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Genetic investigation of XPA gene: high frequency of the c.682C>T mutation in Moroccan XP patients with moderate clinical profile

  • Zineb Kindil1, 2,
  • Mohamed Amine Senhaji1,
  • Amina Bakhchane1,
  • Hicham Charoute1,
  • Soumia Chihab3,
  • Sellama Nadifi2 and
  • Abdelhamid Barakat1Email author
BMC Research Notes201710:704

https://doi.org/10.1186/s13104-017-3042-6

Received: 15 June 2017

Accepted: 30 November 2017

Published: 6 December 2017

Abstract

Objective

Xeroderma pigmentosum (XP) is a genetically and clinically heterogeneous disease, associated with an inherited defect in one of eight different genes (XPA to XPG and XPV). In addition to the early onset of the skin manifestations, the XP group A is marked by the presence of a mild to severe neural disorders which appear tardily and worsens with age. In this study, 9 patients with moderate clinical profile belonging to 6 XP families were recruited to determine the XPA mutational spectrum in Morocco, using the direct sequencing of the whole coding region of the XPA gene.

Results

The genetic investigation of the XPA gene showed that 7 from 9 patients were homozygous for the c.682C>T, p.Arg228X mutation, while all their investigated family members were heterozygous. The frequency of this mutation was estimated to be 83.33% (5/6 families) .The molecular analysis of the 5 other exons of the XPA gene, showed that the 2 negative siblings carried no mutation in the XPA gene. This finding suggests that c.682C>T (p.Arg228X) mutation is relatively associated with moderate phenotype in XP group A Moroccan families; this result will also contribute to improve the molecular diagnosis of XP disease in Moroccan patients.

Keywords

Xeroderma pigmentosum XPA MutationMorocco

Introduction

Xeroderma pigmentosum (XP) is a rare disease inherited under the autosomal recessive mode. It is clinically characterized by sunlight hypersensitivity, pigmentary changes, premature skin ageing, malignant skin and eyes tumor development [1, 2]. This genodermatosis is responsible evenly for a markedly elevated risk of developing other skin and ocular disorders with or without neurological abnormalities [25]. Xeroderma pigmentosum disease shows also a high genetic heterogeneity, it has been classified into eight complementation groups (XPA to XPG and XPV) [6]. The XPC, XPD and XPA complementation groups present the most frequent forms of Xeroderma pigmentosum in Europe, North Africa, Japan and the USA, as they are responsible for about 90% of XP patients worldwide [7]. In Japan about 60% of XP patients belong to group A, moreover, nearly 1% of Japanese population shares the (c.390-1G>C) XPA gene mutation with a founder effect [8]. The XPA human gene ensures a key role in verification of protein damage during the NER, this gene is composed of 6 exons and encodes a 273 amino acid protein which is involved in DNA excision repair pathway [9]. The XPA is a highly mutated gene, and about 32 mutations with different degree of severity have been identified [according to the Human Gene Mutation Database (HGMD): http://www.hgmd.cf.ac.uk/ac/index.php] (Table 1). This explains the fact that the XP group A patients present high heterogeneity of cutaneous and ocular symptoms, which are qualified as moderates comparing with the XPC group, who is known as the most severe form of Xeroderma Pigmentosum. Unlike the XP group C phenotype, XP group A patients present also neurological abnormalities that appeared tardily and independently from UV-induced DNA lesions [10, 11].
Table 1

Number and different type of mutation in XPA human gene (The Human Gene Mutation Database: HGMD)

