Medical genetics
Novel missense mutation in the EDA1 gene identified in a family with hypohidrotic ectodermal dysplasia Glustein Pozo-Molina1,2, MD, Julia Reyes-Reali1, BSC, Marıa Isabel Mendoza-Ramos1, 3 2 MD, Rafael Villalobos-Molina , PHD, Efraın Garrido-Guerrero , PHD, and 1 Adolfo Rene Mendez-Cruz , MD, PHD
1 Laboratorio de Inmunologıa, Facultad de Estudios Superiores Iztacala, Universidad noma de Me xico, Mexico City, Nacional Auto n en Mexico, 2Laboratorio de Investigacio ncer, Biologıa Molecular y Celular del Ca tica y Biologıa Departamento de Gene n y de Molecular, Centro de Investigacio cnico Estudios Avanzados del Instituto Polite Nacional, Mexico City, Mexico, and 3Unidad de Biomedicina, Facultad de Estudios Superiores Iztacala, Universidad Nacional noma de Me xico, Tlalnepantla, Edo. de Auto xico, Me xico, Mexico Me
Correspondence ndez-Cruz, MD, PHD Adolfo Rene Me Laboratorio de Inmunologıa Facultad de Estudios Superiores Iztacala noma de Me xico Universidad Nacional Auto Ave. de los Barrios 1 Los Reyes Iztacala, Tlalnepantla 54090 xico Edo. de Me xico Me E-mail: [email protected]. mx
Abstract Background Hypohidrotic ectodermal dysplasia (HED) is a human genetic disorder that affects structures of ectodermal origin such as hair, teeth, and sweat glands. Although there are autosomal recessive and dominant forms, X-linked (XL) is the most frequent form of the disease. This XL-HED phenotype is associated with mutations in the gene encoding the transmembrane protein ectodysplasin-1 (EDA1). We report the clinical and molecular analysis of a novel mutation in exon 1 affecting the transmembrane domain of the protein. n, Me xico, nine men and Methods We have screened 20 members of a family from Yucata 11 women, searching clinical and histopathological signs of HED. We searched mutations in EDA1 gene from patients with XL-HED, carriers, and controls. Results We identified seven men with clinical characteristics of HED showing short toes and plantar hyperkeratosis not reported previously in patients with HED. A mutational study of the EDA1 gene showed that all seven patients with HED carry a novel missense mutation of the nucleotide 409 (c.409T>C) in exon 1, which changes p.Leu56-Pro in the protein amino acid sequence; five women are heterozygous compatible with carrier status. Conclusions We found a novel missense mutation in exon 1 of the EDA1 gene in a xico with XL-HED. We identified in this population some putative Mayan family from Me novel clinical signs of HED.
Conflicts of interest: None. doi: 10.1111/ijd.12775
Introduction Ectodermal dysplasias are a group of inherited disorders numbering more than 150, which involve developmental abnormalities in one or several structures from ectodermic origin such as hair, teeth, nails, and sweat glands.1,2 Hypohidrotic ectodermal dysplasias (HED) are a subgroup of ectodermal dysplasias related to different modes of Mendelian inheritance. HED phenotype is associated with mutations in the genes that encode ectodysplasin-A (EDA1, also called ED1), EDA receptor (EDAR), EDA receptor-associated death domain (EDARADD), and WNT10, all of them altering the NF-jB signal transduction pathway necessary for initiation, formation, and differentiation of skin appendages.3,4 ª 2015 The International Society of Dermatology
The most frequent form of HED is the X-linked-HED (XL-HED), inherited as a recessive trait. The XL-HED is caused by mutations in the EDA1 gene (MIM 300451) located at Xq12–q13.1, normally expressed in tissues of ectodermal origin such as keratinocytes, hair follicles, and sweat glands. The EDA1 gene constitutes nine exons and encodes the transmembrane type II protein EDA, a member of the tumor necrosis factor (TNF) ligand superfamily. More than 100 mutations in the EDA1 gene associated with XL-HED have been reported,5 most of them affecting functionally important domains of the protein, such as the furin subdomain located in exon 3, collagen-like domain in exon 5, transmembrane domain in exon 1, and TNF-like domain in exon 9. We have reported 20 cases of XL-HED in a rural setting in Yucat an, Mexico.6 Here International Journal of Dermatology 2015
1
2
Medical genetics
Pozo-Molina et al.
