A Founder Large Deletion Mutation in Xeroderma Pigmentosum-Variant Form in Tunisia: Implication for Molecular Diagnosis and Therapy

Research ArticleA Founder Large Deletion Mutation in XerodermaPigmentosum-Variant Form in Tunisia Implication forMolecular Diagnosis and Therapy

Mariem Ben Rekaya1 Nadia Laroussi1 Olfa Messaoud1 Mariem Jones12

Manel Jerbi1 Chokri Naouali1 Yosra Bouyacoub1 Mariem Chargui1 Rym Kefi1

Becima Fazaa12 Mohamed Samir Boubaker13 Hamouda Boussen4 Mourad Mokni5

Sonia Abdelhak1 Mohamed Zghal2 Aida Khaled12 and Houda Yacoub-Youssef1

1 Laboratoire de Genomique Biomedicale et Oncogenetique (LR 11 IPT 05) Institut Pasteur de TunisUniversite de Tunis El Manar El Manar I BP 74 13 Place Pasteur 1002 Tunis Belvedere 2092 Tunis Tunisia

2 Departement de Dermatologie Hopital Charles Nicolle de Tunis 1006 Tunis Tunisia3 Departement drsquoAnatomie-Pathologique Humaine et Experimentale Institut Pasteur de Tunis 1002 Tunis Tunisia4Departement drsquoOncologie Medicale Hopital Abderrahman Mami 2080 Ariana Tunisia5 Departement drsquoOncologie Medicale Hopital La Rabta de Tunis 1007 Tunis Tunisia

Correspondence should be addressed to Mariem Ben Rekaya rekayamariemyahoofr

Received 22 February 2014 Accepted 23 March 2014 Published 4 May 2014

Academic Editor Margit Burmeister

Copyright copy 2014 Mariem Ben Rekaya et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Xeroderma pigmentosumVariant (XP-V) form is characterized by a late onset of skin symptoms Our aim is the clinical and geneticinvestigations of XP-V Tunisian patients in order to develop a simple tool for early diagnosis We investigated 16 suspected XPpatients belonging to ten consanguineous families Analysis of the POLH gene was performed by linkage analysis long range PCRand sequencing Genetic analysis showed linkage to the POLH gene with a founder haplotype in all affected patients Long rangePCR of exon 9 to exon 11 showed a 3926 bp deletion compared to control individuals Sequence analysis demonstrates that thisdeletion has occurred between two Alu-Sq2 repetitive sequences in the same orientation respectively in introns 9 and 10 Wesuggest that this mutation POLH NG 0092521 g36847 40771del3925 is caused by an equal crossover event that occurred betweentwo homologous chromosomes atmeiosisThese results allowed us to develop a simple test based on a simple PCR in order to screensuspected XP-V patients In Tunisia the prevalence of XP-V group seems to be underestimated and clinical diagnosis is usually laterCascade screening of this founder mutation by PCR in regions with high frequency of XP provides a rapid and cost-effective toolfor early diagnosis of XP-V in Tunisia and North Africa

1 Introduction

Xeroderma pigmentosum (XP) is an autosomal recessivecancer prone disease characterized by sensitivity to ultravioletrays (UVR) XP patients are consequently predisposed todevelop skin and eyes cancers [1] This disease is caused byinherited mutations in DNA repair genes encoding proteinsthat protect cells fromUV-induced damage XP is geneticallyheterogeneous with seven XP complementation groups (XP-A to XP-G) defective in nucleotide excision repair (NER)

pathway and an additional ldquovariantrdquo form (XP-V) with nor-mal NER but a deficient translesional synthesis

XP-V patients have a relatively milder phenotype with alate onset of symptoms and delayed progression TypicallyXP-V patients do not have ocular or neurological abnor-malities [2] Many studies suggest that this form of XP isunderdiagnosed [3 4] Therefore XP-V patients representonly 20 to 30 of all XP cases [5]

Cells from XP-V patients are extremely hypermutableafter exposure to UV due to the deficiency of pol eta [6 7]

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 256245 8 pageshttpdxdoiorg1011552014256245

