Gene 533 (2014) 52–56
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Gene journal homepage: www.elsevier.com/locate/gene
Mutational spectrum of Xeroderma pigmentosum group A in Egyptian patients Khalda Amr a, Olfa Messaoud b,c, Mohamad El Darouti d, Sonia Abdelhak b,c, Ghada El-Kamah e,⁎ a
Medical Molecular Genetics Department, Human Genetics & Genome Research Division, National Research Center, Cairo, Egypt Université Tunis El Manar, Tunis 1068, Tunisia Biomedical Genomics and Oncogenetics Laboratory LR11IPT05, Institut Pasteur de Tunis, Tunis 1002, Tunisia d Dermatology Department, Faculty of Medicine, Cairo University, Egypt e Clinical Genetics Department, Human Genetics & Genome Research Division, National Research Center, Cairo, Egypt b c
a r t i c l e
i n f o
Article history: Accepted 30 September 2013 Available online 14 October 2013 Keywords: Xeroderma pigmentosum-group A Novel mutation Clinical correlation to mutation location
a b s t r a c t Xeroderma pigmentosum (XP) is a rare autosomal recessive hereditary disease characterized by hyperphotosensitivity, DNA repair defects and a predisposition to skin cancers. The most frequently occurring type worldwide is the XP group A (XPA). There is a close relationship between the clinical features that ranged from severe to mild form and the mutational site in XPA gene. The aim of this study is to carry out the mutational analysis in Egyptian patients with XP-A. This study was carried out on four unrelated Egyptian XP-A families. Clinical features were examined and direct sequencing of the coding region of XPA gene was performed in patients and their parents. Direct sequencing of the whole coding region of the XPA gene revealed the identification of two homozygous nonsense mutations: (c.553CNT; p.(Gln185*)) and (c.331GNT; p.(Glu111*)), which create premature, stop codon and a homodeletion (c.374delC: p.Thr125Ilefs*15) that leads to frameshift and premature translation termination. We report the identification of one novel XPA gene mutation and two known mutations in four unrelated Egyptian families with Xermoderma pigmentosum. All explored patients presented severe neurological abnormalities and have mutations located in the DNA binding domain. This report gives insight on the mutation spectrum of XP-A in Egypt. This would provide a valuable tool for early diagnosis of this severe disease. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Xeroderma pigmentosum is a rare autosomal recessive genodermatosis characterized by mucocutaneous and ocular hypersensitivity to UV radiation with irreparable DNA damage and subsequent malignant changes. In some subjects, progressive neurological degeneration is also observed. The prevalence of XP is 1:1,000,000 in the United States and Europe, but with higher frequency in Japan 1:22,000 (Hirai et al., 2006) (XP occurs at a frequency of one to four per million live births and its carrier frequency is estimated to be 0.2–0.4% in the general population). The disease is more frequent in populations where marriage between relatives is common and there is no sex or race predilection (RÜnger et al., 2008). Xeroderma pigmentosum group A complementing (XPA) protein is involved in the nucleotide excision repair (NER) pathway. This pathway is controlled by at least 28 genes, Abbreviations: XP, Xeroderma pigmentosum; XP-A, Xeroderma pigmentosum group A; XP-C, Xeroderma pigmentosum group C; XP-G, Xeroderma pigmentosum group G; del, Deletion; DNA, Deoxyribonucleic acid; OMIM, Online Mendelian Inheritance in Man; KD, Kilo Dalton; PCR, Polymerase chain reaction; NER, Nucleotide excision repair; C, Cytosine; T, Thymine; G, Guanine; A, Adenine; RNA, Ribonucleic acid; aa, Amino acid; Fig, Figure; F, Female; M, Male; MR, Mental retardation; +ve, Positive. ⁎ Corresponding author. Tel.: +20 1271225511; fax: +20 2 33370931. E-mail address: [email protected] (G. El-Kamah). 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.09.125
