sharing expected by Mendelian laws of inheritance (figure 5). The null hypothesis is that the sharing is consistent with the expected distribution by Mendelian laws of inheritance and the alternative hypothesis that there is excess sharing of alleles identical by descent (IBD). The affected relative pair LOD score is calculated by the log10 of the ratio between the two likelihoods under the null and the alternative hypothesis. It should be noted that whereas in parametric linkage analysis the alternative hypothesis is that two loci are linked, in the affected relative pair method, the alternative hypothesis is excess sharing of alleles IBD.
Figure 5 According to Mendelian laws of inheritance, the probabilities of a sib pair sharing one marker allele identical by descent (IBD) is 50%, two alleles IBD is 25% and no allele IBD is 25%.
1, 2 3, 4
1, 3 1, 3 25%
1, 4 50% 2, 3
2, 4 25%
Although siblings appear to have inherited identical alleles for a marker, they might just be identical by state (IBS). Parents as well as unaffected siblings can therefore be included in the genotyping to enable the distinction between alleles that are IBD and IBS (Haines and Pericak-Vance 1998). The proportion of genes shared IBD is shown in table 2.
15 Table 2 shows the degree of relationship and corresponding proportion of IBD sharing. It should be emphasized that although sibling pairs and parent-child pairs each share 50% of their genetic material, the type of sharing is different. Sibling pairs share on average 50% of their genetic material, of which some is derived from the mother and some from the father, whereas a parent transmits exactly 50% of his or her genetic material to each offspring. For this reason, parent-child pairs are not informative in an affected relative pair method. Parents are included only to enable the distinction between alleles that are IBD and IBS.
Relationship Degree of Proportion of relationship IBD shared Monozygotic twins 0 1 Dizygotic twins 1 1/2 Sibs 1 1/2 Parent-child 1 1/2 Grandparent-grandchild 2 1/4 Avuncular 2 1/4 Half-sibs 2 1/4 Cousins 3 1/8 Great grandparent-great grandchild 3 1/8 Great avuncular 3 1/8
Power estimations Power is the probability of correctly concluding that linkage exists. Since the underlying genetic model for a complex trait is unknown, as well as the number of susceptibility loci and their λ, it is difficult to accurately predict the power of a non-parametric linkage analysis. It has been estimated that, for a locus with λ 3.0, a 10 cM interval flanked with markers with heterozygosity value of 0.75 and a sample size of 100 affected sibling pairs can generate a LOD score of 3.0 with a power of 0.45. As the number of affected sibling pairs or the λ increases, so does the power. In this example, increasing the sample size to 200 affected sibling pairs results in an increase of power to 0.90 (Hauser et al. 1996). As a rule, any sample of fewer than 40 sib pairs is unlikely to detect even relatively strong genetic effects (Haines and Pericak-Vance 1998).
16 Significance levels The LOD scores achieved from a linkage analysis including many markers must be corrected for multiple testing. This results in different significance levels that must be kept apart. Point-wise significance level is the probability of randomly exceeding the observed value at one specific locus, whereas genome-wide significance level is the probability of randomly exceeding the observed value anywhere in the genome (Lander and Kruglyak 1995). Lander and Kruglyak suggested the following definitions of genome-wide significance levels: Suggestive linkage is defined as statistical evidence expected to randomly occur once per genome-wide linkage analysis. Significant linkage is defined as statistical evidence expected to randomly occur 0.05 times in a genome-wide linkage analysis. Highly significant linkage is defined as statistical evidence expected to randomly occur 0.001 times in a genome-wide linkage analysis.
Many different computer programs, based on different statistical algorithms, are available for non-parametric linkage analysis but LOD scores are not easily comparable between those. It has therefore been suggested that, besides the LOD scores, the appropriate significance thresholds and corresponding p-value should be reported (Nyholt 2000). These recommendations are summarized in table 3.
Table 3 Significance levels and corresponding p-values as suggested by Lander and Kruglyak (1995). LOD scores according to Nyholt (2000) are shown for Allegro (Gudbjartsson et al. 2000) and MAPMAKER/SIBS (MMS, for the X chromosome) (Kruglyak and Lander 1995a) since these programs are used in this thesis. The corresponding p-values denotes allele-sharing methods in sibs or half-sibs. For studies involving a mixture of relative pairs, the thresholds are roughly in the range of 10-3-5x10-4 for suggestive linkage and 5x10-5-10-5 for significant linkage (Lander and Kruglyak 1995).
Significance level Corresponding p-value LOD scores LOD scores (sibs or half-sibs) (Allegro) (MMS) p<0.05 0.59 1.18 p<0.01 1.18 1.90 p<0.005 1.44 2.21 p<0.001 2.07 2.93 Suggestive linkage p<7.4x10-4 2.19 3.06 Significant linkage p<2.2x10-5 3.63 4.62 Highly significant linkage p<3x10-7 5.30 6.52
17 Association mapping The regions that can be identified in a genome-wide linkage analysis of a complex trait are relatively large, often spanning more than 20 cM. A chromosomal region of that size harbors about 200-300 genes. In most cases, the identification of a disease-causing allele will require that the region is restricted using more markers and association mapping (Todd 2001).
Whereas linkage analysis relies on the identification of markers shared between affected individuals due to their proximity to the disease-causing gene, association implies that the specific allele is identical in apparently unrelated affected individuals. The associated allele may be related to the pathogenesis in itself, or it may be in linkage disequilibrium with the disease-causing allele. Linkage disequilibrium refers to the fact that certain alleles in adjacent loci are co-inherited more often than expected by random segregation. This reflects a preserved chromosomal segment through generations and is exploited in association mapping. Association can be found using case-control studies, in which allele frequencies are compared between affected individuals and matched controls, or within families (tests of transmission distortion). In the latter case, problems with selection bias are overcome. Association mapping has been essential in the few examples of successful identification of susceptibility genes for complex traits.
It has been proposed that the identification of SNPs would facilitate association mapping. Although less than one per cent of SNPs have an impact on protein function (Venter et al. 2001) they may be valuable tools in the detection of linkage disequilibrium. However, there are several limitations in the use of SNPs. Since they are biallelic, their informativity is greatly reduced compared to microsatellite markers. This implies the testing of many markers, thus reducing the possibility to reach statistical significance, as corrections for multiple testing must be made. Due to the magnitude of markers needed, high-throughput methodology is required and this is now increasingly being made available to the research community.
18 Success so far and alternative strategies Despite the great expectations cherished in the mid 1990s, there has been but a few identified genes in human complex traits so far. These include the identification of an association of the CAPN10 gene with type 2 diabetes (Horikawa et al. 2000), the NOD2 gene with Crohn´s disease (Hugot et al. 2001; Ogura et al. 2001), the ADAM33 gene with asthma (Van Eerdewegh et al. 2002) and the PCDC1 gene with systemic lupus erythematosus (Prokunina et al. 2002).
Most of these findings have relied on the collection of large materials. In type 2 diabetes, the association with the CAPN10 gene was initially detected in 170 families with Mexican-American origin (Horikawa et al. 2000), but has since then been replicated in other populations as well (Cassell et al. 2002; Malecki et al. 2002). The association of asthma with the ADAM33 gene was found using 460 affected sib-pairs (Van Eerdewegh et al. 2002). The NOD2 gene in Crohn´s disease was identified using 235 and 416 families, respectively (Hugot et al. 2001; Ogura et al. 2001). However, only 78 families were included in the initial linkage analysis identifying the susceptibility locus for Crohn´s disease at chromosome 16 (Hugot et al. 1996). In systemic lupus erythematosus, the association with the PCDC1 gene was detected using 2510 affected individuals, but in the initial linkage analysis no more than 19 multiplex families were included (Lindqvist et al. 2000).
In short segment Hirschsprung disease, three susceptibility loci were recently identified (Gabriel et al. 2002). By estimating each locus-specific risk ratio (λ), the combined effect of the three loci were shown to be both necessary and sufficient for the disease aggregation. Although it was concluded that the susceptibility gene at one of the loci is the RET gene (known to be mutated in long segment Hirschsprung disease), RET mutations could only be identified in 40% of linked families. This suggests that non- coding mutations or mutations in regulatory regions in the RET gene contribute to short segment Hirschsprung disease, illustrating yet another obstacle in the dissection of complex traits.
In Hirschsprung disease, using subphenotypes of the disease turned out to be a valuable strategy. The susceptibility locus for systemic lupus erythematosus at chromosome 2q37 was initially identified using 19 multiplex families in a parametric linkage analysis. Other possible approaches in order to restrict the phenotype are the use of severe cases or cases with early age of onset of disease (Lander and Schork 1994).
19 Genetically homogenous populations, such as the Finnish or the Icelandic populations, may be advantageous for several reasons. It is assumed that the number of segregating susceptibility alleles is reduced compared to a heterogeneous population and that these as a consequence bring about a stronger genetic effect. Also, there is an increased likelihood for linkage disequilibrium in a homogenous population. In particular the Finnish population has the advantage of relatively recent bottlenecks. Although it is without doubt that this facilitates the mapping of monogenic traits by linkage disequilibrium techniques, it has been questioned whether it holds true for complex traits (Kruglyak 1999; Eaves et al. 2000; Johnson and Todd 2000; Altmuller et al. 2001). Nevertheless, evidences for linkage in the Finnish population have been found in for example asthma, multiple sclerosis, schizophrenia and autism-spectrum disorders (Ekelund et al. 2001; Laitinen et al. 2001; Paunio et al. 2001; Auranen et al. 2002; Saarela et al. 2002).
The Icelandic population, although a small and homogenous population, has not undergone recent bottlenecks (Edwards 1999). However, gene mapping in Iceland is carried out in an industrialized fashion using extensive genealogical and medical records and this strategy may be beneficial in the mapping of susceptibility genes (Kong et al. 1999). Evidences for linkage in the Icelandic population have been reported for example in asthma, hypertension, preeclampsia, schizophrenia and stroke (Arngrimsson et al. 1999; Gretarsdottir et al. 2002; Hakonarson et al. 2002; Kristjansson et al. 2002; Stefansson et al. 2002).
In spite of intense efforts in these apparently suitable populations, they have not yet resulted in the identification of susceptibility genes. Although the finding of susceptibility genes in a particular population may not generally be extrapolated to other populations, it would nevertheless give valuable insights in patophysiological mechanisms.
