Phenotypic heterogeneity and mutational spectrum in a cohort of 45 Italian males subjects with X-linked ectodermal dysplasia Guazzarotti Laura 1, Tadini Gianluca 2, Mancini Giovanni Evangelista 3, Giglio Sabrina Willoughby Colin Eric 6, Callea Michele
7,
Sani Ilaria 4, Nannini Pilar 1, Mameli Chiara
1,
4-5
Tenconi
Andrea Angela1, Mauri Silvia 1, Bottero Alessandro 8, Caimi Antonio 9, Morelli Marco 1, Zuccotti Gian Vincenzo 1 1. Department of Pediatrics, University of Milan, Luigi Sacco Hospital, Milan 2. Centre for Inherited Cutaneous Diseases, Pediatric Dermatology Unit, Department of Anesthesiologic and Dermatologic Sciences. IRCCS Ca' Granda Policlinico Hospital, Milan 3. Department of Maxillo-Facial Surgery Orthodontic Unit, IRCCS Galeazzi, Milan 4. Medical Genetics Unit, Meyer Children’s University Hospital, Florence 5. Medical Genetics Section, Department of Clinical Pathophysiology, University of Florence, Florence 6. Department of Eye and Vision Science, Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, UK 7. Institute for Maternal and Child Health - IRCCS “Burlo Garofolo” - Trieste, Italy 8. Department of Otorhinolaryngology, University of Milan, Luigi Sacco Hospital, Milan 9. Department of Ophthalmology, University of Milan, Luigi Sacco Hospital, Milan Corresponding author: Surname, Name: Guazzarotti, Laura Address: via G.B. Grassi, 74, 20157 Milano (Italy) Telephone:+39 02 39042234, +39 02 39042253 Fax: +39 02 39042234 e-mail: [email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/cge.12404 This article is protected by copyright. All rights reserved
Conflict of interest The authors report no conflict of interest.
Acknowledgements The authors acknowledge all patients studied and their families. The National Association of Ectodermal Dysplasia (ANDE) and the Foundation of National Association of Italian Dentists (ANDI).
Abstract Ectodermal dysplasias are a group of genetic disorders characterized by the abnormal development of the ectodermal-derived structures. X-linked hypohidrotic ectodermal dysplasia, resulting from mutations in ED1 gene, is the most common form. The main purpose of this study was to characterize the phenotype spectrum in 45 males harboring ED1 mutations. The study showed that in addition to the involvment of the major ectodermal tissues, the majority of patients also have alterations of several minor ectodermal-derived structures. Characterising the clinical spectrum resulting from ED1 gene mutations improves diagnosis and can direct clinical care.
Introduction
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Ectodermal dysplasias (EDs) represent a genetically heterogeneous group of diseases characterized by the abnormal development of ectodermal derivatives (1). The prevalence of the different EDs altogether is highly variable, the latest estimate is 7/10.000 newborns (2). The most common form is the X-linked Hypohidrotic Ectodermal Dysplasia (XLHED) due to mutations in the ED1 gene, mapped to Xq12-13.1 and encoding the ectodysplasin-A protein. Currently, only one population-based prevalence study is available (3) that reported a prevalence of molecularly-confirmed XLHED as 1.6/100.000 persons. XLHED is characterized by major signs comprising abnormal and missing teeth, inability to sweat, sparse hair associated with several other minor ectodermal manifestations often poorly characterized. A not well-defined risk for neurodevelopmental disorders has been reported over the decades. (4,5). Allergic manifestations have also been reported in EDs for many years (6, 7). Intra and inter-familial variability in clinical manifestations are present and no genotype/phenotype correlations have been reported (8, 9). The aims of our study were to describe the phenotype spectrum of an Italian cohort of males with molecularlyconfirmed XLHED and to investigate potential genotype-phenotype correlations.