Mutation type

Number of mutation

Missense/nonsense

12

Splicing

9

Regulatory

1

Small deletions

6

Small insertions

3

Small indels

1

Gross deletions

0

Gross insertions/duplications

0

Complex rearrangements

0

Repeat variations

0

Total of public mutation

32

In Morocco, prevalence of the xeroderma pigmentosum is approximately 1/80 504, which is higher than that found in Europe and the USA [12]. Soufir et al. have reported that XPC gene is the major cause of xeroderma pigmentosum in North Africa, this study showed also that the founder mutation 1643-1644delTG in XPC gene is responsible for a high proportion of XP cases [13]. This founder mutation was estimated to be responsible of more than 76% XP in Moroccan patients [14]. To the best of our knowledge, there is no study investigating the spectrum of XPA gene mutations in the Moroccan population. Thus, 9 Moroccan patients suspected to be XP group A were analyzed; they present different degrees of severity of neuronal disorders and their age range from 7 to 33. This work aims to describe the mutation spectrum of the XPA gene in Moroccan XP patients, and clarify its involvement in molecular diagnosis of XP group A Moroccan patients.

Main text

Methods

Patients

We recruited 9 XPA patients (5 male and 4 female individuals), belonging to 6 unrelated families to describe the genetic profile of XPA diseases in Moroccan XP patients. The Classification of these patients as XP group A was based on clinical symptoms observed in almost the totality of them, including an abnormal neurological development, which worsens with age and moderate skin lesions which start earlier than neurological signs. One of our patients, a 7 years child (XP43.01) whose recruitment was based in his family history, shows no neurological disorders. Our participants all originated from different regions of Morocco and are all diagnosed and treated at the department of Dermatology in Ibn Rochd University Hospital in Casablanca.

A detailed questionnaire giving all information regarding the clinical data of each patient was filled in. An informed consent was obtained from patients as well as their relatives and was approved by the local committee on research ethics of the Pasteur Institute in Morocco.

Molecular analysis

For all patients and their relatives, the DNA extraction from whole blood was carrying out according to phenol chloroform standard protocol [15]. To disclose the presence of genetic variations in our patients, the six exons of XPA gene were amplified using specific primers (Table 2). The PCRs were done in particular conditions included initial denaturation at 95 °C for 5 min, 35 cycles of denaturation at 94 °C for 30 s, annealing at a specific temperature which change depending of the primer couple for 35 s, extension at 72 °C for 40 s, and final elongation at 72 °C for 7 min.
Table 2

list of primers used in PCR amplification of different exons of the XPA gene

Exon

Size (pb)

Forward primer

Reverse primer

1

430

5-AGAGAGCAGGTAGTTAGGCGGG

5-CGGGGAGAGGGAAGGGGAAAG

2

320

5-TTGTGGACATCCTTGTGTTGTTTG

5-TGGCATTATTTAGCATCACTTTGC

3

417

5-GTCAGGCATTGCATACATGCTG

5-GGCATCCTTCCTATTTTATGGGG

4

337

5-GCTGTGTGTGCCCCTAAGTTGC

5-AGCAAAAGCCAAACCAATTATGAC

5

496

5-AGCATACGTTTACTGACAGTTTCATAGG

5-CTTGAAGACCAACATACTGAGGGC

6a

594

5_-GTGAGGTAAGAAAGTAAGTTTGCCAAG

5_-TCTAGCACTCAGCTCCCATCTCTG

6b

544

5_-GTTTCAGTGAAGGTCACCTGGC

5_-GGTTGGTAAATGCTCAGTAAATGTTAGC

aThe first fragment of exon 6

bThe second fragment of exon 6

PCRs were performed in 15 µl final mixture volume containing 30–50 ng of DNA, 6 pmol of each primer, 200 µM of dNTPs, 3 mM of MgCl2, 1× PCR buffer, 0.75 U of GoTaq polymerase (Promega, Madison, USA). Purified PCR product was sequenced using the BigDye Terminator v 1.1 Standard Kit according to the manufacturer’s recommendations (Applied Biosystems, Foster City, CA, USA) using ABI 3130 Genetic Analyzer.