2 BioMed Research International

The DNA polymerase eta (120578) normally catalyzes translesionsynthesis (TLS) by incorporating dAMP opposite thymineresidues of a cyclobutane thymine dimer (CPD) [8 9] In theabsence of pol eta the highly error-prone pol iota undertakesthis bypass function resulting in the accumulation of UV-induced mutations and an increase in the susceptibility toskin cancer [10 11]

Pol eta is encoded by the POLH gene the humanhomolog of yeast Rad30 [9] Pol 120578 plays an important role inpreventing genome instability after UV or cisplatin-inducedDNA damage [12] Chemoresistance of cancer to cisplatintreatment is due in part to human Pol 120578 Crystal structuresof hPol 120578 complexed with intrastrand cisplatin identified ahydrophobic pocket as a potential drug target for reducingchemoresistance [13]

More than 60mutations have been identified in thePOLHgene in cell lines derived from XP-V patients from differ-ent geographic locations mainly Russia-Armenia ScotlandLebanon Iran Belgium France Japan USA Europe AsiaCayman Islands Turkey Israel Germany Korea Algeria andTunisia [2ndash4 9 14ndash23]

In our study we surveyed POLHmutations in 16 Tunisianpatients with late onset of XP in order to assess the causativemutations of this disorder and to develop a rapid moleculardiagnostic test

2 Patients and Methods

21 Patients Sixteen suspected XP-V patients belonging toten consanguineous Tunisian families originated from differ-ent regions of Tunisia were investigated (Table 2) Their agewas ranging from 4 to 50 years

22 Methods Written informed consent was obtained fromall available family members or from parents of minorchildren Families were interviewed using a structured ques-tionnaire to collect information about family history consan-guinity affected members and associated diseases DNA wasisolated from peripheral blood leukocyte using salting-outmethod [24] or Qiagen kit DNA extraction

221 Genetic Analysis To confirm linkage to POLH geneavailable family members were genotyped using two poly-morphic microsatellite markers spanning a 04Mb inter-val near to POLH locus (cen-D6S207 and D6S1582(POLH-)tel) as previously described [4]Microsatellitemark-ers were selected from the genetic maps available on NCBIbrowsers and the CEPH genotype database (httpwwwcephbfrencephdb) on the basis of their heterozygositypercentage and closeness to the POLH gene Genotyping wasperformed as described elsewhere [25]

222 PCR Long-Range On absence of amplification ofPOLH exon 10 long PCR was performed using the ExpandLong Template PCR System Kit (Expand Long Range dNT-Pack 700 units120583L Roche) PCR was performed using dif-ferent primers (Table 3) The PCR program included 92∘Cfor 2min 10 cycles of 92∘C for 10 sec 60∘C for 15 sec 68∘C

for 10min and 20 cycles of the same program except thatthe extension step was extended by 20 sec per cycle PCRproducts were run on 1 agarose gel with the DNA ladder1 kb molecular size marker (GeneRulerTM)

223 Bioinformatic Analysis As several genomic rearrange-ments are commonly caused by recombination eventsinduced by repetitive elements present in the human genomethe genomic sequence of the POLH gene (NG 0092521) wasobtained and analyzed from chr6 43578281 to 43581387 posi-tion corresponding to exon 9 to exon 11 region Screening forrepetitive elements was performed using the RepeatMaskersoftware available at httpwwwrepeatmaskerorg (Table 1)

224 Sequencing and Mutation Analysis Long range PCRproducts were directly sequenced using the ABI 3130 GeneticAnalyzer by two pairs of primers (Table 3) Mutation analysisand breakpoints of the deletion were determined as thelast nucleotide showing sequence identity between wild andmutated sequences (Figure 3)

225 Mutation Nomenclature The genomic reference of thePOLH gene NG 0117631 was used to annotate the deletionaccording to the HGVS version 20 (Mutalyzer 20beta-26)

3 Results

31 Clinical Findings In this study we investigated sixteenpatients with late onset of XP features These patients belongto ten consanguineous families from different Tunisian geo-graphic areas (Table 2) For all patients skin hyperphotosen-sitivity to UVR began at a mean age of 4 years The mean ageat onset of the first skin cancer was 24 years Nonmelanomaskin cancer (NMSC) occurred in only 10 patients At least 3among them developed only basal cell carcinoma (BCC) and5 developed squamous cell carcinoma (SCC) combined withBCC (Table 2)