20 AIMS
The aim of this thesis was to identify genetic and environmental factors in the pathogenesis of hypospadias. The specific aims of the individual projects were to:
1. Establish the impact of low birth weight in hypospadias (paper I-II) 2. Estimate the role of genetic factors in hypospadias (paper II-III) 3. Localize susceptibility genes for hypospadias (paper IV) 4. Identify the gene responsible for dominant inherited hypospadias in a large family (paper V)
21 MATERIALS
Questionnaires were mailed to 2503 boys admitted for surgical correction of hypospadias at the departments of pediatric surgery in Stockholm, Uppsala, Lund, Gothenburg and the departments of urology and plastic surgery in Umeå and Örebro during the years 1970-1997. A final reply rate of 88% was attained. We asked for additional family members with hypospadias, the number of brothers and birth weight of the boys with hypospadias and their brothers. Patients in whom hypospadias was suspected to be part of a syndrome were excluded (e.g. identifiable syndromes, multiple malformations). All patients that reported relatives with hypospadias were contacted by telephone in order to obtain full information regarding the pedigree. In the present study, familial cases are defined as patients with one or more first, second, or third-degree relatives with hypospadias (table 2). A flow chart overview of the collection of families included in papers II-IV is shown in figure 6.
Overview of included material In paper I, we included 28 twin pairs discordant for hypospadias. They were identified through the questionnaires and through the Department of Pediatric Surgery at Astrid Lindgren Children Hospital, Karolinska Hospital. In paper II, we describe 2138 families with at least one boy with hypospadias ascertained through the departments in Sweden where boys with hypospadias undergo surgery. In eight families, there were four or more affected (pedigrees in figure 7). In paper III, 2005 families with complete information of the pedigree were included, ascertained through the departments in Sweden where boys with hypospadias undergo surgery. In paper IV, 69 families with at least two affected members were included after telephone interviews and blood sampling. They were identified through the questionnaires and through records from the Swedish Malformation Registry. In 58 of the included families both parents were born in Sweden, whereas 11 families originated from Middle Eastern countries (Turkey, Syria, Lebanon, Iran or Iraq). In seven families, there were three affected family members. In paper V, one family with autosomal dominant inheritance of hypospadias was ascertained through the Department of Pediatric Surgery at Astrid Lindgren Children Hospital, Karolinska Hospital (pedigree in figure 8).
22 Figure 6 Flow chart showing the collection of families included in paper II-IV
2503 hypospadias cases in Sweden were mailed a questionnaire regarding additional family members with hypospadias and birth weight
73 refused participation, 2211 (88%) responded stated wrong diagnosis or adoption
2138 included in the analysis 2005 with complete information of heredity, birth weight and on pedigree included in the ethnic origin (paper II) complex segregation analysis (paper III)
144 (7%) familial cases 98 familial cases not included in the genome-wide linkage analysis
DNA from 23 complete families DNA from 46 complete families ascertained through the Swedish Malformation Registry
69 families included in the genome-wide linkage analysis (paper IV)
23 Figure 7 Eight families with four or more affected with hypospadias (black squares) family 40
family 108 family 21
2
family 115
family 45
family 116
family 56
family 124 Figure 8 Pedigree for a family with autosomal dominant inheritance of hypospadias, clinodactyly and feet abnormalities. Phenotypic features as indicated: black shading of the left half: hypospadias in males, urogenital abnormalitites in females; stripes in right half: hand and/or foot abnormalities.
25 ETHICAL PERMISSIONS
The Ethics Committee at the Karolinska Hospital as well as the local Ethics Committees at each included department approved ethical permissions for this project. We obtained permission from the Swedish Data Inspection Board regarding the computer-based filing of the material. This information was publicly spread by advertisements in a national newspaper (Dagens Nyheter) according to the guidelines from the Swedish Data Inspection Board.
26 METHODS
Paper I, IV and V DNA isolation Genomic DNA was extracted from peripheral venous blood, skin or nails using standard protocols.
Paper I Twin zygosity Zygosity of twins can be established by outward resemblance, histopathological evaluation of the placenta and fetal membranes or DNA analysis. An evaluation based on outward resemblance, such as parents describing the twins as “alike as berries”, is considered to correspond to a significance level of 95% (Nancy Pedersen, the Swedish Twin Registry, personal communication). Histopathological examination of the placenta and fetal membranes has an estimated significance level of about 80%, whereas DNA analysis can reach significance levels of 99.999%, if many markers are included.
Here, we used data from the histopathological examination of placenta and fetal membranes in 2 twin pairs and DNA analysis in 15 pairs. In one pair, previous DNA analysis had been performed elsewhere. For the DNA analysis, eleven polymorphic microsatellite markers (on different chromosomes) were used for PCR amplification. PCR products were visualized by autoradiography after size fractionation by electrophoresis. Parents were included to enable the identification of shared alleles. According to allele sharing at each locus, probability ratios for monozygosity were calculated (p<0.005 considered statistically significant). Dizygosity was established when each twin had a different allele for at least two markers. Differences in birth weight were analyzed using a paired Student´s t-test.
Paper II Analysis of heredity, ethnic origin, birth weight, twin rate and phenotype Data concerning the pedigree and country of birth were supplemented from Statistics Sweden. The Swedish National Board of Health and Social Welfare provided information of birth weight and gestational age for 1722 boys with hypospadias and for 1417 of their unaffected brothers, serving as controls. Birth weight of the cases was compared with the mean value of their respective brother(s) using a paired Student´s t- test (n=946). Since birth weight depends on gestational age, we stratified cases and controls based on gestational age in four groups (w. 25-29, w. 30-35, w. 36-39, w. 40- 45). Student´s t-test was used to compare cases and controls within each group.
27 Twin zygosity was either established by DNA analysis, as described in paper I, or by information given by the parents. A χ2 test was used to compare the observed twin rate with the expected.
Experienced pediatric urologists phenotyped 531 index cases and 145 familial cases through medical records. The severity of hypospadias was estimated according to statements in the records regarding the localization of the urethral orifice and the surgical method used. The degree of chordee and the presence of cryptorchidism were also evaluated. Hypospadias was classified as glandular with the orifice on the glans or in the sulcus, penile with the urethral orifice anywhere along the penile shaft or penoscrotal/perineal with the urethral orifice on the scrotum or in the perineum. Differences in phenotype between sporadic and familial cases were analyzed using a χ2 test.
Paper III Complex segregation analysis was performed to evaluate the genetic background in hypospadias in this material. A fundamental problem in segregation analysis involves the ascertainment through affected individuals, resulting in a biased segregation proportion. For this purpose, the probability of an affected individual to be ascertained (π) was estimated to 0.75. Affection status, sex and relationship for each member in 2005 pedigrees, forming 2080 nuclear families, were stated in a file used for the segregation analysis. Liability was set to 0.003 for males. Since hypospadias is a sex-limited trait that only affects males, the liability for females was set to 0. Four different genetic models (additive, multifactorial, dominant and recessive) were tested versus a sporadic model. The sporadic model corresponds to no familial resemblance. The complex segregation analysis involved a maximum likelihood analysis to find the best fit for the included pedigrees. The analysis was performed in POINTER (Lalouel and Morton 1981). Only nuclear families can be analyzed in this program and it therefore involves the usage of pointers, meaning through whom the family was selected.
Paper IV-V Genotyping PCR amplifications of 377 microsatellite markers were carried out as single reactions in 96-well plates. Each forward primer was fluorescently labeled in blue, green or yellow. Since the PCR products also differed in sizes, this enabled the simultaneous size fractionation of several (up to 15) markers on an ABI377 (Applied Biosystems). The resulting genotype data were analyzed with Genescan 2.1 and Genotyper 2.0 software
28 (Applied Biosystems). For each marker, allele numbers were assigned and their sizes standardized using a control DNA with known alleles included on each 96-well plate and on each gel.
The basis for the microsatellite markers was the Weber 6 screening set (Sheffield et al. 1995). In sparsely covered regions, new markers were added from the Genome Database (http://www.gdb.org/) and the Marshfield Medical Research foundation (http://research.marshfieldclinic.org/genetics/). Mean heterozygosity for the autosomal markers was 0.76 and for the X chromosome 0.71. All genotyped markers were analyzed for Mendelian incompatibilities using zGenStat 1.126 software (Henric Zazzi, unpublished). All inconsistencies were reanalyzed and incompatibilities were either resolved unambiguously, or individuals and/or pedigrees were excluded from linkage analyses. To identify markers with allelic dropout or other problems, the expected number of homozygotes was calculated based on the estimated allele frequencies and compared with the observed numbers of homozygotes. For this the Pearson χ2- test as implemented in the zGenStat 1.126 software was used. Any marker showing significant deviation from expected homozygosity frequency (p<0.001) was reanalyzed, resulting in the exclusion of seven markers. A success rate less than 30% resulted in the exclusion of 10 markers. Thus, after quality assessments 17 of the 377 markers were excluded, resulting in 360 markers included in the genotyping with a mean average intermarker distance of 9.5 cM.
Family structures were verified using the SibError software (Ehm and Wagner 1998) based on genotype data of 128 markers spaced at 30 cM interval. We identified one monozygotic twin pair, which was excluded from the linkage analysis.
Linkage analysis (paper IV) As the mode of inheritance is unclear, we used a non-parametric affected relative pair based linkage analysis to detect linkage. This was performed in the Allegro software (Gudbjartsson et al. 2000), which is capable of analyzing allele-sharing between more distant relatives (Nyholt 2000). The analyzed families differ in sizes and each family was therefore power weighted. Since hypospadias is restricted to males, females were coded as unknown. As suggested by Nyholt (2000), linkage analysis for the X chromosome was performed using MAPMAKER/SIBS (Kruglyak and Lander 1995a) through the HGMP web site (http://www.hgmp.mrc.ac.uk/). Corresponding p-values were interpreted according to Lander and Kruglyak (Lander and Kruglyak 1995), as summarized by Nyholt (2000). Allele frequencies were estimated from all genotyped individuals using the zGenStat 1.126 software (Henric Zazzi, unpublished). For the analyses in each
29 subgroup (i.e. families with Swedish and Middle Eastern origin) the allele frequencies were derived from each group. The map distances were based on the Marshfield map (http://research.marshfieldclinic.org/genetics/).
Single-point and multipoint linkage analyses were performed. In single-point analysis, LOD score calculations are performed for each marker in relation to the disease locus, independent of the surrounding markers, whereas multipoint analysis refers to the simultaneous analysis of several markers with known location on a genetic map, which increases information in the region (Ott 1996).
Linkage analysis (paper V) Parametric linkage analysis was used in this family with apparent dominant inheritance of hypospadias. After an initial genome-wide linkage analysis resulting in evidence for linkage to the short arm of chromosome 7, markers D7S2514, D7S641, D7S2464, D7S664, D7S2557, D7S2508, D7S507, D7S503, D7S488, D7S2551, D7S493 and D7S673 were added. Two-point linkage analysis was performed using MLINK in the FASTLINK package through the HGMP web site (http://www.hgmp.mrc.ac.uk/), assuming an autosomal dominant model with full penetrance and the gene frequency 0.001.
Association studies (paper IV) A transmission disequilibrium test was done in the pedigree disequilibrium test (Pdt) which can include several affected individuals in a single pedigree (Martin et al. 2000). The Pdt analyses were performed through the HGMP web site (http://www.hgmp.mrc.ac.uk/).