Results

This study involved 6 unrelated XPA Moroccans families, including 9 patients. All patients developed pokilodermia as first symptom of XP at a mean age of 36 month, this pokilodermia was present in all patients’ sun exposed zones (face and hand), and was more marked in patients with low sun protection, 44.44% (4/9) of our patients showed telangiectasia, and only one woman suffered from malignant tumors, she developed an non melanoma skin cancer (NMSC) at 16 years old, the Pathological analysis demonstrated an 5 × 7.5 × 3 mm basal cell carcinoma (BCC) located in the left side of the base of her nose, a later dermatoscopy examination demonstrated that she has also developed two benign tumors at 20 and 23 years old. No ocular malignancies were noticed in our patients. However five patients had photophobia and three showed keratitis in one or both eyes, one patient XP22.02 had a repetitive eye inflammation which was first diagnosed at the age of 10 years old. All our recruited patients was born with normal size and weight, an neurological abnormalities progression was shown in 8 among them; including a low sensorineural hearing loss observed in two siblings XP16.01 and XP16.02, a 33 years woman XP39.01 showed a severe mental and psychomotor retardation which progress in a loss of ability to walk, speech and motion disorders; according to her family history, two of her sisters had a typical XPA clinical profile, they also developed an progressive intellectual impairment and died in their early adulthood. Those patient’s sisters hadn’t been sequenced for the XPA gene, but their medical history mentioned that during their lives they developed all clinical signs characteristic of the Xeroderma pigmentosum type A. One young boy XP43.01 had a normal neurological development at the moment of his recruitment (Table 3), his family history indicate that two members of his maternal family (aunt and uncle), had a XPA clinical profile, similarly to the XP39.01 sister’s, they developed a severe neurological troubles in their adulthood, before dying from cancer at the age of 29 and 34 years old respectively. The other patients aged between 12 and 18 years old had no similar antecedents in their families.
Table 3

Clinical symptoms of XPA patients

Family code

Number of patient/family

Sex

Age

Age of first consultation (m)

Symptoms

Dermatologic

Ocular

Neurologic

XP16

2

F

31

84

++

+

+

F

32

72

++

+

+

XP22

2

M

15

60

++

+

  

F

18

18

+

+

++

XP31

1

M

12

36

+

+

XP32

2

M

14

72

+

+

+

  

M

16

60

+

+

++

XP39

1

F

33

120

+++

+

+++

XP43

1

M

7

18

+

The screening of the coding region of the XPA gene disclosed the presence in homozygote state of the recurrent mutation c.682C>T (p.Arg228Ter) in 7/9 XP patients. This punctual variation localized in the exon 6 of the XPA gene leads to a premature termination of the encoded protein. Furthermore, all tested parents and healthy relatives of these patients were heterozygous for this mutation (Fig. 1). Additional investigation showed that 2 female siblings had no mutations in the whole XPA gene; even so they present a moderate clinical profile with mild neural retardation, this clinical feature is maybe related with molecular variations in other XP gene (XPB, XPD or XPF) [16, 17].
Fig. 1

a Pedigree of the family XP43. b Localization of the mutation C.682C>T (P.R228X)

Discussion

Xeroderma pigmentosum is genetically and clinically heterogonous disease, its incidence was estimated at 1/1,000,000 in the United States and Europe, 1/20,000–100,000 in Japan [8, 16], and around 1/10,000–50,000 in North Africa and the Middle East [17, 18]. Eight genes were identified as implicated in this rare genodermatosis among them the XPA gene [6], which is characterized by neurological defects [6]. In Morocco, no data concerning the XP group A incidence is available. However in this study a clinical and mutational investigation of XPA complementation group is performed in Moroccan patients, in order to contribute to improve diagnosis for XP group A patients and to establish a genetic counseling for their families.

The majority of our patients 88.88% (8/9) were born from consanguineous parents; they are aged between 7 and 33 years with average age of 16.42 years. Moreover, almost all of them showed moderate skin and ocular symptoms. Neurological disorders were also observed especially in aged patients. The patient XP43.01 too young to show a mental abnormal development had a normal neurological profile.