some of which are also part of the multi-protein basal transcription factor, TFIIH; and others participate in somatic growth and development. Mutations in any of the NER genes XPA, ERCC3, XPC, ERCC2, DDB2, ERCC4, and ERCC5 are responsible of XP (Feller et al., 2010). NER is a DNA-repair pathway responsible of removing many structurally unrelated DNA lesions from the genome and is especially important for protecting the human genome from carcinogenic effects of solar UV. XPA protein appears to play a central and multifunctional role in NER. In addition to preferentially binding damaged over undamaged DNA, XPA maintains an intricate network of contacts with the core repair complex (Gratchev et al., 2003). X pigmentosum is genetically heterogeneous and is classified into seven genetic complementation groups (XP-A to XP-G) that correspond to defects in one of the seven genes involved in NER. An additional group, variant form (XP-V), is resulting from a defect in the translesion synthesis pathway (Wood, 1996). The human XPA gene (XPA: NG_011642.1; OMIM # 278700) is about 25 kilobases length, localized on chromosome 9q34.1 and encodes a 31 kD protein (Tanaka et al., 1990). XPA gene consists of 6 exons that encode 273 amino acid residues (Tanaka et al., 1990). A partial or total loss of the XPA protein prevents cells from repairing DNA damage normally. As a result, damages accumulate in DNA, causing cells to malfunction and eventually become cancerous or die. The incidence of
K. Amr et al. / Gene 533 (2014) 52–56
primary cutaneous neoplasms including melanoma is approximately 10,000-fold higher in XP patients than in normal individuals (Bradford et al., 2011). Neurological symptoms reported in 15% of all XP patients ranged from mild (e.g. isolated hyporeflexia) to severe (e.g. progressive mental retardation) (Kraemer et al., 1987). So far, many mutations have been reported in the XPA gene; the first reported one was a common homozygous transversion (IV3–1GNC) identified in 80% of Japanese XPA patients. This study was performed in order to determine XPA gene alterations in four unrelated Egyptian patients and to analyze whether genetic alterations in XPA gene are correlated to clinical features. 2. Patients and methods Four XP-A patients descending from four unrelated consanguineous Egyptian families were subjected to clinical and molecular evaluation at the Human Genetics & Genome Research Division at the National Research Center in Cairo during the period 2008–2012. Patients' age at presentation ranged from 3 to 12 years; 75% were females. Diagnosis of XP-A was based on clinical features that include photophobia, photosensitivity, poïkiloderma and variable degrees of neurological affection in addition to dermatological malignancies in 50%. After obtaining written informed consent from the patients' parents, we extracted DNA from blood samples of the patients and their parents using standard procedures. All six exons and exon–intron junctions of the XPA gene were amplified and PCR products were sequenced using an ABI Prism 310 automated sequencer (Applied Biosystems, Foster City, CA, USA). PCR reactions were performed in a final volume of 50 μl containing 150 ng of genomic DNA using the following conditions: The PCR steps consisted of 1 cycle of denaturation at 96 °C for 5 min followed by 35 cycles of denaturation at 95 °C for 50 s, annealing at 50 °C, and elongation at 72 °C for 50 s followed by a final elongation at 72 °C for 5 min. Purified PCR products were Cycle sequenced using Big Dye terminator cycle sequencing Kit (Applied Biosystems, Foster City, CA) and analyzed on an ABI Prism 310 automated sequencer (Applied Biosystems). 3. Results In this study, we describe 4 XP patients belonging to 4 unrelated Egyptian families. Among them, 3 families originated from Northern Egypt and the fourth family is from Upper Egypt. The age of disease onset ranged from 2 to 9 months (average: 6.3 months) and examination age ranged from 3 to 12 years (average: 6.7 years). First-degree consanguinity was registered in all families. Clinical data of XP patients are summarized in (Table 1). Clinical manifestations of XP such as skin photosensitivity, poikiloderma and xeroderma were present in all examined patients. Ophthalmological abnormalities as photophobia, conjunctivitis and keratitis were observed in all patients but with variable severity. Skin tumors occurred in 2 cases; XP2UE and XP3NE, for whom recurrence and surgical excision combined with treatment were recorded four times. Variable degrees of neurological symptoms have been observed in all patients suggesting the diagnosis of XP-A.