Simulation analysis (paper IV) We performed simulation analyses to evaluate the results from the genome-wide linkage analysis and to obtain significance levels in all 69 families and in the subgroups consisting of Swedish and Middle Eastern families. Five thousand simulations were generated in Allegro, using the same family structures, the same observed allele frequencies and the same mean success rates for the autosomal markers as in the genome-wide linkage analysis. Genotypes were only generated for individuals that actually were genotyped in the genome-wide linkage analysis. Multipoint analysis was performed using a linear model weighted for each family.
30 Mutation analysis (paper V) HOXA13 is located in the region at chromosome 7p15 (http://www.ncbi.nlm.nih.gov) with evidence for linkage in this family and was therefore subject to mutation analysis. The most sensitive method to detect an unknown mutation is by direct sequence analysis. Here, we used cycle sequencing with fluorescently labeled dideoxynucleotides (ddNTPs). The sequencing reaction involves PCR amplification using one primer and ddNTP chain terminators, resulting in randomly occurring stops in the amplified sequence. The resulting fragments of different lengths are size fractionated by electrophoresis and transferred into a chromatogram, in which the sequence can be interpreted.
We used PCR primers amplifying the whole coding region of HOXA13 (Kosaki et al. 2002). DNA sequence analysis was performed on both strands of amplified and purified PCR products using the ABI PRISM BigDye Terminator CycleSequencing kit 2.0 (Applied Biosystems). The sequencing reactions were carried out according to the manufacturer's recommendations and analyzed on an ABI310 DNA sequencer.
31 RESULTS AND DISCUSSION
Paper I In this study including 28 twin pairs discordant for hypospadias we found 18 monozygotic twin pairs. In 16 of these, the twin with lowest birth weight was affected with hypospadias. The mean intra-pair difference in birth weight was 498 g (p<0.01). This difference was more pronounced than in a normal population of monozygotic twins (p<0.001).
Given the identical genetic background in monozygotic twins, this shows that low birth weight in itself is an important risk factor for hypospadias. Although post-zygotic events may cause discordance in monozygotic twins, this is an unlikely explanation in as many as 16 of 18 discordant twins. Low weight at birth reflects a growth retardation throughout gestation and has been shown to be correlated to sub-optimal first-trimester growth (Smith et al. 1998). In twins, it is well recognized that the twin with lowest birth weight is the smaller throughout gestation, i.e. also at the time of male external genitalia development (T-H Bui, personal communication).
It is here shown that the association with low birth weight is independent of genetic factors, raising some interesting questions. It can be assumed that the twins share intrauterine environmental factors, in all but one respect, the blood supply by the placenta. This suggests an inadequacy of the placenta to provide the fetus with nutrients and/or hormones. A placentary malfunction has previously been implied in hypospadias by the association with a low weight of the placenta (Stoll et al. 1990), severe preeclampsia (Akre et al. 1999) and dystocia (Källén 1988). During the first trimester, fetal androgen production is induced by maternal gonadotropins, preferably hCG produced in the placenta. Thus, a relative lack of hCG in the smaller twin can explain the increased susceptibility for hypospadias. Another explanation may be hypoxia in genital tissue as suggested by the observation of hypospadias in several cases with congenital anemia (e.g. homozygous alpha-thalassemia and hypotransferrinemia) (Dame et al. 1999; Fung et al. 1999; Goldwurm and Biondi 2000). Alternatively, the growth impairment in itself may render the fetus more vulnerable to inadequate endogenous hormone levels (e.g. androgen) or to deleterious exogenous environmental factors (e.g. estrogenic and anti-androgenic chemicals).
32 Paper II In this study of a large number of families with at least one member with hypospadias, we found a familial rate of 7% (to be compared to 3% in the general population). Since we have relied on information given by family members, this is likely to be an underrating and the actual familial rate is probably higher. This finding supports genetic factors in the pathogenesis of hypospadias.
We obtained additional evidence for the impact of low birth weight, since the boys with hypospadias differed significantly in birth weight compared to their brothers (p=5x10-13).
Since hypospadias is a sex-limited trait, we only asked for male siblings and were consequently only able to detect male-male twins. An increased frequency of twins was nevertheless found in this material. Given a twin rate of 1 per 80 pregnancies and the occurrence of male-male twins in 1/3 of these (figure 9), we could expect nine male-male twins. Dizygosity could be expected in six of these and monozygosity in three.
Figure 9 Distribution of zygosity and sex in twins
1/6 monozygotic female-female 1/3 monozygotic 1/6 monozygotic male-male
1/6 dizygotic male-male 1/3 dizygotic same-sex 1/6 dizygotic female-female
1/3 dizygotic unlike sex 1/3 dizygotic male-female
We observed 40 male-male twins (p=4x10-25). Zygosity was established in 33 twins, of which 11 were dizygotic and 22 monozygotic. Thus, we observed an increased frequency of dizygotic as well as monozygotic male-male twins in this material. The finding that two-thirds were monozygotic deviates from the expected 50:50 distribution in twins of same sex (figure 9).
33 An over-representation of monozygotic twins has previously been described and attributed either to a common denominator in monozygosity and hypospadias or to some predisposing factor in association with monozygotic twinning (Roberts and Lloyd 1973). That the risk for hypospadias seems to be higher in male-male twins, regardless of zygosity, again suggests a relative lack of androgen-inducing hormones (i.e. hCG) as one cause for hypospadias.
The high twin rate may be due to the use of assisted reproduction, however this was not investigated here. An increased risk for hypospadias following in vitro fertilization and intracytoplasmic sperm injection has been reported (Macnab and Zouves 1991; Silver et al. 1999b; Wennerholm et al. 2000; Ericson and Källén 2001).
With regards to the distribution of phenotype, we found a higher proportion of intermediately affected cases (i.e. penile) than in most previous studies (table 1) (Sørensen 1953; Roberts and Lloyd 1973; Sweet et al. 1974; Monteleone Neto et al. 1981; Calzolari et al. 1986; Källén et al. 1986). Since we defined hypospadias as penile as soon as the urethra opened anywhere along the penile shaft (including juxta-coronal variants), many of the penile cases are relatively mild variants with the urethral orifice located only a few millimeters proximal to the corona. A similarly high frequency of penile cases was described in the patients studied by Bauer (table 1) (Bauer et al. 1981). However, this population consisted mostly of referrals from pediatricians and urologists, whereas we consider our group of patients to be representative for a general hypospadias population. We found significant differences between familial and sporadic cases with regards to glandular and penoscrotal/perineal variants. Severe variants were less common in familial cases than in sporadic cases. An explanation for this may be a reduced fertility in severe forms of hypospadias.
Interestingly, 6% (n=134) of the cases originated from the Middle Eastern region (Turkey, Syria, Lebanon, Iran or Iraq), to be compared with 2% in the general Swedish population (Susanne Dahllöf, Statistics Sweden, personal communication). Of the 134 subjects from Middle Eastern countries, 22% reported additional family members with hypospadias. In the 144 familial cases, 20% originated from Middle Eastern countries. These observations speak in favor of genetic factors in hypospadias. Consanguinity is frequent in this region, suggesting that recessive genes are involved in hypospadias in this population.
34 Paper III In the complex segregation analysis of 2005 pedigrees we obtained further evidence for a familial aggregation of hypospadias. The high heritability of 0.99 suggests that there are monogenic effects in this material; however, the best fit was achieved for a multifactorial model. This indicates that there are major genes acting in a minor proportion of the families but that there is a multifactorial cause in the majority of cases. Some of these may even be phenocopies, in which hypospadias is caused by environmental factors only (e.g low birth weight). Similar conclusions were drawn from the segregation analysis (performed in POINTER) of 103 Danish families (Harris and Beaty 1993). The heritability in that study was equal to the one in this study (0.99).
In this study, more than 2000 families were ascertained through a nation-wide scan for hypospadias cases. We initially contacted 2500 patients, corresponding to half of all registered cases in Sweden, and included 80% of these in the segregation analysis. There are no simple rules with regards to the size of the material for segregation analysis. The power depends on the structures of the pedigrees and on the underlying genetic model, which by definition is unknown, but a larger material will nevertheless increase the power.
Paper IV In the genome-wide linkage analysis, we found five chromosomal regions with suggestive linkage (figure 10). According to the definition of suggestive linkage, one randomly occurring peak per genome-wide linkage analysis is expected. It is still possible that each of these five peaks represent true susceptibility genes for hypospadias. Interestingly, there were no peaks in regions harboring the genes most frequently found in monogenic variants of hypospadias (e.g. the androgen receptor gene (AR, Xq11-12) and the recessive 5-alpha-reductase deficiency (SRD5A2, 2p23). This suggests that the susceptibility genes for hypospadias may be different from the ones mutated in monogenic variants of hypospadias.
35
How can the search for susceptibility genes be pursued? Lander and Kruglyak (1995) recommend that suggestive linkage should be followed up in extension studies. (The term extension study is preferred for the testing of additional families in the search for significant linkage, whereas replication study should be reserved for situations in which significant linkage has already been obtained). In the extended study, they recommend that data are pooled and the entire dataset reanalyzed (Lander and Kruglyak 1995). An alternative strategy is to study founder populations. Families with several affected may also be useful, as demonstrated in the recent identification of the PCDC1 gene in systemic lupus erythematosus (Prokunina et al. 2002).
Estimates of the desired number of families (i.e. power calculations) are difficult to perform, and to evaluate, since it requires specifications of the model and this cannot be reliably made. It was originally believed that several hundred families would be needed for the identification of susceptibility genes in common traits and that these numbers would be correlated to the relative risk (Risch 1990b). However, it should be noted that only 78 families were included in the original linkage analysis for Crohn´s disease, eventually resulting in the identification of the NOD2 gene (Hugot et al. 1996; Hugot et al. 2001).
Adding more markers has the advantage of increasing information, given that all previously markers were not fully informative (Haines and Pericak-Vance 1998). There is, however, no endpoint equivalent to the situation in the mapping of monogenic traits in which recombinations restrict the region (Kruglyak and Lander 1995b). Instead, association mapping may be used to further narrow the region. Fine mapping of the candidate region, before proceeding with investigations in candidate genes, has been strongly recommended (Lander and Kruglyak 1995).
38 Paper V The identification of hand-foot-genital syndrome (HFGS) in this family with autosomal dominant inherited hypospadias was unexpected, since the skeletal malformations are much less severe than in previously reported HFGS families. However, the finding of a novel polyalanine insertion in the HOXA13 gene clearly speaks in favor of this diagnosis, albeit an atypical variant of the syndrome.
HFGS is an autosomal dominant syndrome characterized by skeletal anomalies in the distal limbs and urogenital malformations (McKusick 1986, 1990). Typical skeletal manifestations in the hand include short, proximally placed thumbs and clinodactyly of the fifth finger and in the feet short, medially deviated great toes and fusion defects of the bones. The radiographic pattern is characteristic for the syndrome (Poznanski et al. 1970). The skeletal manifestations are invariable and highly penetrant, whereas the urogenital abnormalities show reduced penetrance and variable expression. Females have Müllerian duct fusion defects such as uterus bicornis, vaginal septum and ectopic localization of ureteric and urethral orifices. Males often present with hypospadias.