The XPA is one of six XP protein factors overriding in human NER machinery [8]. Nowadays, over thirty mutations affecting the XPA gene had been listed at the Human Gene Mutation Database; the majority of them are Missense variations (http://www.hgmd.cf.ac.uk/ac/index.php). The direct sequencing of the exon 6 of XPA gene showed that about 83% of screened families bear the c.682C>T (p.Arg228X) point mutation, this mutation correspond to C to T transition at position 682 of the coding DNA, and leads to a truncated protein. This nonsense mutation occurs in the C-terminal domain of XPA protein, and this could explain the moderate clinical manifestations of our XP group A patients; since the severity of the clinical phenotype is observed when mutations are located in the DNA binding region (aa 98–219) (Fig. 2). This moderate phenotype was also detected in American, European and Japanese families who are sharing this punctual mutation [19]. In Tunisia, six out of seven XP group A patients had this nonsense mutation with a frequency of 86% [20]. In addition, a previous study has shown that all XP group A Tunisian patients with moderate phenotype had this mutation with founder effect [21]. The same mutation was also identified in Algerian XP group A patients [22]. A study leading by Soufir et al. including 66 unrelated XP families from North Africa, showed that the c.682C>T (p.Arg228X) mutation was present in all XP group A analyzed patients with a frequency of 12% [13]. This suggested that this mutation may have a common founder effect in the North African region.
Fig. 2

Mapping of XPA gene structure showing XPA exons and different XPA reactive domains

(adapted from Ref. [19])

Severe clinical phenotype is usually observed when patients had mutations within exons 3–5, as reported in four Egyptians [23] and one Iranian patient [24]. A very severe abnormalities were also observed in a Japanese patient who present a mutation located at intron 3 in the splice site [25].

On the other hand, the molecular investigation in patients with no mutation in exon 6, has demonstrated that those two young siblings don’t share any molecular variation in the others XPA gene exons. As perspective, those two patients will be analyzed for the XPB, XPD and XPF genes responsible of rare forms of xeroderma pigmentosum with neurological disorders [26, 27].

In conclusion, our finding suggested that the c.682C>T (p.Arg228X) mutation is relatively associated with moderate clinical profile in XP group A Moroccan patients.

Limitations

This work in which the XPA gene were analyzed for the first time in the Moroccan population will provide a basis for the prenatal diagnosis and genetic counseling. However, the sample size is relatively small and further studies are necessary to determine the spectrum of XPA gene mutations is Moroccan patients.

Abbreviations

BCC: 

basal cell carcinoma

NER: 

nucleotide excision repair

NMSC: 

melanoma skin cancer

PCR: 

polymerase chain reaction

XP

xeroderma pigmentosum

XPA

xeroderma pigmentosum, complementation group A

XPC

xeroderma pigmentosum, complementation group C

XPD

xeroderma pigmentosum complementary group D

XPG

xeroderma pigmentosum complementary group G

XPV

xeroderma pigmentosum, variant type

Declarations

Authors’ contributions

AB conceived, designed and coordinated the study. ZK performed the laboratory work. MAS helped in the laboratory work. ZK wrote the paper. AmB, HC, SC and SN participated in interpretation of results. All authors read and approved the final manuscript.

Acknowledgements

The authors would like to express their deep gratitude to patients and their families, as well as the dermatology service members for their contribution to this work.

Competing interests

The authors declare that they have no competing interests.

Availability of data and supporting materials

Please contact Dr. Abdelhamid Barakat (hamid.barakat@pasteur.ma) for data requests.

Consent for publication

Not applicable.

Ethics approval and consent to participate

All participants signed informed consent forms. In the case of children aged less than 16 years old, we obtained parental consent. The study protocol was approved by local Committee on Research Ethics of Pasteur Institut of Morroco.

Funding

This work was supported by Institut Pasteur du Maroc.