53
For the XP4NE patient, she presented a slight delay in psychomotor development at the time of first examination that evolved to a more severe and profound mental retardation. Indeed, at the age of one year, she presented a delayed sitting, followed by a delayed talking till three. Now, she can walk but falls a lot. Direct sequencing of the whole coding region of XPA gene confirmed the diagnosis of Xeroderma pigmentosum in the four investigated families and revealed the presence of three mutations. Among them, one is novel. The first two patients had a new homozygous transition of C to T at position 553 that leads to a nonsense mutation in the last codon of exon 4: p.(Gln185*). This transition was identified in two patients at a homozygous state and in their parents at a heterozygous state. On the other hand, the third patient had a homozygous transversion of G to T at nucleotide 331 that leads to a nonsense mutation at the protein level: p.(Glu111*). The fourth patient presented a deletion of one nucleotide at position 374 (374delC) in exon 3. This mutation leads to a frameshift and causes a premature translation termination: p.Thr125Ilefs*15 (Fig. 1). In silico analysis showed that each mutation alters or creates a new restriction site as follows: for the p.(Gln185*) mutation, transition of C to T at position 553 abolishes the restriction site of the MaeIII enzyme; for the p.(Glu111*) mutation, transversion of G to T at position 331 abolishes the restriction site of the AcsI enzyme and for the p.Thr125Ilefs*15 mutation, deletion of C at nucleotide 374 creates two restriction sites for the MunI and Sse9I enzymes. 4. Discussion Inherited defects in NER can cause Xeroderma pigmentosum (XP), which is characterized by an extreme photosensitivity and a high risk of skin cancer. There is a markedly significant progress in understanding the molecular basis of this rare disease in the last two decades of research (Milota et al., 2011). Our study focused on screening for mutations in XPA gene with trial of correlation to clinical features in Egyptian XP patients (Fig. 2) and comparison to Tunisian patients as part of Northern African populations. Currently, 32 mutations in the XPA gene have been identified. Many previous reports have indicated that XPA gene is highly heterogeneous and that XP-A patients show a wide spectrum of clinical phenotype depending on the mutational site and consequently the residual XPA protein. Patients with typical XP-A phenotype that exhibit both severe neurological abnormalities and extreme photosensitivity have either total mis-splicing or mutations within exons 3, 4 and 5 that result in an alteration of XPA protein (Bartels and Lambert, 2007). In the present study, four Egyptian unrelated consanguineous families with sibs fulfilling the diagnostic criteria of XPA have been investigated in the Clinical Genetics Department. Results indicate the presence of a novel mutation in two XP patients with severe early onset features. For the four families, all identified mutations are located in exons 3 and 4 of the XPA gene (DNA-binding domain). The first and second (XP1GE and XP2UE) XP-A patients are found to have a novel homozygous nonsense mutation, a CNT transition in the first base of the last codon in exon 4 (CAGNTAG). Previously, one mutation that has been reported in this codon is a G to C transversion at the last
Table 1 Clinical manifestations of the four studied Xeroderma pigmentosum group-A patients. Patient
XP1GE XP2UE XP3NE XP4NE
Region of origin
Sex
Gize Upper Egypt North Egypt North Egypt
F F M F
Age (years)
Age of onset (months)
Consanguinity
8 12 4 3
9 9 2 5
+ve +ve +ve +ve
F: female. M: male. MR: mental retardation. +ve: positive.
Clinical symptoms Photosensitivity
Ocular symptoms
Skin lesions
Neurological manifestation
Tumors
+++ +++ +++ +
+++ ++ +++ +
+++ +++ +++ +
Spastic + MR MR Spastic + MR Mild MR
−− +++ ++ −−
XPA mutation
Q185X Q185X E111X 374delC
54
K. Amr et al. / Gene 533 (2014) 52–56
Fig. 1. The sequence electrogram of the sense strand in XP gene. a, DNA sequence analysis of homozygous mutation p.(Gln185*) in patients (XP1GE&XP2UE) and normal control. b, The p.(Glu111*) nonsense mutation in patient (XP3NE) and normal control. c, Normal and mutant sequences of deletion of C at nucleotide 374 (374delC) in patient (XP4NE) are shown.
nucleotide of exon 4 (CAGNCAC) in two Caucasian patients (Satokata et al., 1992a). Also, Satokata et al. (1990), found another mutation, a dinucleotide deletion, in exon 4. This deletion leads to a frameshift and a premature translation of XPA protein. The identified G to C transversion causes almost complete inactivation of the canonical 5′ splice donor site and aberrant RNA splicing termination in exon 4. On the other hand, the other two characterized mutations in this study are both located in exon 3: the nonsense mutation identified in the third patient is a GNT transversion (XP3NE) (GAANTAA) which converts a Glutamic acid by a stop codon (p.(Glu111*)) and a homozygous deletion (374delC) found in the fourth patient (XP4NE). The XPA p.(Glu111*) mutation was previously identified in three Tunisian patients having the same skin and neurological features of the disease as Egyptian patients (Messaoud et al., 2012). This supports the hypothesis of phenotype–genotype correlation and suggests a common ancestor. This is not surprising as Tunisian patients are originating from Kairouan, a city close to Mahdia, which was the capital of the Fatimid dynasty, an empire that encompassed much of North Africa, Sicily and parts of the Middle East and that was reigned from Ifriqiya (between 909 and 969) and from Egypt (between 969 and 1171). A common historical background could explain the common mutational spectrum; hence the hypothesis of a founder effect could be proposed. For the 374delC mutation, it was previously reported once in a Caucasian patient (Satokata et al., 1992b). Having the same mutation between North African and European XP patients is a common event