Novel missense mutation in EDA1 gene in a HED family
Figure 1 Pedigree of a Mexican family from Yucat an affected with X-linked hypohidrotic ectodermal dysplasia. The proband is indicated with a black arrow. Black squares indicate the affected males, and circles with central dot represent the female carriers
we report the clinical and molecular analysis of this family that includes patients with HED and female carriers. We found in this family a novel mutation in exon 1 affecting the transmembrane domain of the protein.
agarose gels, and the bands corresponding to the specific amplicons were cut off from gels, then DNA was extracted using the WizardSV Gel and PCR Clean-Up System (Promega, Madison, WI, USA). Every product was used as a template to conduct a sequencing reaction with the BigDye
Materials and methods Study population Twenty members of a family (nine men and 11 women) from n, Me xico, affected with XL-HED,6 were evaluated. Akil, Yucata Seven males were previously diagnosed with XL-HED. An ethics committee from the Faculty of Higher Studies, Iztacala, xico, approved the study. UNAM, Me Preparation of DNA from buccal cell swabs Porous foam-tipped swabs (Epicentre Biotechnologies, Madison, WI, USA) were used for the collection of cheek epithelial cells from patients and controls (laboratory volunteers). Buccal DNA samples were collected with BuccalAmpTM DNA extraction kits (Epicentre Biotechnologies) and processed according to the manufacturer’s instructions. DNA genotyping The eight exons from EDA1 gene were amplified by polymerase chain reaction (PCR), including intronic sequences flanking the exons using the described primers and PCR conditions.7 The PCR products were electrophoretically migrated on 1.5%
(a)
Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Austin, TX, USA), according to the manufacturer’s procedure. The sequences were analyzed using the ABI PRISM 310 (Applied Biosystems) system.
Results Clinical findings
The seven patients studied (Fig. 1) present the typical clinical manifestations and histopathology findings of this dysplasia (data not shown), similar to those reported in other patients with HED worldwide. Novel clinical manifestations observed in these patients were some features on their feet such as broad toes, short and widely spaced, with wide and thick tips (Fig. 2a, pedigree II-9). The toenails are convex, hypoplastic, unpolished, and thin, and another interesting feature found is the mild or severe plantar-fissured hyperkeratosis, observed in adults and boys with HED (Fig. 2b pedigree II-7). Mutation analysis
The proband was a patient with a very clear phenotype of HED, which based on the observed heredity pattern, (b)
Figure 2 Clinical phenotype of the X-linked hypohidrotic ectodermal dysplasia affected patients. (a) Features of the feet included broad, short, and widely spaced toes with wide and thick ends. Nails are convex, hypoplastic, unpolished, and thin. (b) Severe plantar-fissured hyperkeratosis International Journal of Dermatology 2015
ª 2015 The International Society of Dermatology
Pozo-Molina et al.
Novel missense mutation in EDA1 gene in a HED family
(a)
(c)
(b)
(d)
Medical genetics
Figure 3 Genetic and molecular
analysis of X-linked hypohidrotic ectodermal dysplasia affected patients. Partial nucleotide sequence of the exon 1 from EDA gene in four subjects included in the study. (a) Male included as a control (not belonging to the family studied), without phenotype of the EDA syndrome, this subject shows a normal hemizygous status (409T); (b) proband (II-9) who shows the punctual hemizygous mutation T409C; (c) sister of the proband (II16), without apparent clinical alterations showing a normal homozygous status (409T); (d) sister of the proband (II-12), with mild clinical manifestations showing the punctual heterozygous mutation T409>C, determining a carrier condition
presented the X-linked variety of the disease (II-9 of Fig. 1); then, we conducted a mutational study of the EDA gene. We used the set of primers described,8 and those sequences corresponding to each exon were aligned to the consensus sequence reported in the gene bank (NCBI Reference Sequence: GI: 223941910). We found a punctual mutation in exon 1 present only in affected subjects and carriers; the rest of the patients included in the study have a normal allelic complement. Moreover, the sequence of more than one hundred non-related healthy controls did not show any mutation, for this reason, the mutation found was not considered a polymorphism (Fig. 3, Table 1). This mutation is a T-C transition of the nucleotide 409 (c.409T>C) in the exon ª 2015 The International Society of Dermatology
1 of the EDA gene that conditions a change in the amino acid sequence of the protein, p.Leu56-Pro. The mutation establishes the condition for hemizygous males affected and the heterozygous status for women compatible with the carrier condition with mild clinical manifestations. Discussion HED is a genodermatosis with a low prevalence worldwide. It has been estimated that 1 in 100,000 newborns may have this disease; nevertheless, in a community from Akil, Yucat an, Mexico, we found a considerably higher prevalence of 20 in 7000 newborns.6 International Journal of Dermatology 2015
3
4
Medical genetics
Pozo-Molina et al.