32 Genetic Analysis The genetic examination of XP-Vpatients was initially assessed through routine procedureswhich involved genotyping of all consanguineous XP-Vpatients and available related individuals Haplotype analysisshowed homozygosity for the closest two markers to POLHgene D6S1582 and D6S271 with a founder haplotype (129ndash188) in all investigated patients (Figure 1)

33 PCR Long-Range DNA samples from these patientsrepeatedly failed to yield PCR amplification products forexon 10 Therefore we assumed the presence of a genomicdeletion spanning exon 10 (del exon 10) As del exon 10was previously described in Italian patient at the genomicDNA level with 27 Kb deletion [2] we first screen for thisdeletion Therefore screening of this deletion by PCR didnot yield any amplification product confirming that thereare different breakpoints involved in our XP-V patients Inorder to identify the deletion size we amplified the sequencebetween exon 9 and exon 11 using primers POLH10ΔF andPOLH10ΔR showed in Table 3 Long range PCR revealed

BioMed Research International 3

Table1Sequ

ence

analysisof

repetitivee

lementsof

the9

358b

psequ

ence

ofPO

LHgene43572521ndash43581878

usingrepeatmaskerS

oftware

scorediv

del

ins

Query

sequ

ence

Positionin

query-

C +Matchingrepeat

Repeatcla

ssfa

mily

-Position

inrepeat(le

ft)endbegin

linkage

begin

end

(left)

+repeat

classfa

mily

begin

end

(left)

idgraph

ic1692

135

04

04

chr6POLH

43572521+43581878

657

893

(8465)

CAluJr

SINEAlu

(2)

310

741

2266

104

03

43

chr6POLH

43572521+43581878

925

1240

(8118)

CAluSq

SINEAlu

(9)

304

12

658

263

86

46

chr6POLH

43572521+43581878

1267

1581

(7777)

CL1MA8

LINEL1

(25)

6266

5940

32432

120

00

00

chr6POLH

43572521+43581878

1644

1944

(7414)

CAluSx1

SINEAlu

(10)

302

24

3529

149

69

42

chr6POLH

43572521+43581878

1958

2302

(7056)

CL1MB8

LINEL1

(0)

6178

5821

52464

108

00

00

chr6POLH

43572521+43581878

2303

2609

(6749)

CAluSc5

SINEAlu

(2)

307

16

3529

161

7044

chr6POLH

43572521+43581878

2610

3080

(6278

CL1MB8

LINEL1

(342)

5820

5323

52287

9255

00

chr6POLH

43572521+43581878

3081

3372

(5986)

+AluSq2

SINEAlu

1308

(5)

71739

178

107

18chr6POLH

43572521+43581878

3373

3510

(5848)

CL1MB8

LINEL1

(829)

5333

5170

5810

187

1270

chr6POLH

43572521+43581878

3744

3909

(544

9)C

AluJo

SINEAlu

(19)

293

137

8535

296

9714

chr6POLH

43572521+43581878

3990

4509

(4849)

+L2

aLINEL2

(2804)

3365

(61)

92528

100

00

10chr6POLH

43572521+43581878

5271

5581

(3777)

CAluSx1

SINEAlu

(4)

308

110

13100

59

28

chr6POLH

43572521+43581878

5582

5615

(3743)

+(TCT

TTA)n

Simplerepeat

136

(0)

11684

56

00

00

chr6POLH

43572521+43581878

6162

6233

(3125)

+AluSq10

SINEAlu

172

(241)

122510

103

00

00

chr6POLH

43572521+43581878

6991

7300

(2058)

+AluSq2

SINEAlu

1310

(3)

132679

7000

00

chr6POLH

43572521+43581878

7564

7863

(1495)

CAluSq

SINEAlu

(13)

300

114

2503

84

07

00

chr6POLH

43572521+43581878

8018

8313

(1045)

CAluSg

SINEAlu

(11)

299

215

214

247

186

10chr6POLH

43572521+43581878

8598

8683

(675)

+MER

5ADNAhAT

-Charlie

2102

(87)

16196

161

00

00

chr6POLH

43572521+43581878

8684

8714

(644

)+

MER

5ADNAhAT

-Charlie

(159)

189

(0)