Material and methods
The molecular screening of the ED1 gene was performed in 65 male patients (aged 2 to 20 years) referred to our Clinic with a suspicion of ED and presenting with the impairment of at least one of the following: teeth, hair or sweat glands. Written informed consent was obtained from patients or their parents. The ED1 gene was studied by direct sequence of the entire coding region and multiplex ligation-dependent probe amplification analysis. DNA was extracted from peripheral blood using a standard protocol. Exons and flanking intronic sequences were amplified by PCR. 200 ethnically matched control chromosomes were assessed for each identified ED1 sequence variant. SIFT v.2 and POLYPHEN algorithms were used to predict the pathogenicity of the identified ED1 sequence. This article is protected by copyright. All rights reserved
Dentition was assessed by panoramic radiographs. Hypodontia was defined as agenesis of less than six teeth (+1), oligodontia as agenesis of six or more (+2). The shape of teeth was classified as abnormal if conical or tapered (+2). Sweating was studied with standard starch-jodine method on the palms, both in patients and in a group of healthy subjects. We assumed as moderate dysfunction (+1) a number of pores in a centimeter square between 70% and 20% of normal and as severe dysfunction (+2) a number of pores < 20%. Moderate impairment of hair was defined by the presence of thin and slightly sparse hair (+1) and severe phenotype (+2) by the presence of very sparse thin hair. Moderate involvement (+1) of the skin was defined by the presence of light and thin skin. Severe skin involvement (+2) was defined when visible venous pattern, xerosis, periorbital hyperpigmentation and absence of hair follicles were present. Moderate onychodystrophy (+1) was considered when nails were thin and fragile, severe onychodystrophy (+2) when micronychia and a reduced growth were also present. Eye evaluation included a slit lamp examination of the anterior segment and dilated fundus examination. To study defective meibomain glands and evaporative dry eye all patients underwent measurement of the tear break-up time (TBUT) (10). A global assessment of the ocular surface was performed and graded as described in Table S1 (Supporting information). Hearing was assessed with tuning forks and screening audiograms with tympanometry. A moderate involvement of the respiratory tract function was defined when more than six upper respiratory tract infections (URTIs) per year were present (+1), a severe involvement (+2) when patients suffered from at least 3 lower respiratory tract infections (LRTIs) per year. Allergic disorders were investigated in all patients through clinical and laboratory assessment (total serum IgE) and skin prick tests (SPT). Attentive anamnestic recollection of neurological and psychological development was done. The psychomotor evaluation was performed with Bayley Scales of Infant Development and Cognitive evaluation with Wechsler Scales according to age. Psychiatric evaluation was performed based on clinical suspicion and diagnosis was made according to the Diagnostic and Statistical Manual of Mental Disorders. (Table 1). To study the association between genotype and phenotype, XLHED subjects were divided in two groups: those with missense variations (Group A) and those with all the other more complex structural changes (Group
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B) of the ED1 gene. Total clinical scores of the phenotypic characteristics for the different groups were considered. Comparison between groups was made through Wilcoxon non-parametric test.
Results Molecular analysis of the ED1 gene identified mutations in 45 of 65 studied subjects: 12 novel mutations and 33 previously reported mutations (Table S2 and Figure 1, Supporting Information). None of the 12 novel mutations were detected in a panel of 200 chromosomes from unaffected/unrelated individuals. The phenotypic characteristics of patients carrying ED1 mutations are reported in Table S3 (Supporting information). Dental agenesis were found in 97.5% of patients; 33 (84.6%) had oligodontia while 6 (15%) hypodontia. One patient had the normal total number of teeth without morphological alterations. Abnormal crown morphology of teeth such as conical or tapered was present in 37 patients (92.5%). Most patients (86.7%) showed impairment of sweating, which was severe in 26 (57.8%) and normal in six patients (13.3%). Forty-two patients (93.3%) showed alteration of hair: 8 (17.8%) had total hair alopecia and 34 (75.6%) only mild. Twenty-eight patients (62.2%) showed severe skin abnormalities and 16 (35.6%) moderate. Only one patient had no dermatological abnormalities. Alterations in nails were present in 31 patients (68.8%) but only in 3 were severe (6.7%). Abnormalities of ocular surface, meibomian glands and tear film were found in 86.7% of patients: 21 (46.7%) showed a severe dry eye involvement (+2). Six patients (13.3%) showed minimal or none ocular or tear film alteration (0). A total of 32 patients (71%) suffered from URTIs and LRTIs. In 10 (22.2%) a history of at least three LTRIs per year was present. Two patients showed a neurosensorial hearing loss. Another patient showed a type C tympanogram probably due to endotimpanic transudate and a fourth patient showed a type B tympanogram probably due to recurrent otitis media.
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Thirty-two subjects (71.1%) showed allergic manifestations with at least one positive SPT: atopic dermatitis was the most frequent (66.6%), followed by rhino-conjunctivitis (26.6%) and asthma (17.7%). Food allergy was found only in 1 subject. Seventeen patients (37.7%) showed more than one allergic manifestations. Eleven patients showed psychomotor and/or psychological disorders (Table 1). Seven of the 45 patients (15.%) presented a mild psychomotor delay, 4/7 subjects also had a speech delay that was not identified in a developmental language specific disorder but was due to a severe dental agenesia. Three of seven showed a mild cognitive disorder at the time of primary school that resolved in adolescence. Nine patients of 45 (20 %) showed mild psychiatric disorders, three of them were 2 years old, four were in the first decade of life and two were adolescents. One of the 45 patients showed only mild cognitive delay at the age of 5. The study of the association between the severity of mutation structure and disease severity measured by the total clinical score, showed that subjects with missense variations had a mean total score equal to 11.4±2.4 in Group A vs 10.5±2.4 in Group B. No statistically significant difference was found (p=0.2438).