Novel missense mutation in EDA1 gene in a HED family
Table 1 Mutational analysis and clinical findings of the population studied Pedigree position
Exon
Nucleotide sequence
Clinical status
I-1 I-2 II-2 II-7 II-9 II-12 II-14 II-16 III-8 III-9 III-10 III-13 III-14 III-15 III-16 III-17 III-20 III-21 III-22 III-23
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
Normal T409T/C Normal T409C T409C T409T/C T409T/C Normal Normal T409T/C T409T/C Normal Normal T409C T409C T409C T409C T409C Normal Normal
Healthy Carrier Healthy EDA Sx EDA Sx Carrier Carrier Healthy Healthy Carrier Carrier Healthy Healthy EDA Sx EDA Sx EDA Sx EDA Sx EDA Sx Healthy Healthy
HH
AH
HT
HD
HK
LIE
ST
√
√ √
√ √
√ √
√
√ √
√ √ √ √ √
√ √ √ √ √
√ √ √ √ √
√
√ √ √
√ √
√ √ √ √ √
√ √ √
√ √ √ √ √
√, presence of clinical sign; AH, anhidrosis; empty cell, absence of clinical sign; F, female; HD, hypodontia; HH, hypohidrosis; HK, hyperkeratosis; HT, hypotrichosis; LIE, lower implanted ears; M, male; ST, short toes; Sx, syndrome.
Several striking clinical findings were observed on the feet in the patients with HED of this family: (a) the described plantar hyperkeratosis6 was confirmed in all seven patients with HED still alive, observing severity in this manifestation in adults, and (b) toes are broad, short, and widely spaced. Interestingly, malformation in the feet and hands has been reported in non-linked X chromosome ectodermic dysplasias8,9 but until now, to the best of our knowledge, this is the first report of this alteration in patients with XL-HED. To date, more than 100 different mutations in the EDA1 gene associated with HED have been described,10,11 where the sites of mutations are clustered in functionally important domains of the protein. Hashiguchi et al.12 reviewed 144 mutations reported for the EDA1 gene, many of them present in more than one family. Of these, 27% occur at the furin subdomain located in exon 3, 19% at the collagen-like domain in exon 5, 15% at the transmembrane domain in exon 1, and 13% at the TNF-like domain in exon 9. Given the exon 1 size, its richness in CpG dinucleotides (61% of the coding sequences CpGs, possibly as a part of a CpG island), and the higher mutability of CpG dinucleotides, detection of new mutations in this region may be expected. It is noticeable that most of the mutations in the region of exon 1 encoding the N-terminal cytoplasmic domain result in truncated or no production of EDA because of frameshift, termination, and loss of initiation International Journal of Dermatology 2015
site mutations.13,14 On the contrary, mutations in the region of exon 1 encoding the extracellular domain 1 (EC1) are, most of them, missense mutations.13,15,16 Several reports12,13,16 indicate that nucleotides located in exon 1, that encode for the transmembrane domain (codons 60 and 61) near to the extracellular region of the protein, are frequently mutated either with missense (C60R,12 Y61H,13) or nonsense mutations (Y61X).13 Mutations in the region of exon 1 encoding the transmembrane domain of EDA1 appear at low frequency, as there are few reports, mainly including one family.15,17 The novel mutation we detected is a transition of T by C at position 409 from exon 1 (c.409T>C), changing the codon 56 from leucine to proline located at the transmembrane domain of the protein. This amino acid substitution is localized in a leucine-rich hydrophobic area from the transmembrane domain of the protein and changes the residue from strongly hydrophobic, where it tends to stabilize helical structures, to the neutral nonhydrophobic proline that strongly inhibits the helical structure and quite often promotes turns. The structural changes induced in this region by the amino acid substitution we found, as well as those reported in codons 54, 55, and 56,15,17 may disrupt the transmembrane domain, inducing a conformational modification that affects the proper EDA-EDAR signaling. To date, this is the first mutation in the ED1 gene reported in a putatively Mayan rural family from Yucat an, Mexico. ª 2015 The International Society of Dermatology
Pozo-Molina et al.