17


Table2Clinicalfeatures

ofTu

nisia

nXP

-Vpatie

nts

Patie

nts

Affected

patie

nts

Sex

Age

(years)

Age

aton

seto

fthe

1stX

Pmacules

erythema(

years)

Geographic

Orig

in

Age

aton

seto

f1stTu

mor

inyears(Num

bero

ftum

ors)

Photop

hobia

Radiotherapy

Tumor

post

Radiotherapy

BCC

SCC

Other

tumors

XPV6K

E1

M47

(diedat

50)

4Ke

f22

(6)

20(12)

mdash+minus

+++

+++

XPV15GA

1F

184

Gafsa

15(2)

16(2)

+minus

XPV17-1B

3F

174

Bizerte

00

0mdash

XPV17-2

BF

114

00

0mdash

XPV17-3

BF

45

00

0mdash

XP18G

1F

434

Gafsa

16(8)

21(3)

KA+minus

+++

+++

XPV20G

3F

315

Gafsa

ND(gt10)

ND(gt2)

+++

+

XPV43-1

7M

ND

5Za

ghou

an38

(8)

23(4)

KAand

Actin

icKe

ratosis

+minus

+(lo

cal)

mdash

XPV43-2

F45

541

(1)

+minus

XPV48G

1F

466

Gafsa

047

(1)

+minus

XPV53Z

1M

507

Fahs

Zagh

ouan

37(4)

+(lo

cal)

+minus

XPV79-1

3(1died)

F13

3To

zeurG

afsa

10(3)

mdashKA

+++minus

XPV79-2

F18

3KA

mdash

XPV91-1

3F

296

Tozeur

Actin

icKe

ratosis

mdash

XPV91-2

F21

6mdash

XPV91-3

F24

6mdash

SCC

spinocellcarcinom

aBC

CbasalcellC

arcino

maKA

keratho

acantum(+minus)m

oderatep

heno

type(mdash)a

bsence(++

+)several


Table 3 Complete list of primers used to gDNA amplification of exon 10 and its intronic boundaries

Name Sequence 51015840 rarr 31015840 AnnealingTemperature (∘C)

Suspected PCRproducts size (bp)

PCR Product size forXP-V patients (bp)

XPV10F CCTGGTTCTTTTAATTTCCTCTCCTG 55 459 mdashXPV10R CATTTACCCTTTACCTCATTGAAGGACXPV del 10 F TCATTTGTGCTGTCCTGTTC 60 3012 mdashXPV del 10 R GGTTGCAGTGAGCGGAGATTDel ex10 LR-F AGGTCCTCCCTAGTTACCCTATCACAGCAG 60 4105 mdashDel ex10 LR-R ACTACCTAACCCTGACTGACTTACCACTCTGGPOLH10ΔF AGTGGGTAGGTTTTGGTAGCTGTGGAAG 60 9358 asymp6000 bpPOLH10ΔR GGACACACCCTGGATACTCTGTTGGTAAPOLHFΔ ACCTTGGAGTATAATTTCTGGGTCA 59 5212 asymp1000 bpPOLHRΔ GTCATAAAGTTCCTCATTGTGTCTAAPOLHdelF CATGTGCTTGTTGGACATTTG 60 4526

asymp500 bpPOLHdelR GGTTTCATGCTTTGGGACAG

119886 asymp 6 kb product for XP-V patients versus asymp10 kb in controlindividual corresponding to approximately 4 kb size deletion(Figure 2)

34 Bioinformatic Analysis Screening of repetitive elementspresent in exon 9 to exon 11 using repeat masker soft-ware revealed that 5144 (4814 pb) of the sequence wasinterspersed repeat sequences Among them 11 SINE Alusequences spanned a region of 2908 bp (3108 of all thesequence) and 3 LINE sequences spanned a region of 1789 bp(1912 of all the sequence) These Alu sequences are pre-dicted to promote the occurrence of large deletions (Table 1)

35 Mutation Screening In order to detect the breakpointswith accuracy two internal primer pairs were designed tosequence introns 9 and 10 across the deletion (Table 3)Direct sequencing and analysis of the 6 kb PCR product(Figure 2) of XPV17 and XPV91 patients using primersPOLHdelF and POLHdelR revealed that both the 51015840 and 31015840breakpoints were located within homologous Alu Sq2 (classSINE (short interspersed elements family Alu)) elements inintrons 9 and 10 ofPOLH gene (Figure 3)This deletionPOLHNG 0092521 g32438 36363del3926led to the loss of exon10 (c1370-2567 1539+1188del3925) This mutation has likelyresulted from Alu-Alu equal homologous recombination