Discussion In keeping with previous data this study showed that severe dental agenesis and/or sweating impairment are the most common manifestations in XLHED subjects (11-12). The comprehensive clinical study showed
that the association of the clinical signs was very variable and included several minor ectodermalderived tissue alterations. ED1 mutations are reported to be associated with isolated hypodontia, but none of our patients showed this phenotype (13). In subject ED8 a severe sweating deficiency was the only major clinical sign. This patient carried a nucleotide substitution on exon 3 predicting an arginine to histidine missense mutation at amino acid position 153 in the region corresponding to the furin domain of ED1. Mutations involving Arg 153 position are reported to have a mild effect on the cleavage of ED1 (14) and the c.458G> A mutation has been described in two brothers who had mild dental abnormalities without others signs of florid ED (15). Our observation confirm the mild clinical effect of the c.458G>A mutation despite being in the furin domain. This article is protected by copyright. All rights reserved
Although nail alterations are common among various type of EDs no studies have previously examined their frequency in patients with XLHED. We found that the majority of patients showed nail abnormalities, mostly moderate. No data are present concerning the distribution of hair alterations among ED patients. In most of our XLHED subjects (93%) there was an alteration in the growth or morphology of the hair but mostly mild. Absence or dysfunction of meibomian glands leads to absence in lipid layer of the tear film resulting in impairment of ocular surface. Severe complications can appear as corneal ulceration or pannus (16). Clinical reports have described rare eye alterations associated to EDs (subretinal fibrosis and uveitis, retinal detachment, macular dystrophy, glaucoma) (17-20). In our study one patient had a coloboma of the optic nerve which hasn’t been previously reported. Absence of mucous glands in respiratory tract is associated with susceptibility to develop respiratory infections and hearing problems. One study reported a higher incidence of respiratory tract infections but LRTIs were not analyzed (7). In our study 22% of patients suffered from frequent LRTIs. Otologic problems range from impacted cerumen to recurrent otitis media and hearing loss. A frequency of hearing loss higher than in general population was found in our patients, but lower than reported in a recent study which considered several forms of EDs (21). Prevalence of atopic diseases among our patients is higher than general population, as a result of altered lipid profile in the stratuum corneum (22). The epidermal barrier dysfunction has been proposed as primary event in the development of allergy (23). However, our atopy prevalence is lower than those found in a recent study, where the diagnosis was based only on a questionnaire completed by patients’ families (6). Our data showed that in ED patients mild psychomotor and cognitive disorders may be present during childhood most likely as an expression of frequent and severe episodes of hyperthermia. Our data suggest that these disorders tend to resolve with increasing age being absent in all our patients in the second decade of life when they show a normal IQ. The mild cognitive delay could be a consequence of a negative emotional status of child and parents and improvement and resolution of the delay in adolescence could be due to the child’s cognitive and emotional development. Mild psychiatric disorders may be present as early as in the first years of life and tend to persist into school age and adolescence, most likely as a consequence of their chronic
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illness condition, or as a response to stigmatisation and bullying. Nevertheless, caution is necessary in the interpretation of these data and further studies are needed.
Despite the number of our patients does not allow for specific conclusions we suggest that indication for exhaustive molecular screening of mutations in the ED1 gene, is the involvment of at least one of the major ectodermal derived tissues. This feature may have a variable association with other major and/or minor ectodermal abnormalities, the minor ones should be investigated carefully.