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Authors’ Affiliations

(1)
Human Molecular Genetics Laboratory, Institut Pasteur du Maroc
(2)
Laboratory of Genetics and Molecular Pathology, Faculty of Medicine, Hassan II University
(3)
Department of Dermatology, Hospital University Ibn Rochd

References

  1. Butt FMA, Moshi JR, Owibingire S, Chindia ML. Xeroderma pigmentosum: a review and case series. J Cranio-Maxillo-fac Surg Off Publ Eur Assoc Cranio-Maxillo-fac Surg. 2010;38(7):534–7.Google Scholar
  2. Tamura D, DiGiovanna JJ, Kraemer KH. Founder mutations in xeroderma pigmentosum. J Invest Dermatol. 2010;130(6):1491–3.View ArticlePubMedPubMed CentralGoogle Scholar
  3. Stary A, Sarasin A. The genetics of the hereditary xeroderma pigmentosum syndrome. Biochimie. 2002;84(1):49–60.View ArticlePubMedGoogle Scholar
  4. Kraemer KH, Lee MM, Scotto J. Xeroderma pigmentosum. Cutaneous, ocular, and neurologic abnormalities in 830 published cases. Arch Dermatol. 1987;123(2):241–50.View ArticlePubMedGoogle Scholar
  5. Sugasawa K, Ng JM, Masutani C, Iwai S, van der Spek PJ, Eker AP, et al. Xeroderma pigmentosum group C protein complex is the initiator of global genome nucleotide excision repair. Mol Cell. 1998;2(2):223–32.View ArticlePubMedGoogle Scholar
  6. Lehmann AR. DNA repair-deficient diseases, xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. Biochimie. 2003;85(11):1101–11.View ArticlePubMedGoogle Scholar
  7. Legerski R, Peterson C. Expression cloning of a human DNA repair gene involved in xeroderma pigmentosum group C. Nature. 1992;359(6390):70–3.View ArticlePubMedGoogle Scholar
  8. Hirai Y, Kodama Y, Moriwaki S-I, Noda A, Cullings HM, Macphee DG, et al. Heterozygous individuals bearing a founder mutation in the XPA DNA repair gene comprise nearly 1% of the Japanese population. Mutat Res. 2006;601(1–2):171–8.View ArticlePubMedGoogle Scholar
  9. Legerski RJ, Liu P, Li L, Peterson CA, Zhao Y, Leach RJ, et al. Assignment of xeroderma pigmentosum group C (XPC) gene to chromosome 3p25. Genomics. 1994;21(1):266–9.View ArticlePubMedGoogle Scholar
  10. Laposa RR, Cleaver JE. DNA repair on the brain. Proc Natl Acad Sci USA. 2001;98(23):12860–2.View ArticlePubMedPubMed CentralGoogle Scholar
  11. Ben Rekaya M, Messaoud O, Talmoudi F, Nouira S, Ouragini H, Amouri A, et al. High frequency of the V548A fs X572 XPC mutation in Tunisia: implication for molecular diagnosis. J Hum Genet. 2009;54(7):426–9.View ArticlePubMedGoogle Scholar
  12. Doubaj Y, Laarabi F-Z, Chafai Elalaoui S, Barkat A, Sefiani A. Carrier frequency of the recurrent mutation c.1643_1644delTG in the XPC gene and birth prevalence of the xeroderma pigmentosum in Morocco. J Dermatol. 2012;39(4):382–4.View ArticlePubMedGoogle Scholar
  13. Soufir N, Ged C, Bourillon A, Austerlitz F, Chemin C, Stary A, et al. A prevalent mutation with founder effect in xeroderma pigmentosum group C from north Africa. J Invest Dermatol. 2010;130(6):1537–42.View ArticlePubMedGoogle Scholar
  14. Senhaji MA, Abidi O, Nadifi S, Benchikhi H, Khadir K, Ben Rekaya M, et al. c.1643_1644delTG XPC mutation is more frequent in Moroccan patients with xeroderma pigmentosum. Arch Dermatol Res. 2013;305(1):53–7.View ArticlePubMedGoogle Scholar