36 Screening of Deletion by PCR After identification of thedeletion breakpoints in two patients (XPV91 and XPV17)we screened the following patients for this deletion by PCRusing primers POLHdelF and POLHdelR showed in Table 3In all patients we found a product of 500 pb versus 4500 pbin virtual PCR We then confirmed the presence of the samebreakpoints by direct sequencing

For individuals at a heterozygous state we confirmedtheir profiles by two PCRsThe presence of one allele of exon10was confirmedusingXPV10F andXPV10Rprimers and theabsence of exon 10 on the other allele was confirmed usingPOLHdelF and POLHdelR primers

4 Discussion

We report 16 cases with NMSC BCC and SCC that occurredwith a mean delay of 24 years after XP diagnosis Five of ourpatients (XPV6KE XP18GXPV20G XPV43-1 and XPV53Z)had been treated by skin radiotherapy (Table 2) After cancertreatment many NMSC appeared For example XPV6KEdied after frontal tumor metastasis and XPV18G experienceda metastasis after recurrence on the right cheek Theseconsequences may be explained by the significant role of poleta in cancer radiotherapy response Pol eta-deficient cellsare resistant to ionizing radiationThis radioresistance resultsfrom the increased reparation of double strand breaks byhomologous recombination repair system (HR) [26] Whilefor chemotherapy previous studies demonstrate that pol eta-deficient cells are very sensitive to cisplatin and oxaliplatinand particularly for agents which exert their activities byblocking DNA replication forks [27] Among the roles of poleta is repairing lesions induced by cisplatin Consequentlysystemic chemotherapy using cisplatin will attack healthycells and induce novel cancers on absence of pol etaThis typeof chemotherapy may be very dangerous for XP-V patientsKnowing this important role of pol eta mutation screeningof POLH gene in patients with SCC or BCC could have animpact in guiding treatment choice

Previous studies showed two specific mutations(c1568 1571delGTCA and c660+1GgtA) in three XP-V Tunisian patients [4 20] Deletion of exon 10 has beenpreviously described at the cDNA level in XP-V patients fromdifferent geographic origins It was found at homozygousstate in two Algerian (XP62VI and XP75VI) and in oneAmerican (XP139DC) and at heterozygous state in oneTunisian (XP28VI) XP-V patients [3 16] Also POLH delexon 10 has been described at genomic level in one Italianpatient with 27 Kb deletion occurring between two poly(T) sequences [2] and in one Algerian XP-V patient with3763 bp deletion [22] We report here a novel breakpointof del exon 10 POLH NG 0092521 g32438 36363del3926


43 2 2

Family XPV17B

129188

129188

153168

149194

129188

129188

129188

129188

12918

129188

129188

129188

139186

139184

3

149155

3

3 3 1 3 4 1 2

145190

139186

145190

Family XPV15GA

129188

129188

Family XPV6KE

8 129188

129188

129188

129188

Family XPV18GA D6S271XPV

D6S1582

D6S451

04Mb

036Mb

0051Mb

Figure 1 Pedigree and haplotype analysis for the XPV families (the disease haplotype is indicated by shading) and clinical photograph ofeach affected patient

10000 bp6000 bp

1kbXP-V17-1

XP-V91-1

XP-V(P) Control

Figure 2 Agar gel electrophoretic analysis of the PCR POLH gDNA of exon 10 and its intronic boundaries showed difference in the sizebetween affected individuals (XPV17B-1 and XPV91) compared to healthy parents (XPV(P)) and a healthy control (Marker 1 kb DNA laddermolecular size marker (GeneRuler))


Intron 9 Intron 10 Breakpoint overlap

GCCAACATGGGGAAACCCTGTCTTTACTAAAAATACAAAAATTAGCCCGGCATGGTGGTGGGTGCCTATA

150 160 170 180 190 200 210

(a)