References 1. Visinoni AF, Lisboa-Costa T, Pagnan NA, et al. Ectodermal dysplasias: clinical and molecular review. Am J Med Genet A 2009: 149A: 1980–2002. 2. Wright JT, Morris C, Clements SE, et al. Classifying ectodermal dysplasias: Incorporating the molecular basis and pathways. Am J Med Genet A 2009: 149A: 2062–2067. 3. Nguyen-Nielsen M, Skovbo S, Svaneby D, Pedersen L, Fryzek J. The prevalence of X-linked hypohidrotic ectodermal dysplasia (XLHED) in Denmark, 1995-2010. Eur J Med Genet. 2013; 56:236-42 4. Blüschke G, Nüsken KD, Schneider H. Prevalence and prevention of severe complications of hypohidrotic ectodermal dysplasia in infancy. Early Hum Dev 2010.: 86: 397–399. 5. Tanner BA. Psychological aspects of hypohidrotic ectodermal dysplasia. Birth Defects Orig Artic Ser 1988: 24: 263–275 6. Mark BJ, Becker BA, Halloran DR, et al. Prevalence of atopic disorders and immunodeficiency in patients with ectodermal dysplasia syndromes. Ann Allergy Asthma Immunol 2012: 108:435-438. 7. Clarke A, Phillips DI, Brown R, et al. Clinical aspects of X linked hypohidrotic ectodermal dysplasia. Arch Dis Child 1987: 62: 989‐996. 8. Vincent MC, Biancalana V, Ginisty D, et al. Mutational spectrum of the ED1 gene in X-linked hypohidrotic ectodermal dysplasia. Eur J Hum Genet 2001: 9: 355–363. This article is protected by copyright. All rights reserved
9. Kobielak K, Kobielak A, Roszkiewicz J, Wierzba J, Limon J, Trzeciak WH. Mutations in the EDA gene in three unrelated families reveal no apparent correlation between phenotype and genotype in the patients with an X-linked anhidrotic ectodermal dysplasia. Am J Med Genet 2001;100:191–7. 10. International Dry Eye WorkShop. Ocul Surf. 2007 ;5:75-92. 11. Lexner MO, Bardow A, Hertz JM, et al. Anomalies of tooth formation in hypohidrotic ectodermal dysplasia. Int J Paediatr Dent 2007: 17: 10-18. 12. Wei Y,
XiaoQian Y, Zhuan B. Phenotypic findings in seven Chinese X-Linked hypohidrotic
ectodermal dysplasia HED families. Arch Oral Biol 2012: 57: 1418-1422 13. Fan H, Ye X, Shi L, et al. Mutations in the EDA gene are responsible for X-linked hypohidrotic ectodermal dysplasia and hypodontia in Chinese kindreds. Eur J Oral Sci 2008: 116: 412–417. 14. Schneider P, Street SL, Gaide O, et al. Mutations leading to X-linked hypohidrotic ectodermal dysplasia affect three major functional domains in the tumor necrosis factor family member ectodysplasin-A. J Biol Chem. 2001;276: 18819-18827 15. Keller MD, Petersen M, Ong P, Church J, Risma K, Burham J, Jain A, Stiehm ER, Hanson EP, Uzel G, Deardorff MA, Orange JS. Hypohidrotic ectodermal dysplasia and immunodeficiency with coincident NEMO and EDA mutations. Front Immunol. 2011; 2:61 16. Kaercher T. Ocular symptoms and signs in patients with ectodermal dysplasia syndromes. Graefes Arch Clin Exp Ophthalmol 2004: 242: 495-500. 17. Huffman RI, Huang JJ. Subretinal fibrosis and uveitis syndrome in a patient with ectodermal dysplasia. Ocul Immunol Inflamm 2009: 17: 348-350. 18. Wald KJ, Hirose T. Ectodermal dysplasia, ectrodactyly, and clefting syndrome and bilateral retinal detachment. Arch Ophthalmol 1993: 111:734. 19. Hayakawa M, Yanashima K, Kato K, et al. Association of ectodermal dysplasia, ectrodactyly and macular dystrophy: EEM syndrome (case report). Ophthalmic Paediatr Genet 1989: 10:287-292.
20. Callea M, Vinciguerra A, Willoughby CE, et al. Infantile bilateral glaucoma in a child with ectodermal dysplasia. Ophthalmic Genet. 2013: 34: 58-60. 21. Yildirim M, Yorgancilar E, Gun R, et al. Ectodermal dysplasia: otolaryngologic evaluation of 23 cases. Ear Nose Throat J 2012: 91: 28-33 This article is protected by copyright. All rights reserved
22. Jungersted JM, Høgh JK, Hellgren LI.. Ceramide profile in hypohidrotic ectodermal dysplasia. Clin Exp Dermatol 2012: 37: 153-155 23. Cork MJ, Danby SG, Vasilopoulos Y, et al. Epidermal barrier dysfunction in atopic dermatitis. J Invest Dermatol 2009: 129:1892-1908.
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Legend to Figure 1 Novel mutations are showed in bold TM: predicted transmembrane domain; F: putative furine cleavage site; COL: collagenous domain;TNF: tumor necrosis factor-homologous regions
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Patient code
Age (yrs)
Psychomotor development (BSID) a Cognitive development (Wechsler scales) b
Psychiatric Diagnosis (DSM-IV)
ED 1
9
a
Not available
b
9 yrs 1/12
IQ 102
Attention deficit hyperactivity disorder (314.9) Generalized Anxiety disorder (300.02)
ED 8
18
a
42 months
Mild mental retardation (317)
MDI 69 PDI 76 b
ED 10
3
7 yrs 5/12
b
17 yrs 10/12
a
36 months
IQ 69
IQ 90
MDI 72
Mild mental retardation (317)
Generalized Anxiety disorder (300.02) Mild mental retardation (317)
PDI 66
ED 13
2
a
24 months
Disruptive behavior disorder (312.9)