that could also be explained by a founder effect resulting from migratory flows that took place between the two continents. The same situation was frequently observed for several genetic disorders including XP-C (Ben Rekaya et al., 2009). Of particular interest is that all characterized mutations are located in the DNA binding region (aa 98–219). This could explain the severe phenotype observed in Egyptian patients. Thus, severe skin manifestations associated with neurological abnormalities might be related to a deficiency in the ability of XPA protein to bind to damaged DNA and to act as a processivity factor (Bartels and Lambert, 2007). In addition, neurological abnormalities observed in XP-A patients are due to the insufficient repair of oxidative DNA lesions in the central nervous system, where the production rate of reactive oxygen species is the highest (Rass et al., 2007). It was well established that clinical heterogeneity is associated with the mutational site (Messaoud et al., 2012; Nishigori et al., 1994; Sato et al., 1996; Takahashi et al., 2010). For example, Japanese XP-A patients that have the splicing mutation in intron 3 usually develop very severe neurological abnormalities, which are manifested by the inability to walk unaided and usually die in teens. This is in accordance with XP4NE patient phenotype who bears an exon 3 mutation and presents severe neurological alterations while having the mildest skin symptoms among studied cases (Fig. 2), that could be justified by her young age & high parental awareness. Phenotypic variability was detected among our studied patients even between patients sharing the same mutation (XP1GE and XP2UE) as the
K. Amr et al. / Gene 533 (2014) 52–56
55
Fig. 2. Correlation between detected mutations and clinical severity. Severe skin affection with large achromic spots in XP1GE, XP2UE & XP3NE (black arrows). Malignant lesions (kinked arrows) in XP2UE & XP3NE. Mild skin affection is observed in XP4NE patient.
development of malignancies in XP2UE. This could be essentially due to UV-exposure and age difference. It could also be explained by the level of parental awareness taking into consideration that the most severe dermatological presentation was observed in XP2UE patient (Fig. 2) who resides in the less urban Upper Egypt region. Although XP is a rare hereditary disorder, an accurate early genetic diagnosis might prevent complications associated with unprotected exposure to sunlight and enable early prenatal diagnosis and genetic counseling. Indeed, as each of the identified mutations abolishes or creates a new restriction site, PCR–RFLP could be used as a rapid and relatively cost effective tool for molecular diagnosis and carrier screening. In conclusion, clinical and molecular characterization of XP-A patients is essential to optimize follow-up, genetic counseling and health support of patients and may also pave the way for the development of new therapeutics. Studying common mutations and genetic diversity among neighboring countries from North Africa is an important step in setting diagnostic strategies and prioritizing studies for genetic disorders. Conflict of interest The authors state that the manuscript is not published elsewhere, and is not under consideration elsewhere. All the listed authors have agreed to the submission of the manuscript in its current form. The authors have no commercial affiliations, consultancies, stock or equity interests and patent licensing arrangements that could be considered to pose a conflict of interest regarding the submitted article. Acknowledgments We are very grateful to the families who took part in this study.
References Bartels, C.L., Lambert, M.W., 2007. Domains in the XPA protein important in its role as a processivity factor. Biochem. Biophys. Res. Commun. 356 (1), 219–225. Ben Rekaya, M., et al., 2009. High frequency of the V548A fs X572 XPC mutation in Tunisia: implication for molecular diagnosis. J. Hum. Genet. 54, 426–429. Bradford, P.T., et al., 2011. Cancer and neurologic degeneration in Xeroderma pigmentosum: long term follow-up characterizes the role of DNA repair. J. Med. Genet. 48, 168–176. Feller, L.N.H., Wood, N.H., Motswaledi, M.H., Khammissa, R.A.G., Meyer, M., Lemmer, J., 2010. Xeroderma pigmentosum: a case report and review of the literature. J. Prev. Med. Hyg. 51, 87–91. Gratchev, A., Strein, P., Utikal, J., Goerdt, S., 2003. Molecular genetics of Xeroderma pigmentosum variant. Exp. Dermatol. 12, 529–536. Hirai, Y., et al., 2006. Heterozygous individuals bearing a founder mutation in the XPA DNA repair gene comprise nearly 1% of the Japanese population. Mutat. Res. 601, 171–178. Kraemer, Kh., Lee, M.M., Scotto, J., 1987. Xeroderma pigmentosum. Cutaneous, ocular, and neurologic abnormalities in 830 published cases. Arch. Dermatol. 123, 241–250. Messaoud, O., et al., 2012. Severe phenotypes in two Tunisian families with novel XPA mutations: evidence for a correlation between mutation location and disease severity. Arch. Dermatol. Res. 304, 171–176. Milota, M., Jones, D.L., Cleaver, J., Jamall, L.S., 2011. Xeroderma pigmentosum family support group: helping families and promoting clinical initiatives. DNA Repair (Amst) 10, 792–797. Nishigori, C., Moriwaki, S., Takebe, H., Tanaka, T., Imamura, S., 1994. Gene alterations and clinical characteristics of Xeroderma pigmentosum group A patients in Japan. Arch. Dermatol. 130, 191–197. Rass, U., Ahe, I., West, S.C., 2007. Defective DNA repair and neurodegenerative disease. Cell 130, 991–1004. RÜnger, T.M., DiGiovanna, J.J., Kraemer, K.H., 2008. Hereditary disorders of genome instability and DNA repair, In: Wolff, K.W., Goldsmith, L.A., Katz, S.I., Gilchrest, B.A., Paller, A.S., Leffell, D.J. (Eds.), Fitzpatrick's Dermatology in General Medicine, 7th edn. McGraw-Hill, New York, pp. 1311–1325. Sato, M., Nishigori, C., Yagi, T., Takebe, H., 1996. Aberrant splicing and truncatedprotein expression due to a newly identified XPA gene mutation. Mutat. Res. 362, 199–208. Satokata, I., et al., 1990. Characterization of a splicing mutation in group A Xeroderma pigmentosum. Proc. Natl. Acad. Sci. U. S. A. 87, 9908–9912.