Eight families and four sporadic cases with HFGS have previously been described (Stern et al. 1970; Poznanski et al. 1975; Giedion and Prader 1976; Goeminne 1981; Verp et al. 1983; Halal 1988; Cleveland and Holmes 1990; Donnenfeld et al. 1992; Fryns et al. 1993; Devriendt et al. 1999; Goodman et al. 2000; Utsch et al. 2002). The phenotype varies both within and between these families (table 4).
Three additional families with some of the characteristic features of HFGS have been reported (Longmuir et al. 1986; Hennekam 1989; Guttmacher 1993). The syndrome described by Longmuir (1986) consists mainly of distal skeletal malformations. The family reported by Hennekam (1989) has atypical features of HFGS including Müllerian duct fusion defects and ear malformations, but no hand malformations. The syndrome designated as Guttmacher (1993) show some features of HFGS, such as short thumbs and great toes as well as hypospadias in males, but also includes postaxial polydactyly.
39 Table 4 Phenotype and mutation data in the 9 families and 4 sporadic cases with HFGS reported thus far
Author and year Affected Hand phenotype Foot phenotype Genital phenotype Type of mutation
Stern 1970 18 short thumbs, clinodacyly small feet, tarsal fusion, short calcaneus, double uterus, nonsense short toe 1, hallux varus vaginal septum Poznanski 1975 10 short thumbs, clinodactyly tarsal fusion, short calcaneus, short toe 1 Gideon 1976 3 brothers short thumbs, clinodactyly short calcaneus, hallux varus hypospadias Goeminne 1981 sporadic male clinodactyly brachydactyly toe 2-5 hypospadias Verp 1983 7 affected in short thumbs, clinodactyly short toe 1, hallux varus uterus bicornis, polyalanine insertion (+8) Donnenfeld 1992 4 generations vaginal septum, in third tract hypospadias Halal 1986 6 short thumbs, clinodactyly tarsal fusion, short calcaneus, short toe 1, uterus bicornis, hallux varus, toe syndactyly vaginal septum, ectopic ureter and urethra Cleveland 1990 3 short thumbs, clinodactyly tarsal fusion, short toe 1, hallux varus uterus bicornis nonsense hypospadias Fryns 1993 4 (father short thumbs, clinodactyly small feet, short toe 1 hypospadias nonsense and 3 sons) Devriendt 1999 sporadic male short thumbs, clinodactyly tarsal fusion, short toe 1, hallux valgus cryptochidism, chordee deletion of 7p14 Goodman 2000 (1) sporadic male short thumbs, clinodactyly short toe 1, hallux varus short penis nonsense Goodman 2000 (5) sporadic male extremely short thumbs, tarsal fusion, absence of toe 1, glandular hypospadias missense hypoplasia of phalanges hypoplasia/absence of phalanges Utsch 2002 6 affected in short thumbs, clinodactyly small feet, short toe 1, hallux varus uterus bicornis, polyalanine insertion (+6) 5 generations double cervix, in third tract vaginal septum Frisén 2002 28 affected in clinodactyly small feet, gap between toe 1 and 2, short hypospadias (glandular polyalanine insertion (+6) 6 generations toe 2, hallux varus in 7, penile in 3) in second tract
40 In 1997, it was reported that HFGS is caused by mutations in the HOXA13 gene (Mortlock and Innis 1997). An overview of the mutations that have since then been identified in HFGS is shown in figure 11. The phenotype associated with the previously reported mutations is essentially the same, except for that produced by the missense mutation, which affects the hands and feet much more severely. In addition, an interstitial deletion removing the entire HOXA cluster was found in the family reported by Devriendt (1999). Interestingly, this patient displays a phenotype that is relatively mild, especially with regards to the genital manifestations. In Guttmacher syndrome, both a missense mutation in the homeobox region of the HOXA13 gene and a dinucleotide deletion in the promoter was recently found (Innis et al. 2002).
Figure 11 Mutations in HOXA13 in HFGS families
+6 alanines +8 alanines Q365X W369X (Frisén 2002) (Goodman 2000:family 4, (Goodman 2002:family 3, (Mortlock 1997, Verp 1983, Donnenfeld 1990) Cleveland 1990) Stern 1970) +6 alanines (Utsch 2002)
1 922 923 1167
5´ exon 1 intron exon 2 3´
Q196X S136X (Goodman 2002:family 2, N372H (Goodman 2002:family 1) Fryns 1978) (Goodman 2002:family 5)
polyalanine tract (14, 12, 18 alanines, respectively) homeodomain
The polyalanine insertions consist of cryptic expansions (i.e. GCA, GCC, GCG, GCT) rather than trinucleotide repeats, are stable through generations and are believed to have originated through unequal crossing-over (Warren 1997). Similar polyalanine tract expansions have been described in HOXD13 in several families with synpolydactyly (Muragaki et al. 1996; Goodman et al. 1997; Goodman et al. 2002).
41 HOX genes are highly conserved through evolution and were first identified in Drosophila as key regulators of patterning of the body plan. Mutations in Drosophila result in a certain body segment loosing its positional identity and being transformed to another body structure, i.e. a homeotic transformation. In the C-terminal region of HOX genes there is a 180 bp stretch of highly conserved DNA coding for a 60 amino acids homeodomain. This domain binds to DNA and regulate transcription in interaction with other proteins. The potential target genes and interacting proteins are numerous and not fully characterized (Veraksa et al. 2000).
In all species, genes within a HOX cluster are arranged according to their temporal and spatial expression during development, a phenomenon known as colinearity. In man, there are four paralogous clusters (HOXA, HOXB, HOXC, HOXD), which probably originated through two successive gene duplications. A certain HOX gene is functionally more similar to its paralogue in another cluster than to a neighboring gene in the same cluster. For example, the paralogues HOXA13 and HOXD13 are located at the 5´end of their respective cluster and are both important for the development of the limbs and the lower urogenital tract. Thus, they are likely to have partly overlapping functions with respect to the embryological development of digits and genitalia (Fromental-Ramain et al. 1996; Warot et al. 1997). Both these structures are regions of apical growth and represent morphogenetic end organs. In line with this, it has been suggested that digits and penis have a related phylogenetic history (Kondo et al. 1997).
The mutations in HOXA13 and HOXD13, together with a mutation in HOXA11 resulting in amegakaryocytic thrombocytopenia and radio-ulnar synostosis (Thompson and Nguyen 2000), are the only mutations in HOX genes described in man to date. Characteristically, these abnormalities are discrete and may easily escape medical attention.
The family described here displays a milder phenotype than previously reported HFGS families. Affected individuals are heterozygous for a polyalanine expansion in the second polyalanine tract in the first exon of the HOXA13 gene. This is in contrast to the two previously reported polyalanine expansions in the HOXA13 gene localized in the third tract. It is likely that the discrepancies in phenotype between this and previously reported HFGS families are caused by the different localization of the insertion.
42 It remains unclear whether the mutated HOXA13 protein acts by haploinsufficiency or a dominant negative mechanism. The patient with HFGS carrying a heterozygous deletion of the entire HOXA cluster is an example of haploinsufficiency of the HOXA13 gene (Devriendt et al. 1999). Interestingly, he has a relatively mild variant of HFGS, in particular with regards to the genital malformations that are limited to cryptorchidism and chordee. In contrast, the patient with a missense mutation has an unusually severe phenotype (Goodman et al. 2000), suggesting a dominant negative effect. This is further supported by the finding that mice heterozygous for a minor deletion within the Hoxa13 gene have a more severe limb phenotype than mice homozygous for a Hoxa13 null mutation (Fromental-Ramain et al. 1996; Mortlock et al. 1996).
Several lines of evidence indicate that the polyalanine expansions described in HOXD13 may as well render a dominant negative effect. In synpolydactyly, the severity has been shown to correlate with the size of the polyalanine expansion and in the family with the largest expansion (14 additional alanines) affected males have hypospadias (Goodman et al. 1997). Mice homozygous for a spontaneous polyalanine expansion (expanding the stretch from 15 to 22 alanines) in the Hoxd13 gene have a much more severe phenotype than mice with complete absence of Hoxd13 function (Zakany and Duboule 1996; Johnson et al. 1998). Genetic complementation studies in this mouse model indicate that the mutated protein exerts a ”super” dominant negative effect, by interfering with the function of the remaining wild-type Hoxd13 and other 5´ Hoxd proteins (Bruneau et al. 2001). Moreover, mice with homozygous deletions of Hoxd11, Hoxd12 and Hoxd13 have a less severe phenotype than mice and humans with polyalanine tract expansions in HOXD13 (Zakany and Duboule 1996; Johnson et al. 1998).
43 GENERAL DISCUSSION
In this thesis, data suggesting a significant impact of low birth weight on the pathogenesis of hypospadias is presented. Hypospadias is known to be strongly associated with low birth weight, but it remains unclear whether it is the low birth weight in itself that renders the fetus more susceptible for other predisposing factors (genetic or environmental) or if there is a common denominator in the genesis of the two conditions. Here, we show that the association for hypospadias with low birth weight is independent of genetic factors in discordant monozygotic twins. Since monozygotic twins are genetically identical and can be assumed to share intrauterine environmental factors, this points to a role of the placenta in the pathogenesis of hypospadias. This exemplifies a situation in which hypospadias results from environmental factors only, although it is still possible that the condition arose in a genetically predisposed individual surpassing a threshold.
Several findings in this thesis also suggest a genetic cause for isolated hypospadias: a 7% familial rate, a high rate of sporadic as well as familial hypospadias in Middle Eastern populations (in which consanguinity is frequent) and a heritability of 0.99. However, there is little support for monogenic effects in hypospadias, at least in the majority of this material. Nevertheless, in one family, we identified a mutation in a HOX gene causing autosomal dominant inheritance of hypospadias. This illustrates the extreme end of the spectrum in which a single genetic alteration is sufficient to cause the malformation. It is likely that the background for hypospadias constitutes a continuum from environmental factors only to single genetic effects, with the majority of cases caused by the interaction of several susceptibility genes with or without environmental factors.
With the aim to identify the susceptibility genes for hypospadias, a genome-wide linkage analysis was performed. Five loci with suggestive evidence for linkage were identified. These regions need to be investigated further, preferably in an extended population. But it is not improbable that there are in fact five genes behind the susceptibility for hypospadias and that these eventually will be identified. Two different models may explain the genetic background for hypospadias as well as other complex traits. One theory is that common alleles at a few loci interact to cause the disease, i.e. the common disease/common variant hypothesis. The other concept is that rare alleles at numerous loci each on its own cause the disease, i.e. genetic heterogeneity (Pritchard and Cox 2002; Smith and Lusis 2002). To date, the available sample of known complex disease genes is too small to draw any general conclusions. In the end, both these viewpoints may turn out to be accurate, within a disease as well as in different diseases. The results from the
44 genome-wide linkage analysis presented here may suggest a common disease/common variant model in hypospadias, since it is unlikely that we would have detected five loci unless they all contribute to the phenotype in some way.