MDI 85 PDI 68 BRS 68 ED 18
2
a
24 months
Disruptive behavior disorder (312.9)
MDI 86 PDI 70 BRS 74 ED 21
2
a
24 months
MDI 90 PDI 68
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BRS 78 ED 22
11
a
Not done
Disruptive behavior disorder (312.9)
b
11 yrs
IQ 103
Dysthymic disorder (300.4) (CDI 24)
ED 24
ED 25
11
9
a
Not done
b
10 yrs 10/12 IQ 99
Dysthymic disorder (300.4) Generalized Anxiety disorder (300.02)
a
48 months
Mild mental retardation (317)
MDI 70 PDI 68 BRS 74 b
8 yrs 10/12
IQ 69
Mild mental retardation (317) Dysthymic disorder (300.4) (CDI 20)
ED 29
17
a
Mild mental retardation (317)
48 months
MDI 67 PDI 63 b
7 yrs 2/12
IQ 68
Mild mental retardation (317)
b
16 yrs 10/12
IQ 102
Dysthymic disorder (300.4) (CDI 23)
ED 38
5
b
4 yrs 10/12
IQ 69
Mild mental retardation (317)
Table 1: Characterization of Neurodevelopmental and Psychological disorders in XLHED subjects
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7,
Sani Ilaria 4, Nannini Pilar 1, Mameli Chiara
1,
4-5
Tenconi
Andrea Angela1, Mauri Silvia 1, Bottero Alessandro 8, Caimi Antonio 9, Morelli Marco 1, Zuccotti Gian Vincenzo 1 1. Department of Pediatrics, University of Milan, Luigi Sacco Hospital, Milan 2. Centre for Inherited Cutaneous Diseases, Pediatric Dermatology Unit, Department of Anesthesiologic and Dermatologic Sciences. IRCCS Ca' Granda Policlinico Hospital, Milan 3. Department of Maxillo-Facial Surgery Orthodontic Unit, IRCCS Galeazzi, Milan 4. Medical Genetics Unit, Meyer Children’s University Hospital, Florence 5. Medical Genetics Section, Department of Clinical Pathophysiology, University of Florence, Florence 6. Department of Eye and Vision Science, Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, UK 7. Institute for Maternal and Child Health - IRCCS “Burlo Garofolo” - Trieste, Italy 8. Department of Otorhinolaryngology, University of Milan, Luigi Sacco Hospital, Milan 9. Department of Ophthalmology, University of Milan, Luigi Sacco Hospital, Milan Corresponding author: Surname, Name: Guazzarotti, Laura Address: via G.B. Grassi, 74, 20157 Milano (Italy) Telephone:+39 02 39042234, +39 02 39042253 Fax: +39 02 39042234 e-mail: [email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/cge.12404 This article is protected by copyright. All rights reserved
Conflict of interest The authors report no conflict of interest.
Acknowledgements The authors acknowledge all patients studied and their families. The National Association of Ectodermal Dysplasia (ANDE) and the Foundation of National Association of Italian Dentists (ANDI).
Abstract Ectodermal dysplasias are a group of genetic disorders characterized by the abnormal development of the ectodermal-derived structures. X-linked hypohidrotic ectodermal dysplasia, resulting from mutations in ED1 gene, is the most common form. The main purpose of this study was to characterize the phenotype spectrum in 45 males harboring ED1 mutations. The study showed that in addition to the involvment of the major ectodermal tissues, the majority of patients also have alterations of several minor ectodermal-derived structures. Characterising the clinical spectrum resulting from ED1 gene mutations improves diagnosis and can direct clinical care.
Introduction
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Ectodermal dysplasias (EDs) represent a genetically heterogeneous group of diseases characterized by the abnormal development of ectodermal derivatives (1). The prevalence of the different EDs altogether is highly variable, the latest estimate is 7/10.000 newborns (2). The most common form is the X-linked Hypohidrotic Ectodermal Dysplasia (XLHED) due to mutations in the ED1 gene, mapped to Xq12-13.1 and encoding the ectodysplasin-A protein. Currently, only one population-based prevalence study is available (3) that reported a prevalence of molecularly-confirmed XLHED as 1.6/100.000 persons. XLHED is characterized by major signs comprising abnormal and missing teeth, inability to sweat, sparse hair associated with several other minor ectodermal manifestations often poorly characterized. A not well-defined risk for neurodevelopmental disorders has been reported over the decades. (4,5). Allergic manifestations have also been reported in EDs for many years (6, 7). Intra and inter-familial variability in clinical manifestations are present and no genotype/phenotype correlations have been reported (8, 9). The aims of our study were to describe the phenotype spectrum of an Italian cohort of males with molecularlyconfirmed XLHED and to investigate potential genotype-phenotype correlations.