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Satokata, I., Tanaka, K., Yuba, S., Okada, Y., 1992a. Identification of splicing mutations of the last nucleotides of exons, a nonsense mutation, and a missense mutation of the XPAC gene as causes of group A Xeroderma pigmentosum. Mutat. Res. 273, 203–212. Satokata, I., Tanaka, K., Okada, Y., 1992b. Molecular basis of group A Xeroderma pigmentosum: a missense mutation and two deletions located in a zinc finger consensus sequence of the XPAC gene. Hum. Genet. 88, 603–607.
Takahashi, Y., et al., 2010. XPA gene mutations resulting in subtle truncation of protein in Xeroderma pigmentosum group A patients with mild skin symptoms. J. Invest. Dermatol. 130, 2481–2488. Tanaka, K., et al., 1990. Analysis of a human DNA excision repair gene involved in group A Xeroderma pigmentosum and containing a zinc finger domain. Nature 348, 73–76. Wood, R.D., 1996. DNA repair in eukaryotes. Annu. Rev. Biochem. 65, 135–167.
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Mutational spectrum of Xeroderma pigmentosum group A in Egyptian patients Khalda Amr a, Olfa Messaoud b,c, Mohamad El Darouti d, Sonia Abdelhak b,c, Ghada El-Kamah e,⁎ a
Medical Molecular Genetics Department, Human Genetics & Genome Research Division, National Research Center, Cairo, Egypt Université Tunis El Manar, Tunis 1068, Tunisia Biomedical Genomics and Oncogenetics Laboratory LR11IPT05, Institut Pasteur de Tunis, Tunis 1002, Tunisia d Dermatology Department, Faculty of Medicine, Cairo University, Egypt e Clinical Genetics Department, Human Genetics & Genome Research Division, National Research Center, Cairo, Egypt b c
a r t i c l e
i n f o
Article history: Accepted 30 September 2013 Available online 14 October 2013 Keywords: Xeroderma pigmentosum-group A Novel mutation Clinical correlation to mutation location
a b s t r a c t Xeroderma pigmentosum (XP) is a rare autosomal recessive hereditary disease characterized by hyperphotosensitivity, DNA repair defects and a predisposition to skin cancers. The most frequently occurring type worldwide is the XP group A (XPA). There is a close relationship between the clinical features that ranged from severe to mild form and the mutational site in XPA gene. The aim of this study is to carry out the mutational analysis in Egyptian patients with XP-A. This study was carried out on four unrelated Egyptian XP-A families. Clinical features were examined and direct sequencing of the coding region of XPA gene was performed in patients and their parents. Direct sequencing of the whole coding region of the XPA gene revealed the identification of two homozygous nonsense mutations: (c.553CNT; p.(Gln185*)) and (c.331GNT; p.(Glu111*)), which create premature, stop codon and a homodeletion (c.374delC: p.Thr125Ilefs*15) that leads to frameshift and premature translation termination. We report the identification of one novel XPA gene mutation and two known mutations in four unrelated Egyptian families with Xermoderma pigmentosum. All explored patients presented severe neurological abnormalities and have mutations located in the DNA binding domain. This report gives insight on the mutation spectrum of XP-A in Egypt. This would provide a valuable tool for early diagnosis of this severe disease. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Xeroderma pigmentosum is a rare autosomal recessive genodermatosis characterized by mucocutaneous and ocular hypersensitivity to UV radiation with irreparable DNA damage and subsequent malignant changes. In some subjects, progressive neurological degeneration is also observed. The prevalence of XP is 1:1,000,000 in the United States and Europe, but with higher frequency in Japan 1:22,000 (Hirai et al., 2006) (XP occurs at a frequency of one to four per million live births and its carrier frequency is estimated to be 0.2–0.4% in the general population). The disease is more frequent in populations where marriage between relatives is common and there is no sex or race predilection (RÜnger et al., 2008). Xeroderma pigmentosum group A complementing (XPA) protein is involved in the nucleotide excision repair (NER) pathway. This pathway is controlled by at least 28 genes, Abbreviations: XP, Xeroderma pigmentosum; XP-A, Xeroderma pigmentosum group A; XP-C, Xeroderma pigmentosum group C; XP-G, Xeroderma pigmentosum group G; del, Deletion; DNA, Deoxyribonucleic acid; OMIM, Online Mendelian Inheritance in Man; KD, Kilo Dalton; PCR, Polymerase chain reaction; NER, Nucleotide excision repair; C, Cytosine; T, Thymine; G, Guanine; A, Adenine; RNA, Ribonucleic acid; aa, Amino acid; Fig, Figure; F, Female; M, Male; MR, Mental retardation; +ve, Positive. ⁎ Corresponding author. Tel.: +20 1271225511; fax: +20 2 33370931. E-mail address: [email protected] (G. El-Kamah). 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.09.125