It has been suggested that the polarized view of monogenic versus complex traits is outdated (Badano and Katsanis 2002). Some classical monogenic diseases have turned out to be not so simple (e.g. lack of genotype-phenotype correlation in some families with cystic fibrosis) (Dipple and McCabe 2000). This has in part been attributed to the influence of modifier genes. In other diseases, an unexpected mode of inheritance has been found (e.g. triallelic inheritance in Bardet-Biedl syndrome) (Katsanis et al. 2001). Although complex traits are generally believed to result from the interaction between several genes and environmental factors, in some individuals the phenotype is caused by mutations in one gene. These observations speak in favor of a transition from a segmented view of human genetic diseases to a conceptual continuum between monogenic and complex traits.
The overall aim of this thesis was to gain an increased understanding of the pathogenesis for hypospadias. An additional benefit would be to shed light on key mechanisms in sex differentiation. In that respect, this is basic research with no immediate clinical application, although the importance of increased information to affected families should not be undervalued. It is nevertheless possible that the future identification of the molecular basis for hypospadias may enable causal treatment.
45 SAMMANFATTNING PÅ SVENSKA
Hypospadi är en av de vanligaste missbildningarna med en förekomst av cirka 0.3 % och innebär att urinrörsmynningen är lokaliserad på undersidan av penis. Mycket talar för att hypospadi har en genetisk bakgrund i kombination med miljöfaktorer. Den övergripande målsättningen med denna avhandling var att identifiera orsaker till hypospadi.
Flera grundläggande genetiska metoder utnyttjades i ett landsomfattande material, motsvarande cirka hälften av alla registrerade fall i Sverige. Drygt 2500 individer med hypospadi erhöll en enkät bestående av frågor om ytterligare fall av hypospadi i släkten och födelsevikt. Detta material utgör grunden för fem delarbeten i avhandlingen.
I. Tvillingstudie i syfte att utvärdera miljöfaktorers inverkan vid uppkomsten av hypospadi. Enäggstvillingar är genetiskt identiska, medan tvåäggstvillingar delar hälften av det genetiska materialet, precis som syskon. Studier av enäggstvillingar där bara den ena är sjuk kan ge värdefull information om miljöfaktorers inverkan. I materialet identifierades 18 enäggstvillingpar, där enbart en av tvillingarna hade hypospadi. I 16 av dessa par var det den mindre tvillingen som drabbats. Detta visar att låg födelsevikt, oberoende av den genetiska bakgrunden, har betydelse för uppkomsten av hypospadi.
II. Epidemiologisk studie i syfte att dels undersöka andelen familjära fall i materialet, dels att fortsatt analysera sambandet mellan hypospadi och låg födelsevikt. Ett eller flera ytterligare fall av hypospadi i släkten rapporterades av 7 % (n=144). Det förelåg en signifikant skillnad i födelsevikt hos pojkar med hypospadi jämfört med sina respektive bröder (p=5x10-13). Vi fann en ökad frekvens av såväl enägg- som tvåäggstvillingar. Dessutom kartlade vi den etniska bakgrunden i hela materialet och karaktäriserade graden av hypospadi i en tredjedel av materialet.
III. Segregationsanalys i syfte att definiera nedärvningsmönstret för hypospadi. En multifaktoriell modell var mest förenlig med nedärvningsmönstret i vårt material. Heritabiliteten beräknades till 0.99. Detta tolkas som att hypospadi har en multifaktoriell bakgrund i de flesta fall, medan mutationer i enskilda gener orsakar en mindre andel.
IV. Syskonparanalys med målsättning att identifiera gener som bidrar till uppkomsten av hypospadi. Syskonparanalys innebär att man jämför markörer mellan sjuka individer inom många familjer för att identifiera kromosomregioner som de delar i större utsträckning än förväntat. I denna studie inkluderades 69 familjer med två eller flera pojkar med hypospadi. Alla familjemedlemmar inklusive föräldrar genotypades med
46 360 fluorescensmärkta polymorfa mikrosatellitmarkörer spridda över hela genomet med i genomsnitt 9.5 cM avstånd. Vi fann fem intressanta kromosomregioner som är föremål för utvidgade studier.
V. Mutationsanalys i en familj med autosomalt dominant nedärvning av hypospadi. Med kopplingsanalys lokaliserades genen till ett mindre område på kromosom 7, där HOXA13 genen är belägen. Mutationer i HOXA13 ger upphov till ett mycket ovanligt syndrom, hand-foot-genital syndrome (HFGS). Endast 12 familjer i världen finns beskrivna tidigare och hos 8 av dessa har man hittat en mutation i HOXA13 genen. Vi identifierade en ny mutation i HOXA13 genen i denna familj med atypisk variant av HFGS. Det är den hittills största beskrivna HFGS-familjen med 28 fall i 6 generationer. HFGS karaktäriseras av milda utvecklingsstörningar i skelett och genitalia. Typiska skelettavvikelser är små fötter, små stortår, små tummar och krokiga lillfingrar. Kvinnor med HFGS kan ha hjärtformad livmoder, skiljevägg i vagina, inkontinens och upprepade urinvägsinfektioner på grund av felplacerad mynning av urinrör och urinledare. Män med HFGS har ibland hypospadi. I denna familj är hypospadi ett vanligare fenomen än i tidigare beskrivna familjer och skelettavvikelserna är betydligt mildare. Mutationen utgörs av en insertion av sex extra alanin inom ett område där inga tidigare mutationer finns beskrivna. Den atypiska bilden i denna familj kan förklaras av mutationens lokalisation.
Slutsats Arbetet som presenteras i denna avhandling har inneburit att epidemiologiska och genetiska mekanismer vid hypospadi kunnat kartläggas. Resultaten från tvillingstudien och den epidemiologiska studien visar att låg födelsevikt är en viktig riskfaktor för hypospadi. Dessutom stöder flera fynd i den epidemiologiska studien att gener medverkar till uppkomsten av hypospadi, liksom resultaten från segregationsanalysen och syskonparanalysen. I syskonparanalysen identifierades fem kromosomregioner med indikationer på gener för hypospadi. I en familj med autosomalt dominant nedärvning av hypospadi identifierades en ny mutation i HOXA13 genen. Det visade sig alltså att denna familj har en atypisk variant av HFGS med hypospadi som huvudsaklig manifestation.
Den övergripande målsättningen med arbetet var att öka förståelsen för bakgrunden till hypospadi, vilket dessutom kan ge insikter i grundläggande mekanismer för könsutvecklingen. Det kan också medföra ökad information till drabbade familjer och framtida identifiering av den molekylära orsaken till hypospadi kan möjliggöra riktad behandling.
47 ACKNOWLEDGEMENTS
I have been encouraged and supported by many people, to whom I am very thankful:
Agneta Nordenskjöld, my supervisor, for being so caring and patient during all these years and for limitless stamina, numerous ideas and vast knowledge on hypospadias
Holger Luthman, my co-supervisor, for encouragement and consideration, for enthusiasm and many stimulating discussions
Ingrid Kockum, my almost-supervisor, for lots of assistance, for always being helpful and for patiently explaining statistics in complex traits
Margareta Tapper-Persson for being the laboratory technician every PhD student dream of, for being so caring and kind since the very first day and for unbelievable amount of practical help
Magnus Nordenskjöld for creating an enjoyable atmosphere as well as a thorough research education and for engagement in this project, but most of all for being such a considerate person and for bringing many laughs into the lab
Lennart Iselius for generous and competent collaboration in the segregation analysis
Paul Lichtenstein and Nancy Pedersen for cooperation in the twin study and for their excellent research course in genetic epidemiology
Einar Hansson, Staffan Holmner, Lars Lundquist, Göran Läckgren, Jörgen Pedersen, Arne Stenberg and Gunnar Westbacke for providing records of their patients with hypospadias
Kicki Lagerstedt for being so energetic and helpful, in particular for invaluable sequencing assistance
Cilla Söderhäll for lots of practical advice along the way and many fruitful discussions, for valuable comments on this thesis and for being a good friend
Maria Bradley for shared struggles with complex genetics and for continuous close friendship
My fellow PhD students throughout the years: Britt-Marie Anderlid, Erik Björck, Lovisa Bylund, Kim Ericson, Heléne Fischer, Pernilla Holm, Erik Iwarsson, Shideh Khodaei- O'Brien, Charlotta Lindvall, Ann Nordgren, Sofia Persson, Tiina Robins, Jacqueline Schoumans and Emma Tham for lots of fun and friendship
Magdalena Fossum and Mina Kalbasi for good spirits and continued work in the hypospadias field
Former and present group leaders and post docs in the department: Maria Anvret, Jan Dumanski, Catharina Larsson, Nils-Göran Larsson, Annika Lindblom, Helena Malmgren, Kevin O´Brien, Martin Schalling, Keng-Ling Wallin, Günther Weber, Anna Wedell and Gabrielle Åhlberg for being such nice and helpful people and for showing interest in this work
48 Yvonne Cowan, Ann-Mari Dumanski and Kerstin Florell for lots of help with administrative matters
Laboratory technicians Sigrid Sahlén, Margareta Tapper-Persson, Anki Thelander and Barbro Werelius for providing everything including the joyful atmosphere
Sivonne Arvidsson and Ingrid Delin for teaching me ABI gel running and the interpretation of genotypes
Annika Röhl for invaluable help in the preparation of the sex differentiation presentation and for sharing numerous pictures as well as advice on layout
Lennart Nilsson for generously letting me use his extraordinary photographs
Susanne Dahllöf at Statistics Sweden for efficiently providing invaluable information
I am indebted to several people for helping out with computer-related obstacles: Carl Bruder for once introducing me to the world of computers, The-Hung Bui for patiently converting personal computer based items, Jacci Fredell, Godfather and uncle, for always taking time to help and in addition being better at it than any Excel-manual and Lennart Helleday, Rudolf Matousek and Dagmar Vejsicka for patience with a computer-illiterate and for being so nice and helpful
Komplexa gruppen for struggling with basic concepts in complex traits, in particular Ingrid Kockum for guidance
Karin Kindberg and colleagues at the Clinical Genetics laboratory for being helpful and friendly, in particular Ulla Grandell for practical help
Clinical geneticists Elisabeth Blennow for willingly answering questions and The-Hung Bui for valuable comments on the twin study
Barbro Högrell for advice and help during my first year in the lab
Summer students Enes Efendic, Madeleine Frederich, Linn Hjortsberg, Åsa Kallas, Philip Waldenström and Lisa Örtqvist for hard work
Anna Lundh and Carl Johan Sundberg for providing me with all kinds of projects inside KI, outside the lab, and for being good friends and fellow teachers
Rolf Heuman and colleagues at the surgical clinic, Mora lasarett, for giving me the opportunity to spend holidays and weekends on call and for being clinical role models
Anna Barnéus, Per Borgström and Tony Frisk for great times in medical school and for continued close friendship since then
Isabelle Dahlborg Lidström for lots of practical help and advice on layout
All friends outside the lab for being so good at having fun and for sweeping me away too
49 Ann Erling and my parents in law Marianne Frisén and Lars Frisén for showing surprisingly much interest in this work, for many good advice and helpful comments and for welcoming me so warmly into the family
All cousins, aunts and uncles for life long friendship and for many enjoyable family gatherings
Ebba von Rosen, Godmother and close friend throughout life, for generosity and support and for companionship at the Karolinska
Lottie and Nicolas for being the best imaginable siblings, for many laughs and lessons along the way and for being such good advisers
My parents for continuous support and trust, for being immensely caring, generous and fun and my role models on the brighter side of life
Jonas for innumerable lunches during the first part of this thesis and for marriage in the end of it, for being a wonderful life-companion and Krakel´s master, for providing inspiration in science as well as in all other aspects of life and for infinite patience and support
My deep gratitude to all boys with hypospadias and their families for participation
This work was supported by scholarships and grants from HRH Crown Princess Lovisa Foundation, the Swedish Research Council, Åke Wiberg Foundation, Magnus Bergvall Foundation, Marcus Borgström Foundation, Förenade Liv, Karolinska Institutet, Ingabritt och Arne Lundbergs forskningsstiftelse, Ronald McDonald Child Foundation, Erik Rönnbergs donationer (Riksbankens jubileumsfond), Stiftelsen Frimurare Barnhuset, Stiftelsen Samariten, the Swedish Society of Medicine and Sällskapet Barnavård.