Material and methods
The molecular screening of the ED1 gene was performed in 65 male patients (aged 2 to 20 years) referred to our Clinic with a suspicion of ED and presenting with the impairment of at least one of the following: teeth, hair or sweat glands. Written informed consent was obtained from patients or their parents. The ED1 gene was studied by direct sequence of the entire coding region and multiplex ligation-dependent probe amplification analysis. DNA was extracted from peripheral blood using a standard protocol. Exons and flanking intronic sequences were amplified by PCR. 200 ethnically matched control chromosomes were assessed for each identified ED1 sequence variant. SIFT v.2 and POLYPHEN algorithms were used to predict the pathogenicity of the identified ED1 sequence. This article is protected by copyright. All rights reserved
Dentition was assessed by panoramic radiographs. Hypodontia was defined as agenesis of less than six teeth (+1), oligodontia as agenesis of six or more (+2). The shape of teeth was classified as abnormal if conical or tapered (+2). Sweating was studied with standard starch-jodine method on the palms, both in patients and in a group of healthy subjects. We assumed as moderate dysfunction (+1) a number of pores in a centimeter square between 70% and 20% of normal and as severe dysfunction (+2) a number of pores < 20%. Moderate impairment of hair was defined by the presence of thin and slightly sparse hair (+1) and severe phenotype (+2) by the presence of very sparse thin hair. Moderate involvement (+1) of the skin was defined by the presence of light and thin skin. Severe skin involvement (+2) was defined when visible venous pattern, xerosis, periorbital hyperpigmentation and absence of hair follicles were present. Moderate onychodystrophy (+1) was considered when nails were thin and fragile, severe onychodystrophy (+2) when micronychia and a reduced growth were also present. Eye evaluation included a slit lamp examination of the anterior segment and dilated fundus examination. To study defective meibomain glands and evaporative dry eye all patients underwent measurement of the tear break-up time (TBUT) (10). A global assessment of the ocular surface was performed and graded as described in Table S1 (Supporting information). Hearing was assessed with tuning forks and screening audiograms with tympanometry. A moderate involvement of the respiratory tract function was defined when more than six upper respiratory tract infections (URTIs) per year were present (+1), a severe involvement (+2) when patients suffered from at least 3 lower respiratory tract infections (LRTIs) per year. Allergic disorders were investigated in all patients through clinical and laboratory assessment (total serum IgE) and skin prick tests (SPT). Attentive anamnestic recollection of neurological and psychological development was done. The psychomotor evaluation was performed with Bayley Scales of Infant Development and Cognitive evaluation with Wechsler Scales according to age. Psychiatric evaluation was performed based on clinical suspicion and diagnosis was made according to the Diagnostic and Statistical Manual of Mental Disorders. (Table 1). To study the association between genotype and phenotype, XLHED subjects were divided in two groups: those with missense variations (Group A) and those with all the other more complex structural changes (Group
This article is protected by copyright. All rights reserved
B) of the ED1 gene. Total clinical scores of the phenotypic characteristics for the different groups were considered. Comparison between groups was made through Wilcoxon non-parametric test.
Results Molecular analysis of the ED1 gene identified mutations in 45 of 65 studied subjects: 12 novel mutations and 33 previously reported mutations (Table S2 and Figure 1, Supporting Information). None of the 12 novel mutations were detected in a panel of 200 chromosomes from unaffected/unrelated individuals. The phenotypic characteristics of patients carrying ED1 mutations are reported in Table S3 (Supporting information). Dental agenesis were found in 97.5% of patients; 33 (84.6%) had oligodontia while 6 (15%) hypodontia. One patient had the normal total number of teeth without morphological alterations. Abnormal crown morphology of teeth such as conical or tapered was present in 37 patients (92.5%). Most patients (86.7%) showed impairment of sweating, which was severe in 26 (57.8%) and normal in six patients (13.3%). Forty-two patients (93.3%) showed alteration of hair: 8 (17.8%) had total hair alopecia and 34 (75.6%) only mild. Twenty-eight patients (62.2%) showed severe skin abnormalities and 16 (35.6%) moderate. Only one patient had no dermatological abnormalities. Alterations in nails were present in 31 patients (68.8%) but only in 3 were severe (6.7%). Abnormalities of ocular surface, meibomian glands and tear film were found in 86.7% of patients: 21 (46.7%) showed a severe dry eye involvement (+2). Six patients (13.3%) showed minimal or none ocular or tear film alteration (0). A total of 32 patients (71%) suffered from URTIs and LRTIs. In 10 (22.2%) a history of at least three LTRIs per year was present. Two patients showed a neurosensorial hearing loss. Another patient showed a type C tympanogram probably due to endotimpanic transudate and a fourth patient showed a type B tympanogram probably due to recurrent otitis media.
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Thirty-two subjects (71.1%) showed allergic manifestations with at least one positive SPT: atopic dermatitis was the most frequent (66.6%), followed by rhino-conjunctivitis (26.6%) and asthma (17.7%). Food allergy was found only in 1 subject. Seventeen patients (37.7%) showed more than one allergic manifestations. Eleven patients showed psychomotor and/or psychological disorders (Table 1). Seven of the 45 patients (15.%) presented a mild psychomotor delay, 4/7 subjects also had a speech delay that was not identified in a developmental language specific disorder but was due to a severe dental agenesia. Three of seven showed a mild cognitive disorder at the time of primary school that resolved in adolescence. Nine patients of 45 (20 %) showed mild psychiatric disorders, three of them were 2 years old, four were in the first decade of life and two were adolescents. One of the 45 patients showed only mild cognitive delay at the age of 5. The study of the association between the severity of mutation structure and disease severity measured by the total clinical score, showed that subjects with missense variations had a mean total score equal to 11.4±2.4 in Group A vs 10.5±2.4 in Group B. No statistically significant difference was found (p=0.2438).