Alu-Sq2 intron 9

Alu-Sq2 intron 10

Alu-Sq2 intron 9

Alu-Sq2 intron 10

Alu-Sq2 intron 9

Alu-Sq2 intron 10

Alu-Sq2 intron 9

Alu-Sq2 intron 10

23 TTTTGGAGGCCGAGGTGGGCAGATCACCTGAGGTCAGGAGTTCGAGGGTAGCCTGGCCAA 82||| ||||||||||| |||||||||||||||||||||||||| || | | || |||||

38 TTTCGGAGGCCGAGGCAGGCAGATCACCTGAGGTCAGGAGTTCAAGAGCACCCCAGCCAA 97

83 CATGGCGAAACCCTGTCTCTACTAAAAATACAAAAATTAGCTGGGCGTGGTGGCGGGCAC 142||||| ||||||||||||||||||||||||||||||||||| ||| |||||| || |

98 CATGGTGAAACCCTGTCTCTACTAAAAATACAAAAATTAGCCAGGCATGGTGGTGGATGC 157

143 CTGTAATCCCAGCTACGCAGGAGGCTGAGGCAAGAGAATCGCTTGAACCT-GGAGGTGGA 201-|| ||||||||||||| ||||||||||||| |||||| |||||||||| |||||||||

158 CTATAATCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATTGCTTGAACCTGGGAGGTGGA 217

202 GGTTGCAGTGAGCCGAGATTGCGCCATTGCACTCCAGCATGAGCAACAAGAGC-AAA 257-|||||| ||||||||||||||||||||||||||||||| || ||||||||||| |||

218 GGTTGCGGTGAGCCGAGATTGCGCCATTGCACTCCAGCCTGGGCAACAAGAGCGAAA 274

(b)

Exon 9 Exon 11Exon 10Alu-Sq2

Alu-Sq2 Alu-Sq2

Alu-Sq2

Exon 9 Exon 11

GAAACCCTGTCTCTACTAAAAATACAAAAATTAGC

3925bp

1

2

(c)

Figure 3 Characterization of the deletion breakpoints (a) Electropherogram demonstrating the junction fragment resulting from the largedeletion in the XP-V patients Partial representation of introns 9 and 10 with the 35 bp breakpoint overlap framed in red (b) Nucleotidesequence alignment of the genomic sequence of introns 9 and 10 of the POLH gene Short vertical lines indicate matched bases between bothintrons (c) Schematic representation of the deletion breakpoints and their flanking Alu Sq2 elements (1) represents a normal gDNA fragmentand (2) schematizes the mutated gDNA with a deletion of 3925 bp

that presents in 16 XP-V Tunisian patients belonging to 10unrelated families This deletion can be screened by a simplePCR without confirming by sequencing This rapid tool mayfacilitate molecular investigation of XP-V patient

This mutation is probably a founder variation because itwas carried by a particular haplotype (129ndash188 or 129ndash186)Del exon 10 is common in the world and probably it may bedue to different founder effects Repetitive sequences are theprimary candidates to generate stable abnormal secondarystructures producing large deletion during replication [28]Alu elements are normally located within introns and 31015840untranslated regions of genes which are considered muta-tional ldquohotspotsrdquo for large gene rearrangements [29] Largedeletions in POLH gene have been previously described inexons 5 and 6 [3 16] Similar founder mutations in thePOLH gene have been reported in other populations such asJapanese and Korean Therefore 87 of the Japanese XP-Vpatients shared one of the four founder mutations describedin Japan [3 18]

5 Conclusion

The presence of this founder mutation reported in our studycould simplify genetic screening of XP patients in Tunisian

population by implementing presymptomatic tests and henceearly UV protection Before treatment of patientsrsquo skin can-cers XP status should be verified to avoid cancer recurrenceIt is also important to consider the possible existence ofsuch large deletion at heterozygous state Consequently wepropose systematic screening of this mutation in all XP-Vpatients by two PC reactions the 1st will amplify exon 10while the 2nd will amplify across deletion breakpoints Afterconfirmation at a large scale in XP Tunisian patients the testwill be proposed for patients from Southern Mediterraneanand Middle East countries

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Theauthorswould like to thank the patients and their familiesas well as the patientsrsquo support group ldquoHelping XerodermaPigmentosum Childrenrdquo (httpwwwxp-tunisieorgtn) fortheir collaborationThis work was supported by the Tunisian