Novel missense mutation in EDA1 gene in a HED family
Acknowledgments This work was financially supported by the FES-Iztacala UNAM institutional program PAPCA 2010-2011 (project 63), and a grant from CONACyT (project no. 105174).
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 Salinas CF, Jorgenson RJ, Wright JT, et al. 2008International Conference on Ectodermal Dysplasias Classification: conference report. Am J Med Genet A 2009; 149A: 1958–1969. 3 Cluzeau C, Hadj-Rabia S, Jambou M, et al. Only four genes (EDA1, EDAR, EDARADD, and WNT10A) account for 90% of hypohidrotic/anhidrotic ectodermal dysplasia cases. Hum Mutat 2011; 32: 70–72. 4 Lobito AA, Gabriel TL, Medema JP, et al. Disease causing mutations in the TNF and TNFR superfamilies: focus on molecular mechanisms driving disease. Trends Mol Med 2011; 17: 494–505. 5 Mikkola ML. Molecular aspects of hypohidrotic ectodermal dysplasia. Am J Med Genet A 2009; 149A: 2031–2036. 6 Reyes-Chicuellar N, De Haro R, Jimenez-Flores R, et al. Higher-than-expected prevalence of hypohidrotic ectodermal dysplasia in a rural Mexican setting: report of 20 cases. Int J Dermatol 2010; 49: 1050–1055. 7 Zhang XJ, Chen JJ, Song YX, et al. Mutation analysis of the ED1 gene in two Chinese Han families with X-linked hypohidrotic ectodermal dysplasia. Arch Dermatol Res 2003; 295: 38–42. 8 Penchaszadeh VB, de Negrotti TC. Ectrodactyly-ectodermal dysplasia-clefting (EEC)
ª 2015 The International Society of Dermatology
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11
12
13
14
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17
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syndrome: dominant inheritance and variable expression. J Med Genet 1976; 13: 281–284. Thomson J, Aberd M. Malformations of hands and feet. Br Med J 1892; 1: 1188–1190. Monreal AW, Zonana J, Ferguson B. Identification of a new splice form of the EDA1 gene permits detection of nearly all X-linked hypohidrotic ectodermal dysplasia mutations. Am J Hum Genet 1998; 63: 380–389. Swee LK, Ingold-Salamin K, Tardivel A, et al. Biological activity of ectodysplasin A is conditioned by its collagen and heparan sulfate proteoglycan-binding domains. J Biol Chem 2009; 284: 27567–27576. Hashiguchi T, Yotsumoto S, Kanzaki T. Mutations in the ED1 gene in Japanese families with X-linked hypohidrotic ectodermal dysplasia. Exp Dermatol 2003; 12: 518–522. Kere J, Srivastava AK, Montonen O, et al. X-linked anhidrotic (hypohidrotic) ectodermal dysplasia is caused by mutation in a novel transmembrane protein. Nat Genet 1996; 13: 409–416. Bayes M, Hartung AJ, Ezer S, et al. The anhidrotic ectodermal dysplasia gene (EDA) undergoes alternative splicing and encodes ectodysplasin-A with deletion mutations in collagenous repeats. Hum Mol Genet 1998; 7: 1661–1669. Martinez F, Millan JM, Orellana C, et al. X-linked anhidrotic (hypohidrotic) ectodermal dysplasia caused by a novel mutation in EDA1 gene: 406T > G (Leu55Arg). J Invest Dermatol 1999; 113: 285–286. 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. Hertz JM, Norgaard Hansen K, Juncker I, et al. A novel missense mutation (402C–>T) in exon 1 in the EDA gene in a family with X-linked hypohidrotic ectodermal dysplasia. Clin Genet 1998; 53: 205–209.