Discussion In keeping with previous data this study showed that severe dental agenesis and/or sweating impairment are the most common manifestations in XLHED subjects (11-12). The comprehensive clinical study showed
that the association of the clinical signs was very variable and included several minor ectodermalderived tissue alterations. ED1 mutations are reported to be associated with isolated hypodontia, but none of our patients showed this phenotype (13). In subject ED8 a severe sweating deficiency was the only major clinical sign. This patient carried a nucleotide substitution on exon 3 predicting an arginine to histidine missense mutation at amino acid position 153 in the region corresponding to the furin domain of ED1. Mutations involving Arg 153 position are reported to have a mild effect on the cleavage of ED1 (14) and the c.458G> A mutation has been described in two brothers who had mild dental abnormalities without others signs of florid ED (15). Our observation confirm the mild clinical effect of the c.458G>A mutation despite being in the furin domain. This article is protected by copyright. All rights reserved
Although nail alterations are common among various type of EDs no studies have previously examined their frequency in patients with XLHED. We found that the majority of patients showed nail abnormalities, mostly moderate. No data are present concerning the distribution of hair alterations among ED patients. In most of our XLHED subjects (93%) there was an alteration in the growth or morphology of the hair but mostly mild. Absence or dysfunction of meibomian glands leads to absence in lipid layer of the tear film resulting in impairment of ocular surface. Severe complications can appear as corneal ulceration or pannus (16). Clinical reports have described rare eye alterations associated to EDs (subretinal fibrosis and uveitis, retinal detachment, macular dystrophy, glaucoma) (17-20). In our study one patient had a coloboma of the optic nerve which hasn’t been previously reported. Absence of mucous glands in respiratory tract is associated with susceptibility to develop respiratory infections and hearing problems. One study reported a higher incidence of respiratory tract infections but LRTIs were not analyzed (7). In our study 22% of patients suffered from frequent LRTIs. Otologic problems range from impacted cerumen to recurrent otitis media and hearing loss. A frequency of hearing loss higher than in general population was found in our patients, but lower than reported in a recent study which considered several forms of EDs (21). Prevalence of atopic diseases among our patients is higher than general population, as a result of altered lipid profile in the stratuum corneum (22). The epidermal barrier dysfunction has been proposed as primary event in the development of allergy (23). However, our atopy prevalence is lower than those found in a recent study, where the diagnosis was based only on a questionnaire completed by patients’ families (6). Our data showed that in ED patients mild psychomotor and cognitive disorders may be present during childhood most likely as an expression of frequent and severe episodes of hyperthermia. Our data suggest that these disorders tend to resolve with increasing age being absent in all our patients in the second decade of life when they show a normal IQ. The mild cognitive delay could be a consequence of a negative emotional status of child and parents and improvement and resolution of the delay in adolescence could be due to the child’s cognitive and emotional development. Mild psychiatric disorders may be present as early as in the first years of life and tend to persist into school age and adolescence, most likely as a consequence of their chronic
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illness condition, or as a response to stigmatisation and bullying. Nevertheless, caution is necessary in the interpretation of these data and further studies are needed.
Despite the number of our patients does not allow for specific conclusions we suggest that indication for exhaustive molecular screening of mutations in the ED1 gene, is the involvment of at least one of the major ectodermal derived tissues. This feature may have a variable association with other major and/or minor ectodermal abnormalities, the minor ones should be investigated carefully.