Ministry of Higher Education and Scientific Research (Labo-ratory on Biomedical Genomics and Oncogenetics no LR 11IPT 05) and the Tunisian Ministry of Public Health

References

[1] K H Kraemer N J Patronas R Schiffmann B P BrooksD Tamura and J J DiGiovanna ldquoXeroderma pigmento-sum trichothiodystrophy and Cockayne syndrome a complexgenotype-phenotype relationshiprdquo Neuroscience vol 145 no 4pp 1388ndash1396 2007

[2] A Gratchev P Strein J Utikal and G Sergij ldquoMoleculargenetics of Xeroderma pigmentosum variantrdquo ExperimentalDermatology vol 12 no 5 pp 529ndash536 2003

[3] H Inui K S Oh C Nadem et al ldquoXeroderma pigmentosum-variant patients from America Europe and Asiardquo Journal ofInvestigative Dermatology vol 128 no 8 pp 2055ndash2068 2008

[4] M B Rekaya O Messaoud A Mebazaa et al ldquoA novelPOLHgenemutation in aXeroderma pigmentosum-VTunisianpatient phenotype-genotype correlationrdquo Journal of Geneticsvol 90 no 3 pp 483ndash487 2011

[5] S Moriwaki and K H Kraemer ldquoXeroderma pigmentosummdashbridging a gap between clinic and laboratoryrdquo Photodermatol-ogy Photoimmunology and Photomedicine vol 17 no 2 pp 47ndash54 2001

[6] P Kannouche and A Stary ldquoXeroderma pigmentosum variantand error-prone DNA polymerasesrdquo Biochimie vol 85 no 11pp 1123ndash1132 2003

[7] A Stary P Kannouche A R Lehmann and A Sarasin ldquoRoleof DNA polymerase 120578 in the UV mutation spectrum in humancellsrdquo The Journal of Biological Chemistry vol 278 no 21 pp18767ndash18775 2003

[8] C Masutani M Araki A Yamada et al ldquoXeroderma pig-mentosum variant (XP-V) correcting protein from HeLa cellshas a thymine dimer bypass DNA polymerase activityrdquo EMBOJournal vol 18 no 12 pp 3491ndash3501 1999

[9] C Masutani R Kusumoto A Yamada et al ldquoThe XPV(Xeroderma pigmentosum variant) gene encodes human DNApolymerase 120578rdquo Nature vol 399 no 6737 pp 700ndash704 1999

[10] C A Dumstorf A B Clark Q Lin et al ldquoParticipation ofmouse DNA polymerase 120580 in strand-biasedmutagenic bypass ofUV photoproducts and suppression of skin cancerrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 103 no 48 pp 18083ndash18088 2006

[11] Q Gueranger A Stary S Aoufouchi et al ldquoRole of DNApolymerases 120578 120580 and 120577 in UV resistance and UV-inducedmutagenesis in a human cell linerdquo DNA Repair vol 7 no 9 pp1551ndash1562 2008

[12] S Cruet-Hennequart K Gallagher A M Sokol S Villalan AM Prendergast and M P Carty ldquoDNA polymerase 120578 a keyprotein in translesion synthesis in human cellsrdquo Sub-CellularBiochemistry vol 50 pp 189ndash209 2010

[13] Y Zhao C Biertumpfel M T Gregory Y J Hua F Hanaokaand W Yang ldquoStructural basis of human DNA polymerase120578-mediated chemoresistance to cisplatinrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 109 no 19 pp 7269ndash7274 2012

[14] T Itoh and S Linn ldquoXP43TO previously classified as Xero-derma pigmentosum group E should be reclassified as Xero-derma pigmentosum variantrdquo Journal of Investigative Derma-tology vol 117 no 6 pp 1672ndash1674 2001

[15] R E Johnson C M Kondratick S Prakash and L PrakashldquohRAD30mutations in the variant form of Xeroderma pigmen-tosumrdquo Science vol 285 no 5425 pp 263ndash265 1999

[16] B C Broughton A Cordonnier W J Kleijer et al ldquoMolecularanalysis of mutations in DNA polymerase 120578 in Xerodermapigmentosum-variant patientsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 99 no2 pp 815ndash820 2002