International Journal of Dermatology 2015
5
Novel missense mutation in the EDA1 gene identified in a family with hypohidrotic ectodermal dysplasia Glustein Pozo-Molina1,2, MD, Julia Reyes-Reali1, BSC, Marıa Isabel Mendoza-Ramos1, 3 2 MD, Rafael Villalobos-Molina , PHD, Efraın Garrido-Guerrero , PHD, and 1 Adolfo Rene Mendez-Cruz , MD, PHD
1 Laboratorio de Inmunologıa, Facultad de Estudios Superiores Iztacala, Universidad noma de Me xico, Mexico City, Nacional Auto n en Mexico, 2Laboratorio de Investigacio ncer, Biologıa Molecular y Celular del Ca tica y Biologıa Departamento de Gene n y de Molecular, Centro de Investigacio cnico Estudios Avanzados del Instituto Polite Nacional, Mexico City, Mexico, and 3Unidad de Biomedicina, Facultad de Estudios Superiores Iztacala, Universidad Nacional noma de Me xico, Tlalnepantla, Edo. de Auto xico, Me xico, Mexico Me
Correspondence ndez-Cruz, MD, PHD Adolfo Rene Me Laboratorio de Inmunologıa Facultad de Estudios Superiores Iztacala noma de Me xico Universidad Nacional Auto Ave. de los Barrios 1 Los Reyes Iztacala, Tlalnepantla 54090 xico Edo. de Me xico Me E-mail: [email protected]. mx
Abstract Background Hypohidrotic ectodermal dysplasia (HED) is a human genetic disorder that affects structures of ectodermal origin such as hair, teeth, and sweat glands. Although there are autosomal recessive and dominant forms, X-linked (XL) is the most frequent form of the disease. This XL-HED phenotype is associated with mutations in the gene encoding the transmembrane protein ectodysplasin-1 (EDA1). We report the clinical and molecular analysis of a novel mutation in exon 1 affecting the transmembrane domain of the protein. n, Me xico, nine men and Methods We have screened 20 members of a family from Yucata 11 women, searching clinical and histopathological signs of HED. We searched mutations in EDA1 gene from patients with XL-HED, carriers, and controls. Results We identified seven men with clinical characteristics of HED showing short toes and plantar hyperkeratosis not reported previously in patients with HED. A mutational study of the EDA1 gene showed that all seven patients with HED carry a novel missense mutation of the nucleotide 409 (c.409T>C) in exon 1, which changes p.Leu56-Pro in the protein amino acid sequence; five women are heterozygous compatible with carrier status. Conclusions We found a novel missense mutation in exon 1 of the EDA1 gene in a xico with XL-HED. We identified in this population some putative Mayan family from Me novel clinical signs of HED.
Conflicts of interest: None. doi: 10.1111/ijd.12775
Introduction Ectodermal dysplasias are a group of inherited disorders numbering more than 150, which involve developmental abnormalities in one or several structures from ectodermic origin such as hair, teeth, nails, and sweat glands.1,2 Hypohidrotic ectodermal dysplasias (HED) are a subgroup of ectodermal dysplasias related to different modes of Mendelian inheritance. HED phenotype is associated with mutations in the genes that encode ectodysplasin-A (EDA1, also called ED1), EDA receptor (EDAR), EDA receptor-associated death domain (EDARADD), and WNT10, all of them altering the NF-jB signal transduction pathway necessary for initiation, formation, and differentiation of skin appendages.3,4 ª 2015 The International Society of Dermatology
The most frequent form of HED is the X-linked-HED (XL-HED), inherited as a recessive trait. The XL-HED is caused by mutations in the EDA1 gene (MIM 300451) located at Xq12–q13.1, normally expressed in tissues of ectodermal origin such as keratinocytes, hair follicles, and sweat glands. The EDA1 gene constitutes nine exons and encodes the transmembrane type II protein EDA, a member of the tumor necrosis factor (TNF) ligand superfamily. More than 100 mutations in the EDA1 gene associated with XL-HED have been reported,5 most of them affecting functionally important domains of the protein, such as the furin subdomain located in exon 3, collagen-like domain in exon 5, transmembrane domain in exon 1, and TNF-like domain in exon 9. We have reported 20 cases of XL-HED in a rural setting in Yucat an, Mexico.6 Here International Journal of Dermatology 2015
1
2
Medical genetics
Pozo-Molina et al.