References 1. Visinoni AF, Lisboa-Costa T, Pagnan NA, et al. Ectodermal dysplasias: clinical and molecular review. Am J Med Genet A 2009: 149A: 1980–2002. 2. Wright JT, Morris C, Clements SE, et al. Classifying ectodermal dysplasias: Incorporating the molecular basis and pathways. Am J Med Genet A 2009: 149A: 2062–2067. 3. Nguyen-Nielsen M, Skovbo S, Svaneby D, Pedersen L, Fryzek J. The prevalence of X-linked hypohidrotic ectodermal dysplasia (XLHED) in Denmark, 1995-2010. Eur J Med Genet. 2013; 56:236-42 4. Blüschke G, Nüsken KD, Schneider H. Prevalence and prevention of severe complications of hypohidrotic ectodermal dysplasia in infancy. Early Hum Dev 2010.: 86: 397–399. 5. Tanner BA. Psychological aspects of hypohidrotic ectodermal dysplasia. Birth Defects Orig Artic Ser 1988: 24: 263–275 6. Mark BJ, Becker BA, Halloran DR, et al. Prevalence of atopic disorders and immunodeficiency in patients with ectodermal dysplasia syndromes. Ann Allergy Asthma Immunol 2012: 108:435-438. 7. Clarke A, Phillips DI, Brown R, et al. Clinical aspects of X linked hypohidrotic ectodermal dysplasia. Arch Dis Child 1987: 62: 989‐996. 8. Vincent MC, Biancalana V, Ginisty D, et al. Mutational spectrum of the ED1 gene in X-linked hypohidrotic ectodermal dysplasia. Eur J Hum Genet 2001: 9: 355–363. This article is protected by copyright. All rights reserved
9. Kobielak K, Kobielak A, Roszkiewicz J, Wierzba J, Limon J, Trzeciak WH. Mutations in the EDA gene in three unrelated families reveal no apparent correlation between phenotype and genotype in the patients with an X-linked anhidrotic ectodermal dysplasia. Am J Med Genet 2001;100:191–7. 10. International Dry Eye WorkShop. Ocul Surf. 2007 ;5:75-92. 11. Lexner MO, Bardow A, Hertz JM, et al. Anomalies of tooth formation in hypohidrotic ectodermal dysplasia. Int J Paediatr Dent 2007: 17: 10-18. 12. Wei Y,
XiaoQian Y, Zhuan B. Phenotypic findings in seven Chinese X-Linked hypohidrotic
ectodermal dysplasia HED families. Arch Oral Biol 2012: 57: 1418-1422 13. Fan H, Ye X, Shi L, et al. Mutations in the EDA gene are responsible for X-linked hypohidrotic ectodermal dysplasia and hypodontia in Chinese kindreds. Eur J Oral Sci 2008: 116: 412–417. 14. Schneider P, Street SL, Gaide O, et al. Mutations leading to X-linked hypohidrotic ectodermal dysplasia affect three major functional domains in the tumor necrosis factor family member ectodysplasin-A. J Biol Chem. 2001;276: 18819-18827 15. Keller MD, Petersen M, Ong P, Church J, Risma K, Burham J, Jain A, Stiehm ER, Hanson EP, Uzel G, Deardorff MA, Orange JS. Hypohidrotic ectodermal dysplasia and immunodeficiency with coincident NEMO and EDA mutations. Front Immunol. 2011; 2:61 16. Kaercher T. Ocular symptoms and signs in patients with ectodermal dysplasia syndromes. Graefes Arch Clin Exp Ophthalmol 2004: 242: 495-500. 17. Huffman RI, Huang JJ. Subretinal fibrosis and uveitis syndrome in a patient with ectodermal dysplasia. Ocul Immunol Inflamm 2009: 17: 348-350. 18. Wald KJ, Hirose T. Ectodermal dysplasia, ectrodactyly, and clefting syndrome and bilateral retinal detachment. Arch Ophthalmol 1993: 111:734. 19. Hayakawa M, Yanashima K, Kato K, et al. Association of ectodermal dysplasia, ectrodactyly and macular dystrophy: EEM syndrome (case report). Ophthalmic Paediatr Genet 1989: 10:287-292.
20. Callea M, Vinciguerra A, Willoughby CE, et al. Infantile bilateral glaucoma in a child with ectodermal dysplasia. Ophthalmic Genet. 2013: 34: 58-60. 21. Yildirim M, Yorgancilar E, Gun R, et al. Ectodermal dysplasia: otolaryngologic evaluation of 23 cases. Ear Nose Throat J 2012: 91: 28-33 This article is protected by copyright. All rights reserved
22. Jungersted JM, Høgh JK, Hellgren LI.. Ceramide profile in hypohidrotic ectodermal dysplasia. Clin Exp Dermatol 2012: 37: 153-155 23. Cork MJ, Danby SG, Vasilopoulos Y, et al. Epidermal barrier dysfunction in atopic dermatitis. J Invest Dermatol 2009: 129:1892-1908.
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Legend to Figure 1 Novel mutations are showed in bold TM: predicted transmembrane domain; F: putative furine cleavage site; COL: collagenous domain;TNF: tumor necrosis factor-homologous regions
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Patient code
Age (yrs)
Psychomotor development (BSID) a Cognitive development (Wechsler scales) b
Psychiatric Diagnosis (DSM-IV)
ED 1
9
a
Not available
b
9 yrs 1/12
IQ 102
Attention deficit hyperactivity disorder (314.9) Generalized Anxiety disorder (300.02)
ED 8
18
a
42 months
Mild mental retardation (317)
MDI 69 PDI 76 b
ED 10
3
7 yrs 5/12
b
17 yrs 10/12
a
36 months
IQ 69
IQ 90
MDI 72
Mild mental retardation (317)
Generalized Anxiety disorder (300.02) Mild mental retardation (317)
PDI 66
ED 13
2
a
24 months
Disruptive behavior disorder (312.9)
MDI 85 PDI 68 BRS 68 ED 18
2
a
24 months
Disruptive behavior disorder (312.9)
MDI 86 PDI 70 BRS 74 ED 21
2
a
24 months
MDI 90 PDI 68
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BRS 78 ED 22
11
a
Not done
Disruptive behavior disorder (312.9)
b
11 yrs
IQ 103
Dysthymic disorder (300.4) (CDI 24)
ED 24
ED 25
11
9
a
Not done
b
10 yrs 10/12 IQ 99
Dysthymic disorder (300.4) Generalized Anxiety disorder (300.02)
a
48 months
Mild mental retardation (317)
MDI 70 PDI 68 BRS 74 b
8 yrs 10/12
IQ 69
Mild mental retardation (317) Dysthymic disorder (300.4) (CDI 20)
ED 29
17
a
Mild mental retardation (317)
48 months
MDI 67 PDI 63 b
7 yrs 2/12
IQ 68
Mild mental retardation (317)
b
16 yrs 10/12
IQ 102
Dysthymic disorder (300.4) (CDI 23)
ED 38
5
b
4 yrs 10/12
IQ 69
Mild mental retardation (317)
Table 1: Characterization of Neurodevelopmental and Psychological disorders in XLHED subjects
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