some of which are also part of the multi-protein basal transcription factor, TFIIH; and others participate in somatic growth and development. Mutations in any of the NER genes XPA, ERCC3, XPC, ERCC2, DDB2, ERCC4, and ERCC5 are responsible of XP (Feller et al., 2010). NER is a DNA-repair pathway responsible of removing many structurally unrelated DNA lesions from the genome and is especially important for protecting the human genome from carcinogenic effects of solar UV. XPA protein appears to play a central and multifunctional role in NER. In addition to preferentially binding damaged over undamaged DNA, XPA maintains an intricate network of contacts with the core repair complex (Gratchev et al., 2003). X pigmentosum is genetically heterogeneous and is classified into seven genetic complementation groups (XP-A to XP-G) that correspond to defects in one of the seven genes involved in NER. An additional group, variant form (XP-V), is resulting from a defect in the translesion synthesis pathway (Wood, 1996). The human XPA gene (XPA: NG_011642.1; OMIM # 278700) is about 25 kilobases length, localized on chromosome 9q34.1 and encodes a 31 kD protein (Tanaka et al., 1990). XPA gene consists of 6 exons that encode 273 amino acid residues (Tanaka et al., 1990). A partial or total loss of the XPA protein prevents cells from repairing DNA damage normally. As a result, damages accumulate in DNA, causing cells to malfunction and eventually become cancerous or die. The incidence of
K. Amr et al. / Gene 533 (2014) 52–56
primary cutaneous neoplasms including melanoma is approximately 10,000-fold higher in XP patients than in normal individuals (Bradford et al., 2011). Neurological symptoms reported in 15% of all XP patients ranged from mild (e.g. isolated hyporeflexia) to severe (e.g. progressive mental retardation) (Kraemer et al., 1987). So far, many mutations have been reported in the XPA gene; the first reported one was a common homozygous transversion (IV3–1GNC) identified in 80% of Japanese XPA patients. This study was performed in order to determine XPA gene alterations in four unrelated Egyptian patients and to analyze whether genetic alterations in XPA gene are correlated to clinical features. 2. Patients and methods Four XP-A patients descending from four unrelated consanguineous Egyptian families were subjected to clinical and molecular evaluation at the Human Genetics & Genome Research Division at the National Research Center in Cairo during the period 2008–2012. Patients' age at presentation ranged from 3 to 12 years; 75% were females. Diagnosis of XP-A was based on clinical features that include photophobia, photosensitivity, poïkiloderma and variable degrees of neurological affection in addition to dermatological malignancies in 50%. After obtaining written informed consent from the patients' parents, we extracted DNA from blood samples of the patients and their parents using standard procedures. All six exons and exon–intron junctions of the XPA gene were amplified and PCR products were sequenced using an ABI Prism 310 automated sequencer (Applied Biosystems, Foster City, CA, USA). PCR reactions were performed in a final volume of 50 μl containing 150 ng of genomic DNA using the following conditions: The PCR steps consisted of 1 cycle of denaturation at 96 °C for 5 min followed by 35 cycles of denaturation at 95 °C for 50 s, annealing at 50 °C, and elongation at 72 °C for 50 s followed by a final elongation at 72 °C for 5 min. Purified PCR products were Cycle sequenced using Big Dye terminator cycle sequencing Kit (Applied Biosystems, Foster City, CA) and analyzed on an ABI Prism 310 automated sequencer (Applied Biosystems). 3. Results In this study, we describe 4 XP patients belonging to 4 unrelated Egyptian families. Among them, 3 families originated from Northern Egypt and the fourth family is from Upper Egypt. The age of disease onset ranged from 2 to 9 months (average: 6.3 months) and examination age ranged from 3 to 12 years (average: 6.7 years). First-degree consanguinity was registered in all families. Clinical data of XP patients are summarized in (Table 1). Clinical manifestations of XP such as skin photosensitivity, poikiloderma and xeroderma were present in all examined patients. Ophthalmological abnormalities as photophobia, conjunctivitis and keratitis were observed in all patients but with variable severity. Skin tumors occurred in 2 cases; XP2UE and XP3NE, for whom recurrence and surgical excision combined with treatment were recorded four times. Variable degrees of neurological symptoms have been observed in all patients suggesting the diagnosis of XP-A.