Novel missense mutation in EDA1 gene in a HED family
Figure 1 Pedigree of a Mexican family from Yucat an affected with X-linked hypohidrotic ectodermal dysplasia. The proband is indicated with a black arrow. Black squares indicate the affected males, and circles with central dot represent the female carriers
we report the clinical and molecular analysis of this family that includes patients with HED and female carriers. We found in this family a novel mutation in exon 1 affecting the transmembrane domain of the protein.
agarose gels, and the bands corresponding to the specific amplicons were cut off from gels, then DNA was extracted using the WizardSV Gel and PCR Clean-Up System (Promega, Madison, WI, USA). Every product was used as a template to conduct a sequencing reaction with the BigDye
Materials and methods Study population Twenty members of a family (nine men and 11 women) from n, Me xico, affected with XL-HED,6 were evaluated. Akil, Yucata Seven males were previously diagnosed with XL-HED. An ethics committee from the Faculty of Higher Studies, Iztacala, xico, approved the study. UNAM, Me Preparation of DNA from buccal cell swabs Porous foam-tipped swabs (Epicentre Biotechnologies, Madison, WI, USA) were used for the collection of cheek epithelial cells from patients and controls (laboratory volunteers). Buccal DNA samples were collected with BuccalAmpTM DNA extraction kits (Epicentre Biotechnologies) and processed according to the manufacturer’s instructions. DNA genotyping The eight exons from EDA1 gene were amplified by polymerase chain reaction (PCR), including intronic sequences flanking the exons using the described primers and PCR conditions.7 The PCR products were electrophoretically migrated on 1.5%
(a)
Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Austin, TX, USA), according to the manufacturer’s procedure. The sequences were analyzed using the ABI PRISM 310 (Applied Biosystems) system.
Results Clinical findings
The seven patients studied (Fig. 1) present the typical clinical manifestations and histopathology findings of this dysplasia (data not shown), similar to those reported in other patients with HED worldwide. Novel clinical manifestations observed in these patients were some features on their feet such as broad toes, short and widely spaced, with wide and thick tips (Fig. 2a, pedigree II-9). The toenails are convex, hypoplastic, unpolished, and thin, and another interesting feature found is the mild or severe plantar-fissured hyperkeratosis, observed in adults and boys with HED (Fig. 2b pedigree II-7). Mutation analysis
The proband was a patient with a very clear phenotype of HED, which based on the observed heredity pattern, (b)
Figure 2 Clinical phenotype of the X-linked hypohidrotic ectodermal dysplasia affected patients. (a) Features of the feet included broad, short, and widely spaced toes with wide and thick ends. Nails are convex, hypoplastic, unpolished, and thin. (b) Severe plantar-fissured hyperkeratosis International Journal of Dermatology 2015
ª 2015 The International Society of Dermatology
Pozo-Molina et al.
Novel missense mutation in EDA1 gene in a HED family
(a)
(c)
(b)
(d)
Medical genetics
Figure 3 Genetic and molecular
analysis of X-linked hypohidrotic ectodermal dysplasia affected patients. Partial nucleotide sequence of the exon 1 from EDA gene in four subjects included in the study. (a) Male included as a control (not belonging to the family studied), without phenotype of the EDA syndrome, this subject shows a normal hemizygous status (409T); (b) proband (II-9) who shows the punctual hemizygous mutation T409C; (c) sister of the proband (II16), without apparent clinical alterations showing a normal homozygous status (409T); (d) sister of the proband (II-12), with mild clinical manifestations showing the punctual heterozygous mutation T409>C, determining a carrier condition
presented the X-linked variety of the disease (II-9 of Fig. 1); then, we conducted a mutational study of the EDA gene. We used the set of primers described,8 and those sequences corresponding to each exon were aligned to the consensus sequence reported in the gene bank (NCBI Reference Sequence: GI: 223941910). We found a punctual mutation in exon 1 present only in affected subjects and carriers; the rest of the patients included in the study have a normal allelic complement. Moreover, the sequence of more than one hundred non-related healthy controls did not show any mutation, for this reason, the mutation found was not considered a polymorphism (Fig. 3, Table 1). This mutation is a T-C transition of the nucleotide 409 (c.409T>C) in the exon ª 2015 The International Society of Dermatology
1 of the EDA gene that conditions a change in the amino acid sequence of the protein, p.Leu56-Pro. The mutation establishes the condition for hemizygous males affected and the heterozygous status for women compatible with the carrier condition with mild clinical manifestations. Discussion HED is a genodermatosis with a low prevalence worldwide. It has been estimated that 1 in 100,000 newborns may have this disease; nevertheless, in a community from Akil, Yucat an, Mexico, we found a considerably higher prevalence of 20 in 7000 newborns.6 International Journal of Dermatology 2015
3
4
Medical genetics
Pozo-Molina et al.