  15. Grimberg J, Nawoschik S, Belluscio L, McKee R, Turck A, Eisenberg A. A simple and efficient non-organic procedure for the isolation of genomic DNA from blood. Nucleic Acids Res. 1989;17(20):8390.View ArticlePubMedPubMed CentralGoogle Scholar
  16. Moriwaki S, Kraemer KH. Xeroderma pigmentosum–bridging a gap between clinic and laboratory. Photodermatol Photoimmunol Photomed. 2001;17(2):47–54.View ArticlePubMedGoogle Scholar
  17. Cartault F, Nava C, Malbrunot A-C, Munier P, Hebert J-C, N’Guyen P, et al. A new XPC gene splicing mutation has lead to the highest worldwide prevalence of xeroderma pigmentosum in black Mahori patients. DNA Repair Amst. 2011;10(6):577–85.View ArticlePubMedGoogle Scholar
  18. Zghal M, El-Fekih N, Fazaa B, Fredj M, Zhioua R, Mokhtar I, et al. Xeroderma pigmentosum. Cutaneous, ocular, and neurologic abnormalities in 49 Tunisian cases. Tunis Méd. 2005;83(12):760–3.PubMedGoogle Scholar
  19. States J, McDuffie E, Myrand S, McDowell M, Cleaver J. Distribution of mutations in the human xeroderma pigmentosum group A gene and their relationships to the functional regions of the DNA damage recognition protein. Hum Mutat. 1998;12(2):103–13.View ArticlePubMedGoogle Scholar
  20. Nishigori C, Zghal M, Yagi T, Imamura S, Komoun MR, Takebe H. High prevalence of the point mutation in exon 6 of the xeroderma pigmentosum group A-complementing (XPAC) gene in xeroderma pigmentosum group A patients in Tunisia. Am J Hum Genet. 1993;53(5):1001–6.PubMedPubMed CentralGoogle Scholar
  21. Messaoud O, Ben Rekaya M, Cherif W, Talmoudi F, Boussen H, Mokhtar I, et al. Genetic homogeneity of mutational spectrum of group-A xeroderma pigmentosum in Tunisian patients. Int J Dermatol. 2010;49(5):544–8.View ArticlePubMedGoogle Scholar
  22. Bensenouci S, Louhibi L, De Verneuil H, Mahmoudi K, Saidi-Mehtar N. Diagnosis of xeroderma pigmentosum Groups A and C by detection of two prevalent mutations in West Algerian population: a rapid genotyping tool for the frequent XPC mutation c.1643_1644delTG. BioMed Res Int. 2016. Disponible sur: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4931069/. Accessed 18 Jul 2017.
  23. Amr K, Messaoud O, El Darouti M, Abdelhak S, El-Kamah G. Mutational spectrum of xeroderma pigmentosum group A in Egyptian patients. Gene. 2014;533(1):52–6.View ArticlePubMedGoogle Scholar
  24. Ghafouri-Fard S, Fardaei M, Miryounesi M. A novel 5 nucleotide deletion in XPA gene is associated with severe neurological abnormalities. Gene. 2016;576(1, Part 2):379–80.View ArticlePubMedGoogle Scholar
  25. Nishigori C, Moriwaki S, Takebe H, Tanaka T, Imamura S. Gene alterations and clinical characteristics of xeroderma pigmentosum group A patients in Japan. Arch Dermatol. 1994;130(2):191–7.View ArticlePubMedGoogle Scholar
  26. Dupuy A, Sarasin A. DNA damage and gene therapy of xeroderma pigmentosum, a human DNA repair-deficient disease. Mutat Res. 2015;776:2–8.View ArticlePubMedGoogle Scholar
  27. Moriwaki S, Nishigori C, Imamura S, Yagi T, Takahashi C, Fujimoto N, et al. A case of xeroderma pigmentosum complementation group F with neurological abnormalities. Br J Dermatol. 1993;128(1):91–4.View ArticlePubMedGoogle Scholar

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