53
For the XP4NE patient, she presented a slight delay in psychomotor development at the time of first examination that evolved to a more severe and profound mental retardation. Indeed, at the age of one year, she presented a delayed sitting, followed by a delayed talking till three. Now, she can walk but falls a lot. Direct sequencing of the whole coding region of XPA gene confirmed the diagnosis of Xeroderma pigmentosum in the four investigated families and revealed the presence of three mutations. Among them, one is novel. The first two patients had a new homozygous transition of C to T at position 553 that leads to a nonsense mutation in the last codon of exon 4: p.(Gln185*). This transition was identified in two patients at a homozygous state and in their parents at a heterozygous state. On the other hand, the third patient had a homozygous transversion of G to T at nucleotide 331 that leads to a nonsense mutation at the protein level: p.(Glu111*). The fourth patient presented a deletion of one nucleotide at position 374 (374delC) in exon 3. This mutation leads to a frameshift and causes a premature translation termination: p.Thr125Ilefs*15 (Fig. 1). In silico analysis showed that each mutation alters or creates a new restriction site as follows: for the p.(Gln185*) mutation, transition of C to T at position 553 abolishes the restriction site of the MaeIII enzyme; for the p.(Glu111*) mutation, transversion of G to T at position 331 abolishes the restriction site of the AcsI enzyme and for the p.Thr125Ilefs*15 mutation, deletion of C at nucleotide 374 creates two restriction sites for the MunI and Sse9I enzymes. 4. Discussion Inherited defects in NER can cause Xeroderma pigmentosum (XP), which is characterized by an extreme photosensitivity and a high risk of skin cancer. There is a markedly significant progress in understanding the molecular basis of this rare disease in the last two decades of research (Milota et al., 2011). Our study focused on screening for mutations in XPA gene with trial of correlation to clinical features in Egyptian XP patients (Fig. 2) and comparison to Tunisian patients as part of Northern African populations. Currently, 32 mutations in the XPA gene have been identified. Many previous reports have indicated that XPA gene is highly heterogeneous and that XP-A patients show a wide spectrum of clinical phenotype depending on the mutational site and consequently the residual XPA protein. Patients with typical XP-A phenotype that exhibit both severe neurological abnormalities and extreme photosensitivity have either total mis-splicing or mutations within exons 3, 4 and 5 that result in an alteration of XPA protein (Bartels and Lambert, 2007). In the present study, four Egyptian unrelated consanguineous families with sibs fulfilling the diagnostic criteria of XPA have been investigated in the Clinical Genetics Department. Results indicate the presence of a novel mutation in two XP patients with severe early onset features. For the four families, all identified mutations are located in exons 3 and 4 of the XPA gene (DNA-binding domain). The first and second (XP1GE and XP2UE) XP-A patients are found to have a novel homozygous nonsense mutation, a CNT transition in the first base of the last codon in exon 4 (CAGNTAG). Previously, one mutation that has been reported in this codon is a G to C transversion at the last
Table 1 Clinical manifestations of the four studied Xeroderma pigmentosum group-A patients. Patient
XP1GE XP2UE XP3NE XP4NE
Region of origin
Sex
Gize Upper Egypt North Egypt North Egypt
F F M F
Age (years)
Age of onset (months)
Consanguinity
8 12 4 3
9 9 2 5
+ve +ve +ve +ve
F: female. M: male. MR: mental retardation. +ve: positive.