50 REFERENCES
Adams F (1844-1847) The Seven Books of Paulus Aegineta. Translated from the Greek. With a Commentary Embracing a Complete View of the Knowledge Possessed by the Greeks, Romans and Arabians on All Subjects Connected with Medicine and Surgery. vol 2, London, pp 347
Akre O, Lipworth L, Cnattingius S, Sparen P, Ekbom A (1999) Risk factor patterns for cryptorchidism and hypospadias. Epidemiology 10: 364-9
Allera A, Herbst MA, Griffin JE, Wilson JD, Schweikert HU, McPhaul MJ (1995) Mutations of the androgen receptor coding sequence are infrequent in patients with isolated hypospadias. J Clin Endocrinol Metab 80: 2697-9
Altmuller J, Palmer LJ, Fischer G, Scherb H, Wjst M (2001) Genomewide scans of complex human diseases: true linkage is hard to find. Am J Hum Genet 69: 936-50
Arngrimsson R, Sigurardottir S, Frigge ML, Bjarnadottir RI, Jonsson T, Stefansson H, Baldursdottir A, Einarsdottir AS, Palsson B, Snorradottir S, et al. (1999) A genome-wide scan reveals a maternal susceptibility locus for pre-eclampsia on chromosome 2p13. Hum Mol Genet 8: 1799-805
Auranen M, Vanhala R, Varilo T, Ayers K, Kempas E, Ylisaukko-Oja T, Sinsheimer JS, Peltonen L, Jarvela II (2002) A genomewide screen for autism-spectrum disorders: Evidence for a major susceptibility locus on chromosome 3q25-27. Am J Hum Genet 71: 777-790
Badano JL, Katsanis N (2002) Human genetics and disease: Beyond Mendel: an evolving view of human genetic disease transmission. Nat Rev Genet 3: 779-89
Bauer SB, Retik AB, Colodny AH (1981) Genetic aspects of hypospadias. Urol Clin North Am 8: 559-64
Bergh T, Ericson A, Hillensjö T, Nygren K-G, Wennerholm U-B (1999) Deliveries and children born after in-vitro fertilisation in Sweden 1982-95: a retrospective cohort study. The Lancet 354: 1579-85
Botstein D, White RL, Skolnick M, Davis RW (1980) Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am J Hum Genet 32: 314-31
Bracka A (1999) Sexuality after hypospadias repair. BJU International 83, suppl. 3: 29-33
Bruneau S, Johnson KR, Yamamoto M, Kuroiwa A, Duboule D (2001) The mouse Hoxd13(spdh) mutation, a polyalanine expansion similar to human type II synpolydactyly (SPD), disrupts the function but not the expression of other Hoxd genes. Dev Biol 237: 345-53
Calzolari E, Contiero MR, Roncarati E, Mattiuz PL, Volpato S (1986) Aetiological factors in hypospadias. J Med Genet 23: 333-7
Cassar P (1974) A medico-legal report of the 16th century from Malta. Med Hist 18: 354-359
Cassell PG, Jackson AE, North BV, Evans JC, Syndercombe-Court D, Phillips C, Ramachandran A, Snehalatha C, Gelding SV, Vijayaravaghan S, et al. (2002) Haplotype combinations of calpain 10 gene polymorphisms associate with increased risk of impaired glucose tolerance and type 2 diabetes in South Indians. Diabetes 51: 1622-8
Chen YC, Woolley PV, Jr. (1971) Genetic studies on hypospadias in males. J Med Genet 8: 153-9
51 Cleveland RH, Holmes LB (1990) Hand-foot-genital syndrome: the importance of hallux varus. Pediatr Radiol 20: 339-43
Czeizel A, Toth J, Erodi E (1979) Aetiological studies of hypospadias in Hungary. Hum Hered 29: 166-71
Dame C, Albers N, Hasan C, Bode U, Eigel A, Hansmann M, Brenner R, Bartmann P (1999) Homozygous alpha-thalassaemia and hypospadias--common aetiology or incidental association? Long-term survival of Hb Bart's hydrops syndrome leads to new aspects for counselling of alpha- thalassaemic traits. Eur J Pediatr 158: 217-20
Davits RJ, van den Aker ES, Scholtmeijer RJ, de Muinck Keizer-Schrama SM, Nijman RJ (1993) Effect of parenteral testosterone therapy on penile development in boys with hypospadias. Br J Urol 71: 593-5
Devriendt K, Jaeken J, Matthijs G, Van Esch H, Debeer P, Gewillig M, Fryns JP (1999) Haploinsufficiency of the HOXA gene cluster, in a patient with hand-foot-genital syndrome, velopharyngeal insufficiency, and persistent patent Ductus botalli. Am J Hum Genet 65: 249-51
Dipple KM, McCabe ER (2000) Modifier genes convert "simple" Mendelian disorders to complex traits. Mol Genet Metab 71: 43-50
Donnenfeld AE, Schrager DS, Corson SL (1992) Update on a family with hand-foot-genital syndrome: hypospadias and urinary tract abnormalities in two boys from the fourth generation. Am J Med Genet 44: 482-4
Eaves IA, Merriman TR, Barber RA, Nutland S, Tuomilehto-Wolf E, Tuomilehto J, Cucca F, Todd JA (2000) The genetically isolated populations of Finland and Sardinia may not be a panacea for linkage disequilibrium mapping of common disease genes. Nat Genet 25: 320-3
Edwards JH (1999) Unifactorial models are not appropriate for multifactorial disease. Bmj 318: 1353-4
Ehm M, Wagner M (1998) A test statistic to detect errors in sib-pair relationships. Am J Hum Genet 62: 181-8
Ekelund J, Hovatta I, Parker A, Paunio T, Varilo T, Martin R, Suhonen J, Ellonen P, Chan G, Sinsheimer JS, et al. (2001) Chromosome 1 loci in Finnish schizophrenia families. Hum Mol Genet 10: 1611-7
Ericson A, Källén B (2001) Congenital malformations in infants born after IVF: a population-based study. Hum Reprod 16: 504-9
Falconer DS (1965) The inheritance of liability to certain disease estimated from the incidence among relatives. Ann Hum Genet 29: 51-76
Fisch H, Golden RJ, Libersen GL, Hyun GS, Madsen P, New MI, Hensle TW (2001) Maternal age as a risk factor for hypospadias. J Urol 165: 934-6
Fromental-Ramain C, Warot X, Messadecq N, LeMeur M, Dolle P, Chambon P (1996) Hoxa-13 and Hoxd-13 play a crucial role in the patterning of the limb autopod. Development 122: 2997- 3011
52 Frydman M, Greiber C, Cohen HA (1985) Uncomplicated familial hypospadias: evidence for autosomal recessive inheritance. Am J Med Genet 21: 51-60
Fryns JP, Vogels A, Decock P, van den Berghe H (1993) The hand-foot-genital syndrome: on the variable expression in affected males. Clin Genet 43: 232-4
Fung TY, Kin LT, Kong LC, Keung LC (1999) Homozygous alpha-thalassemia associated with hypospadias in three survivors. Am J Med Genet 82: 225-7
Gabriel SB, Salomon R, Pelet A, Angrist M, Amiel J, Fornage M, Attie-Bitach T, Olson JM, Hofstra R, Buys C, et al. (2002) Segregation at three loci explains familial and population risk in Hirschsprung disease. Nat Genet 31: 89-93
Galen (c. 130-201 A.D.) Opera Omnia, vol 10. C.G. Kühn, Leipzig, pp 1001
Gatti JM, Kirsch AJ, Troyer WA, Perez-Brayfield MR, Smith EA, Scherz HC (2001) Increased incidence of hypospadias in small-for-gestational age infants in a neonatal intensive-care unit. BJU Int 87: 548-50
Giedion A, Prader A (1976) Hand-foot-uterus-(HFU) syndrome with hypospadias: the hand-foot- genital- (HFG) syndrome. Pediatr Radiol 4: 96-102
Goeminne L (1981) Syndroom van Poznanski-Stern. Tijdschr. voor Geneeskunde 37: 1461-1465
Goldwurm S, Biondi A (2000) Case of congenital hypotransferrinemia suggests that tissue hypoxia during fetal development may cause hypospadias. Am J Med Genet 95: 287-90
Goodman FR, Bacchelli C, Brady AF, Brueton LA, Fryns JP, Mortlock DP, Innis JW, Holmes LB, Donnenfeld AE, Feingold M, et al. (2000) Novel HOXA13 mutations and the phenotypic spectrum of hand-foot-genital syndrome. Am J Hum Genet 67: 197-202
Goodman FR, Majewski F, Collins AL, Scambler PJ (2002) A 117-kb microdeletion removing HOXD9-HOXD13 and EVX2 causes synpolydactyly. Am J Hum Genet 70: 547-55
Goodman FR, Mundlos S, Muragaki Y, Donnai D, Giovannucci-Uzielli ML, Lapi E, Majewski F, McGaughran J, McKeown C, Reardon W, et al. (1997) Synpolydactyly phenotypes correlate with size of expansions in HOXD13 polyalanine tract. Proc Natl Acad Sci U S A 94: 7458-63
Gretarsdottir S, Sveinbjörnsdottir S, Jonsson HH, Jakobsson F, Einarsdottir E, Agnarsson U, Shkolny D, Einarsson G, Gudjonsdottir HM, Valdimarsson EM, et al. (2002) Localization of a susceptibility gene for common forms of stroke to 5q12. Am J Hum Genet 70: 593-603
Gudbjartsson DF, Jonasson K, Frigge ML, Kong A (2000) Allegro, a new computer program for multipoint linkage analysis. Nat Genet 25: 12-3
Guttmacher AE (1993) Autosomal dominant preaxial deficiency, postaxial polydactyly, and hypospadias. Am J Med Genet 46: 219-22
Haines JL, Pericak-Vance MA (1998) Approaches to gene mapping in complex human diseases. Wiley-Liss, New York, USA
Hakonarson H, Bjornsdottir US, Halapi E, Palsson S, Adalsteinsdottir E, Gislason D, Finnbogason G, Gislason T, Kristjansson K, Arnason T, et al. (2002) A major susceptibility gene for asthma maps to chromosome 14q24. Am J Hum Genet 71: 483-91
53 Halal F (1988) The hand-foot-genital (hand-foot-uterus) syndrome: family report and update. Am J Med Genet 30: 793-803
Harris EL, Beaty TH (1993) Segregation analysis of hypospadias: a reanalysis of published pedigree data. Am J Med Genet 45: 420-5