Novel missense mutation in EDA1 gene in a HED family
Table 1 Mutational analysis and clinical findings of the population studied Pedigree position
Exon
Nucleotide sequence
Clinical status
I-1 I-2 II-2 II-7 II-9 II-12 II-14 II-16 III-8 III-9 III-10 III-13 III-14 III-15 III-16 III-17 III-20 III-21 III-22 III-23
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
Normal T409T/C Normal T409C T409C T409T/C T409T/C Normal Normal T409T/C T409T/C Normal Normal T409C T409C T409C T409C T409C Normal Normal
Healthy Carrier Healthy EDA Sx EDA Sx Carrier Carrier Healthy Healthy Carrier Carrier Healthy Healthy EDA Sx EDA Sx EDA Sx EDA Sx EDA Sx Healthy Healthy
HH
AH
HT
HD
HK
LIE
ST
√
√ √
√ √
√ √
√
√ √
√ √ √ √ √
√ √ √ √ √
√ √ √ √ √
√
√ √ √
√ √
√ √ √ √ √
√ √ √
√ √ √ √ √
√, presence of clinical sign; AH, anhidrosis; empty cell, absence of clinical sign; F, female; HD, hypodontia; HH, hypohidrosis; HK, hyperkeratosis; HT, hypotrichosis; LIE, lower implanted ears; M, male; ST, short toes; Sx, syndrome.
Several striking clinical findings were observed on the feet in the patients with HED of this family: (a) the described plantar hyperkeratosis6 was confirmed in all seven patients with HED still alive, observing severity in this manifestation in adults, and (b) toes are broad, short, and widely spaced. Interestingly, malformation in the feet and hands has been reported in non-linked X chromosome ectodermic dysplasias8,9 but until now, to the best of our knowledge, this is the first report of this alteration in patients with XL-HED. To date, more than 100 different mutations in the EDA1 gene associated with HED have been described,10,11 where the sites of mutations are clustered in functionally important domains of the protein. Hashiguchi et al.12 reviewed 144 mutations reported for the EDA1 gene, many of them present in more than one family. Of these, 27% occur at the furin subdomain located in exon 3, 19% at the collagen-like domain in exon 5, 15% at the transmembrane domain in exon 1, and 13% at the TNF-like domain in exon 9. Given the exon 1 size, its richness in CpG dinucleotides (61% of the coding sequences CpGs, possibly as a part of a CpG island), and the higher mutability of CpG dinucleotides, detection of new mutations in this region may be expected. It is noticeable that most of the mutations in the region of exon 1 encoding the N-terminal cytoplasmic domain result in truncated or no production of EDA because of frameshift, termination, and loss of initiation International Journal of Dermatology 2015
site mutations.13,14 On the contrary, mutations in the region of exon 1 encoding the extracellular domain 1 (EC1) are, most of them, missense mutations.13,15,16 Several reports12,13,16 indicate that nucleotides located in exon 1, that encode for the transmembrane domain (codons 60 and 61) near to the extracellular region of the protein, are frequently mutated either with missense (C60R,12 Y61H,13) or nonsense mutations (Y61X).13 Mutations in the region of exon 1 encoding the transmembrane domain of EDA1 appear at low frequency, as there are few reports, mainly including one family.15,17 The novel mutation we detected is a transition of T by C at position 409 from exon 1 (c.409T>C), changing the codon 56 from leucine to proline located at the transmembrane domain of the protein. This amino acid substitution is localized in a leucine-rich hydrophobic area from the transmembrane domain of the protein and changes the residue from strongly hydrophobic, where it tends to stabilize helical structures, to the neutral nonhydrophobic proline that strongly inhibits the helical structure and quite often promotes turns. The structural changes induced in this region by the amino acid substitution we found, as well as those reported in codons 54, 55, and 56,15,17 may disrupt the transmembrane domain, inducing a conformational modification that affects the proper EDA-EDAR signaling. To date, this is the first mutation in the ED1 gene reported in a putatively Mayan rural family from Yucat an, Mexico. ª 2015 The International Society of Dermatology
Pozo-Molina et al.
Novel missense mutation in EDA1 gene in a HED family
Acknowledgments This work was financially supported by the FES-Iztacala UNAM institutional program PAPCA 2010-2011 (project 63), and a grant from CONACyT (project no. 105174).
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