Clinical symptoms Photosensitivity
Ocular symptoms
Skin lesions
Neurological manifestation
Tumors
+++ +++ +++ +
+++ ++ +++ +
+++ +++ +++ +
Spastic + MR MR Spastic + MR Mild MR
−− +++ ++ −−
XPA mutation
Q185X Q185X E111X 374delC
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Fig. 1. The sequence electrogram of the sense strand in XP gene. a, DNA sequence analysis of homozygous mutation p.(Gln185*) in patients (XP1GE&XP2UE) and normal control. b, The p.(Glu111*) nonsense mutation in patient (XP3NE) and normal control. c, Normal and mutant sequences of deletion of C at nucleotide 374 (374delC) in patient (XP4NE) are shown.
nucleotide of exon 4 (CAGNCAC) in two Caucasian patients (Satokata et al., 1992a). Also, Satokata et al. (1990), found another mutation, a dinucleotide deletion, in exon 4. This deletion leads to a frameshift and a premature translation of XPA protein. The identified G to C transversion causes almost complete inactivation of the canonical 5′ splice donor site and aberrant RNA splicing termination in exon 4. On the other hand, the other two characterized mutations in this study are both located in exon 3: the nonsense mutation identified in the third patient is a GNT transversion (XP3NE) (GAANTAA) which converts a Glutamic acid by a stop codon (p.(Glu111*)) and a homozygous deletion (374delC) found in the fourth patient (XP4NE). The XPA p.(Glu111*) mutation was previously identified in three Tunisian patients having the same skin and neurological features of the disease as Egyptian patients (Messaoud et al., 2012). This supports the hypothesis of phenotype–genotype correlation and suggests a common ancestor. This is not surprising as Tunisian patients are originating from Kairouan, a city close to Mahdia, which was the capital of the Fatimid dynasty, an empire that encompassed much of North Africa, Sicily and parts of the Middle East and that was reigned from Ifriqiya (between 909 and 969) and from Egypt (between 969 and 1171). A common historical background could explain the common mutational spectrum; hence the hypothesis of a founder effect could be proposed. For the 374delC mutation, it was previously reported once in a Caucasian patient (Satokata et al., 1992b). Having the same mutation between North African and European XP patients is a common event
that could also be explained by a founder effect resulting from migratory flows that took place between the two continents. The same situation was frequently observed for several genetic disorders including XP-C (Ben Rekaya et al., 2009). Of particular interest is that all characterized mutations are located in the DNA binding region (aa 98–219). This could explain the severe phenotype observed in Egyptian patients. Thus, severe skin manifestations associated with neurological abnormalities might be related to a deficiency in the ability of XPA protein to bind to damaged DNA and to act as a processivity factor (Bartels and Lambert, 2007). In addition, neurological abnormalities observed in XP-A patients are due to the insufficient repair of oxidative DNA lesions in the central nervous system, where the production rate of reactive oxygen species is the highest (Rass et al., 2007). It was well established that clinical heterogeneity is associated with the mutational site (Messaoud et al., 2012; Nishigori et al., 1994; Sato et al., 1996; Takahashi et al., 2010). For example, Japanese XP-A patients that have the splicing mutation in intron 3 usually develop very severe neurological abnormalities, which are manifested by the inability to walk unaided and usually die in teens. This is in accordance with XP4NE patient phenotype who bears an exon 3 mutation and presents severe neurological alterations while having the mildest skin symptoms among studied cases (Fig. 2), that could be justified by her young age & high parental awareness. Phenotypic variability was detected among our studied patients even between patients sharing the same mutation (XP1GE and XP2UE) as the
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Fig. 2. Correlation between detected mutations and clinical severity. Severe skin affection with large achromic spots in XP1GE, XP2UE & XP3NE (black arrows). Malignant lesions (kinked arrows) in XP2UE & XP3NE. Mild skin affection is observed in XP4NE patient.
development of malignancies in XP2UE. This could be essentially due to UV-exposure and age difference. It could also be explained by the level of parental awareness taking into consideration that the most severe dermatological presentation was observed in XP2UE patient (Fig. 2) who resides in the less urban Upper Egypt region. Although XP is a rare hereditary disorder, an accurate early genetic diagnosis might prevent complications associated with unprotected exposure to sunlight and enable early prenatal diagnosis and genetic counseling. Indeed, as each of the identified mutations abolishes or creates a new restriction site, PCR–RFLP could be used as a rapid and relatively cost effective tool for molecular diagnosis and carrier screening. In conclusion, clinical and molecular characterization of XP-A patients is essential to optimize follow-up, genetic counseling and health support of patients and may also pave the way for the development of new therapeutics. Studying common mutations and genetic diversity among neighboring countries from North Africa is an important step in setting diagnostic strategies and prioritizing studies for genetic disorders. Conflict of interest The authors state that the manuscript is not published elsewhere, and is not under consideration elsewhere. All the listed authors have agreed to the submission of the manuscript in its current form. The authors have no commercial affiliations, consultancies, stock or equity interests and patent licensing arrangements that could be considered to pose a conflict of interest regarding the submitted article. Acknowledgments We are very grateful to the families who took part in this study.
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