Hauser ER, Boehnke M, Guo SW, Risch N (1996) Affected-sib-pair interval mapping and exclusion for complex genetic traits: sampling considerations. Genet Epidemiol 13: 117-37
Hellbom K (2002) Allergimedel kan skada foster. Dagens Nyheter, Stockholm, Sweden
Hennekam RC (1989) Acral-genital anomalies combined with ear anomalies. Am J Med Genet 34: 454-5
Hiort O, Klauber G, Cendron M, Sinnecker GH, Keim L, Schwinger E, Wolfe HJ, Yandell DW (1994) Molecular characterization of the androgen receptor gene in boys with hypospadias. Eur J Pediatr 153: 317-21
Horikawa Y, Oda N, Cox NJ, Li X, Orho-Melander M, Hara M, Hinokio Y, Lindner TH, Mashima H, Schwarz PE, et al. (2000) Genetic variation in the gene encoding calpain-10 is associated with type 2 diabetes mellitus. Nat Genet 26: 163-75
Hugot JP, Chamaillard M, Zouali H, Lesage S, Cezard JP, Belaiche J, Almer S, Tysk C, O'Morain CA, Gassull M, et al. (2001) Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease. Nature 411: 599-603
Hugot JP, Laurent-Puig P, Gower-Rousseau C, Olson JM, Lee JC, Beaugerie L, Naom I, Dupas JL, Van Gossum A, Orholm M, et al. (1996) Mapping of a susceptibility locus for Crohn's disease on chromosome 16. Nature 379: 821-3
Hussain N, Chaghtai A, Herndon CD, Herson VC, Rosenkrantz TS, McKenna PH (2002) Hypospadias and early gestation growth restriction in infants. Pediatrics 109: 473-8
Imperato-McGinley J, Guerrero L, Gautier T, Peterson RE (1974) Steroid 5alpha-reductase deficiency in man: an inherited form of male pseudohermaphroditism. Science 186: 1213-5
Innis JW, Goodman FR, Bacchelli C, Williams TM, Mortlock DP, Sateesh P, Scambler PJ, McKinnon W, Guttmacher AE (2002) A HOXA13 allele with a missense mutation in the homeobox and a dinucleotide deletion in the promoter underlies Guttmacher syndrome. Hum Mutat 19: 573-4
Iselius L, Slack J, Littler M, Morton NE (1991) Genetic epidemiology of breast cancer in Britain. Ann Hum Genet 55: 151-9
Johnson GC, Todd JA (2000) Strategies in complex disease mapping. Curr Opin Genet Dev 10: 330-4
Johnson KR, Sweet HO, Donahue LR, Ward-Bailey P, Bronson RT, Davisson MT (1998) A new spontaneous mouse mutation of Hoxd13 with a polyalanine expansion and phenotype similar to human synpolydactyly. Hum Mol Genet 7: 1033-8
Katsanis N, Ansley SJ, Badano JL, Eichers ER, Lewis RA, Hoskins BE, Scambler PJ, Davidson WS, Beales PL, Lupski JR (2001) Triallelic inheritance in Bardet-Biedl syndrome, a Mendelian recessive disorder. Science 293: 2256-9
54 Khoury MJ, Beaty TH, Cohen BH (1993) Fundamentals of genetic epidemiology. Oxford University Press, New York, USA
Knight HML, Phillips NJ, Mouriquand PDE (1995) Female hypospadias: a case report. Journal of Pediatric Surgery 30: 1738-1740
Koff SA, Jayanthi VR (1999) Preoperative treatment with human chorionic gonadotropin in infancy decreases the severity of proximal hypospadias and chordee. J Urol 162: 1435-9
Kondo T, Zakany J, Innis JW, Duboule D (1997) Of fingers, toes and penises. Nature 390: 29
Kong A, Gulcher J, Stefansson K (1999) Genealogy certainly matters for multifactorial genetic disease. Bmj 319: 578-9
Konig IR, Schafer H, Muller HH, Ziegler A (2001) Optimized group sequential study designs for tests of genetic linkage and association in complex diseases. Am J Hum Genet 69: 590-600
Kosaki K, Kosaki R, Suzuki T, Yoshihashi H, Takahashi T, Sasaki K, Tomita M, McGinnis W, Matsuo N (2002) Complete mutation analysis panel of the 39 human HOX genes. Teratology 65: 50-62
Kristjansson K, Manolescu A, Kristinsson A, Hardarson T, Knudsen H, Ingason S, Thorleifsson G, Frigge ML, Kong A, Gulcher JR, et al. (2002) Linkage of essential hypertension to chromosome 18q. Hypertension 39: 1044-9
Kruglyak L (1999) Prospects for whole-genome linkage disequilibrium mapping of common disease genes. Nat Genet 22: 139-44
Kruglyak L, Lander ES (1995a) Complete multipoint sib-pair analysis of qualitative and quantitative traits. Am J Hum Genet 57: 439-54
Kruglyak L, Lander ES (1995b) High-resolution genetic mapping of complex traits. Am J Hum Genet 56: 1212-23
Källén B (1988) Case control study of hypospadias, based on registry information. Teratology 38: 45-50
Källén B, Bertollini R, Castilla E, Czeizel A, Knudsen LB, Martinez-Frias ML, Mastroiacovo P, Mutchinick O (1986) A joint international study on the epidemiology of hypospadias. Acta Paediatr Scand Suppl 324: 1-52
Källén B, Mastroiacovo P, Lancaster PA, Mutchinick O, Kringelbach M, Martinez-Frias ML, Robert E, Castilla EE (1991) Oral contraceptives in the etiology of isolated hypospadias. Contraception 44: 173-82
Källén B, Winberg J (1982) An epidemiological study of hypospadias in Sweden. Acta Paediatr Scand Suppl 293: 1-21
Laitinen T, Daly MJ, Rioux JD, Kauppi P, Laprise C, Petays T, Green T, Cargill M, Haahtela T, Lander ES, et al. (2001) A susceptibility locus for asthma-related traits on chromosome 7 revealed by genome-wide scan in a founder population. Nat Genet 28: 87-91
Lalouel JM, Morton NE (1981) Complex segregation analysis with pointers. Hum Hered 31: 312- 21
55 Lamy M (1952) Lecture. University Institute of Human Genetics, Copenhagen, Denmark
Lander E, Kruglyak L (1995) Genetic dissection of complex traits: guidelines for interpreting and reporting linkage results. Nat Genet 11: 241-7
Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar K, Doyle M, FitzHugh W, et al. (2001) Initial sequencing and analysis of the human genome. Nature 409: 860-921
Lander ES, Schork NJ (1994) Genetic dissection of complex traits. Science 265: 2037-48
Lindqvist AK, Steinsson K, Johanneson B, Kristjansdottir H, Arnasson A, Grondal G, Jonasson I, Magnusson V, Sturfelt G, Truedsson L, et al. (2000) A susceptibility locus for human systemic lupus erythematosus (hSLE1) on chromosome 2q. J Autoimmun 14: 169-78
Longmuir GA, Conley RN, Nicholson DL, Whitehead M (1986) The hand-foot-uterus syndrome: a case study. J Manipulative Physiol Ther 9: 213-7
Lowry RB, Kliman MR (1976) Hypospadias in successive generations - possible dominant gene inheritance. Clin Genet 9: 285-8
Macnab AJ, Zouves C (1991) Hypospadias after assisted reproduction incorporating in vitro fertilization and gamete intrafallopian transfer. Fertil Steril 56: 918-22
Malecki MT, Moczulski DK, Klupa T, Wanic K, Cyganek K, Frey J, Sieradzki J (2002) Homozygous combination of calpain 10 gene haplotypes is associated with type 2 diabetes mellitus in a Polish population. Eur J Endocrinol 146: 695-9
Martin ER, Monks SA, Warren LL, Kaplan NL (2000) A test for linkage and association in general pedigrees: the pedigree disequilibrium test. Am J Hum Genet 67: 146-54
McKusick VA (1986) OMIM # 140000 Hand-foot-uterus syndrome. http://www3.ncbi.nlm.nih.gov/htbin-post/Omim/dispmim?140000
McKusick VA (1990) OMIM # 142959 Hand-foot-uterus syndrome. http://www3.ncbi.nlm.nih.gov/htbin-post/Omim/dispmim?142959
McKusick VA (2002) OMIM. http://www.ncbi.nlm.nih.gov/omim
Meizner I, Mashiach R, Shalev J, Efrat Z, Feldberg D (2002) The 'tulip sign': a sonographic clue for in-utero diagnosis of severe hypospadias. Ultrasound Obstet Gynecol 19: 250-3
Merriam-Webster (2002) Merriam-Webster Collegiate Dictionary OnLine. http://www.m- w.com/cgi-bin/dictionary
Miki Y, Swensen J, Shattuck-Eidens D, Futreal PA, Harshman K, Tavtigian S, Liu Q, Cochran C, Bennett LM, Ding W, et al. (1994) A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science 266: 66-71
Monteleone Neto R, Castilla EE, Paz JE (1981) Hypospadias: an epidemiological study in Latin America. Am J Med Genet 10: 5-19
Mortlock DP, Innis JW (1997) Mutation of HOXA13 in hand-foot-genital syndrome. Nat Genet 15: 179-80
56 Mortlock DP, Post LC, Innis JW (1996) The molecular basis of hypodactyly (Hd): a deletion in Hoxa 13 leads to arrest of digital arch formation. Nat Genet 13: 284-9
Morton NE (1955) Sequential tests for the detection of linkage. Am J Hum Genet 7: 277-318
Muragaki Y, Mundlos S, Upton J, Olsen BR (1996) Altered growth and branching patterns in synpolydactyly caused by mutations in HOXD13. Science 272: 548-51
North K, Golding J (2000) A maternal vegetarian diet in pregnancy is associated with hypospadias. The ALSPAC Study Team. Avon Longitudinal Study of Pregnancy and Childhood. BJU Int 85: 107-13
Nyholt DR (2000) All LODs are not created equal. Am J Hum Genet 67: 282-8
Ogura Y, Bonen DK, Inohara N, Nicolae DL, Chen FF, Ramos R, Britton H, Moran T, Karaliuskas R, Duerr RH, et al. (2001) A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease. Nature 411: 603-6
Ott J (1991) Analysis of human genetic linkage. Johns Hopkins University Press, Baltimore, USA
Ott J (1996) Complex traits on the map. Nature 379: 772-3
Page LA (1979) Inheritance of uncomplicated hypospadias. Pediatrics 63: 788-90
Paulozzi LJ (1999) International trends in rates of hypospadias and cryptorchidism. Environ Health Perspect 107: 297-302
Paulozzi LJ, Erickson JD, Jackson RJ (1997) Hypospadias trends in two US surveillance systems. Pediatrics 100: 831-4
Paunio T, Ekelund J, Varilo T, Parker A, Hovatta I, Turunen JA, Rinard K, Foti A, Terwilliger JD, Juvonen H, et al. (2001) Genome-wide scan in a nationwide study sample of schizophrenia families in Finland reveals susceptibility loci on chromosomes 2q and 5q. Hum Mol Genet 10: 3037-48
Peltonen L, McKusick VA (2001) Genomics and medicine. Dissecting human disease in the postgenomic era. Science 291: 1224-9
Penrose LS (1935) The detection of autosomal linkage in data which consist of pairs of brothers and sisters of unspecified parentage. Annual Eugenics 6: 133-8
Pierik FH, Burdorf A, Nijman JM, de Muinck Keizer-Schrama SM, Juttmann RE, Weber RF (2002) A high hypospadias rate in The Netherlands. Hum Reprod 17: 1112-5
Poznanski AK, Kuhns LR, Lapides J, Stern AM (1975) A new family with the hand-foot-genital syndrome--a wider spectrum of the hamd-foot-uterus syndrome. Birth Defects Orig Artic Ser 11: 127-35
Poznanski AK, Stern AM, Gall JC, Jr. (1970) Radiographic findings in the hand-foot-uterus syndrome (HFUS). Radiology 95: 129-34
Pritchard JK, Cox NJ (2002) The allelic architecture of human disease genes: common disease- common variant. or not? Hum Mol Genet 11: 2417-23
57 Prokunina L, Castillejo-Lopez C, Oberg F, Gunnarsson I, Berg L, Magnusson V, Brookes AJ, Tentler D, Kristjansdottir H, Grondal G, et al. (2002) A regulatory polymorphism in PDCD1 is associated with susceptibility to systemic lupus erythematosus in humans. Nat Genet
Quigley CA, De Bellis A, Marschke KB, el-Awady MK, Wilson EM, French FS (1995) Androgen receptor defects: historical, clinical, and molecular perspectives. Endocr Rev 16: 271-321
Ramos-Arroyo MA (1991) Birth defects in twins: study in a Spanish population. Acta Genet Med Gemellol (Roma) 40: 337-44
Risch N (1990a) Linkage strategies for genetically complex traits. I. Multilocus models. Am J Hum Genet 46: 222-8
Risch N (1990b) Linkage strategies for genetically complex traits. II. The power of affected relative pairs. Am J Hum Genet 46: 229-41
Roberts CJ, Lloyd S (1973) Observations on the epidemiology of simple hypospadias. Br Med J 1: 768-70
Ronzoni G, De Giovanni L, Weir JM, Pasqui F, Menchinelli P (2001) Transposing the urethral meatus in the treatment of recurrent and postcoital cystitis in women with hypospadias. BJU Int 87: 894-6
Saarela J, Schoenberg Fejzo M, Chen D, Finnila S, Parkkonen M, Kuokkanen S, Sobel E, Tienari PJ, Sumelahti ML, Wikstrom J, et al. (2002) Fine mapping of a multiple sclerosis locus to 2.5 Mb on chromosome 17q22-q24. Hum Mol Genet 11: 2257-67
Sadler TW (1990) Langman´s medical embryology, Sixth edn. Williams & Wilkins, Baltimore, Maryland, USA
Sandberg DE, Meyer-Bahlburg HF, Hensle TW, Levitt SB, Kogan SJ, Reda EF (2001) Psychosocial adaptation of middle childhood boys with hypospadias after genital surgery. J Pediatr Psychol 26: 465-75
Sandberg DE, Meyer-Bahlburg HFL, Yager TJ, Hensle TW, Levitt SB, Kogan SJ, Reda EF (1995) Gender development in boys born with hypospadias. Psychoneuroendocrinology 20: 693-709
Sheffield VC, Weber JL, Buetow KH, Murray JC, Even DA, Wiles K, Gastier JM, Pulido JC, Yandava C, Sunden SL, et al. (1995) A collection of tri- and tetranucleotide repeat markers used to generate high quality, high resolution human genome-wide linkage maps. Hum Mol Genet 4: 1837- 44
Sides D, Goldstein RB, Baskin L, Kleiner BC (1996) Prenatal diagnosis of hypospadias. J Ultrasound Med 15: 741-746
Silver RI, Rodriguez R, Chang TS, Gearhart JP (1999a) In vitro fertilization is associated with an increased risk of hypospadias. J Urol 161: 1954-7
Silver RI, Rodriguez R, Chang TSK, Gearhart JP (1999b) In vitro fertilization is associated with an increased risk of hypospadias. The Journal of Urology 161: 1954-1957
Silver RI, Russell DW (1999) 5alpha-reductase type 2 mutations are present in some boys with isolated hypospadias. J Urol 162: 1142-5
58 Smith DJ, Lusis AJ (2002) The allelic structure of common disease. Hum Mol Genet 11: 2455-61
Smith ED (1997) The history of hypospadias. Pediatr Surg Int 12: 81-85
Smith GC, Smith MF, McNay MB, Fleming JE (1998) First-trimester growth and the risk of low birth weight. N Engl J Med 339: 1817-22
Stefansson H, Sigurdsson E, Steinthorsdottir V, Bjornsdottir S, Sigmundsson T, Ghosh S, Brynjolfsson J, Gunnarsdottir S, Ivarsson O, Chou TT, et al. (2002) Neuregulin 1 and susceptibility to schizophrenia. Am J Hum Genet 71: 877-92
Stern AM, Gall JC, Jr., Perry BL, Stimson CW, Weitkamp LR, Poznanski AK (1970) The hand- food-uterus syndrome: a new hereditary disorder characterized by hand and foot dysplasia, dermatoglyphic abnormalities, and partial duplication of the female genital tract. J Pediatr 77: 109- 16
Stoll C, Alembik Y, Roth MP, Dott B (1990) Genetic and environmental factors in hypospadias. J Med Genet 27: 559-63
Sweet RA, Schrott HG, Kurland R, Culp OS (1974) Study of the incidence of hypospadias in Rochester, Minnesota, 1940- 1970, and a case-control comparison of possible etiologic factors. Mayo Clin Proc 49: 52-8
Sørensen HR (1953) Hypospadias with special reference to etiology. Thesis. Munksgaard, Copenhagen.
Thompson AA, Nguyen LT (2000) Amegakaryocytic thrombocytopenia and radio-ulnar synostosis are associated with HOXA11 mutation. Nat Genet 26: 397-8
Todd JA (2001) Human genetics. Tackling common disease. Nature 411: 537, 539
Toppari J, Larsen JC, Christiansen P, Giwercman A, Grandjean P, Guillette LJ, Jr., Jegou B, Jensen TK, Jouannet P, Keiding N, et al. (1996) Male reproductive health and environmental xenoestrogens. Environ Health Perspect 104 Suppl 4: 741-803
Tsur M, Linder N, Cappis S (1987) Hypospadias in a consanguineous family. Am J Med Genet 27: 487-9
Utsch B, Becker K, Brock D, Lentze MJ, Bidlingmaier F, Ludwig M (2002) A novel stable polyalanine [poly(A)] expansion in the HOXA13 gene associated with hand-foot-genital syndrome: proper function of poly(A)-harbouring transcription factors depends on a critical repeat length? Hum Genet 110: 488-94
Van Eerdewegh P, Little RD, Dupuis J, Del Mastro RG, Falls K, Simon J, Torrey D, Pandit S, McKenny J, Braunschweiger K, et al. (2002) Association of the ADAM33 gene with asthma and bronchial hyperresponsiveness. Nature 418: 426-30
Venter JC, Adams MD, Myers EW, Li PW, Mural RJ, Sutton GG, Smith HO, Yandell M, Evans CA, Holt RA, et al. (2001) The sequence of the human genome. Science 291: 1304-51
Veraksa A, Del Campo M, McGinnis W (2000) Developmental patterning genes and their conserved functions: from model organisms to humans. Mol Genet Metab 69: 85-100
59 Verp MS, Simpson JL, Elias S, Carson SA, Sarto GE, Feingold M (1983) Heritable aspects of uterine anomalies. I. Three familial aggregates with Mullerian fusion anomalies. Fertil Steril 40: 80-5
Virtanen HE, Kaleva M, Haavisto AM, Schmidt IM, Chellakooty M, Main KM, Skakkebaek NE, Toppari J (2001) The birth rate of hypospadias in the Turku area in Finland. Apmis 109: 96-100
Warot X, Fromental-Ramain C, Fraulob V, Chambon P, Dolle P (1997) Gene dosage-dependent effects of the Hoxa-13 and Hoxd-13 mutations on morphogenesis of the terminal parts of the digestive and urogenital tracts. Development 124: 4781-91
Warren ST (1997) Polyalanine expansion in synpolydactyly might result from unequal crossing-over of HOXD13. Science 275: 408-9
Weidner IS, Moller H, Jensen TK, Skakkebaek NE (1998) Cryptorchidism and hypospadias in sons of gardeners and farmers. Environ Health Perspect 106: 793-6
Weidner IS, Moller H, Jensen TK, Skakkebaek NE (1999) Risk factors for cryptorchidism and hypospadias. J Urol 161: 1606-9
Wennerholm U-B, Bergh C, Hamberger L, Lundin K, Nilsson L, Wikland M, Källén B (2000) Incidence of congenital malformations in children born after ICSI. Human Reproduction 15: 944- 948
Wilson JD, Griffin JE, Russell DW (1993) Steroid 5 alpha-reductase 2 deficiency. Endocr Rev 14: 577-93
Wooster R, Bignell G, Lancaster J, Swift S, Seal S, Mangion J, Collins N, Gregory S, Gumbs C, Micklem G (1995) Identification of the breast cancer susceptibility gene BRCA2. Nature 378: 789- 92
Zakany J, Duboule D (1996) Synpolydactyly in mice with a targeted deficiency in the HoxD complex. Nature 384: 69-71
60