[17] M Tanioka T Masaki R Ono et al ldquoMolecular analysis ofDNA polymerase eta gene in Japanese patients diagnosed asXeroderma pigmentosum variant typerdquo Journal of InvestigativeDermatology vol 127 no 7 pp 1745ndash1751 2007

[18] T Masaki R Ono M Tanioka et al ldquoFour types of possiblefounder mutations are responsible for 87 of Japanese patientswith Xeroderma pigmentosum variant typerdquo Journal of Derma-tological Science vol 52 no 2 pp 144ndash148 2008

[19] X Liu X Zhang J Qiao and H Fang ldquoIdentification of anovel nonsense mutation in POLH in a Chinese pedigree withXeroderma pigmentosum variant typerdquo International Journalof Medical Sciences vol 10 no 6 pp 766ndash770 2013

[20] O Messaoud ldquoNovel mutation in POLH gene responsible ofsevere phenotype of XP-VrdquoClinical Dermatology vol 1 pp 125ndash129 2013

[21] R Ono T Masaki S Takeuchi et al ldquoThree school-age casesof Xeroderma pigmentosum variant typerdquo PhotodermatologyPhotoimmunology and Photomedicine vol 29 no 3 pp 132ndash1392013

[22] K Opletalova A Bourillon W Yang et al ldquoCorrelation of phe-notypegenotype in a cohort of 23 Xeroderma pigmentosum-variant patients reveals 12 new disease-causing POLH muta-tionsrdquo Human Mutation vol 35 no 1 pp 117ndash128 2014

[23] O Ortega-Recalde J I Vergara D J Fonseca et al ldquoWhole-exome sequencing enables rapid determination of Xerodermapigmentosum molecular etiologyrdquo PLoS ONE vol 8 no 6Article ID e64692 2013

[24] S A Miller D D Dykes and H F Polesky ldquoA simple saltingout procedure for extracting DNA from human nucleated cellsrdquoNucleic Acids Research vol 16 no 3 article 1215 1988

[25] C Bouchlaka S Abdelhak A Amouri et al ldquoFanconi anemiain Tunisia high prevalence of group A and identification ofnew FANCAmutationsrdquo Journal of HumanGenetics vol 48 pp352ndash361 2003

[26] N H Nicolay R Carter S B Hatch et al ldquoHomologousrecombination mediates S-phase-dependent radioresistance incells deficient in DNA polymerase etardquo Carcinogenesis vol 33no 11 pp 2026ndash2034 2012

[27] Y W Chen J E Cleaver F Hanaoka C F Chang and K MChou ldquoA novel role of DNA polymerase 120578 in modulating cel-lular sensitivity to chemotherapeutic agentsrdquoMolecular CancerResearch vol 4 no 4 pp 257ndash265 2006

[28] D Gebow N Miselis and H L Liber ldquoHomologous andnonhomologous recombination resulting in deletion effects ofp53 status microhomology and repetitive DNA length andorientationrdquo Molecular and Cellular Biology vol 20 no 11 pp4028ndash4035 2000

[29] P L Deininger and M A Batzer ldquoAlu repeats and humandiseaserdquo Molecular Genetics and Metabolism vol 67 no 3 pp183ndash193 1999

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


The DNA polymerase eta (120578) normally catalyzes translesionsynthesis (TLS) by incorporating dAMP opposite thymineresidues of a cyclobutane thymine dimer (CPD) [8 9] In theabsence of pol eta the highly error-prone pol iota undertakesthis bypass function resulting in the accumulation of UV-induced mutations and an increase in the susceptibility toskin cancer [10 11]

Pol eta is encoded by the POLH gene the humanhomolog of yeast Rad30 [9] Pol 120578 plays an important role inpreventing genome instability after UV or cisplatin-inducedDNA damage [12] Chemoresistance of cancer to cisplatintreatment is due in part to human Pol 120578 Crystal structuresof hPol 120578 complexed with intrastrand cisplatin identified ahydrophobic pocket as a potential drug target for reducingchemoresistance [13]

More than 60mutations have been identified in thePOLHgene in cell lines derived from XP-V patients from differ-ent geographic locations mainly Russia-Armenia ScotlandLebanon Iran Belgium France Japan USA Europe AsiaCayman Islands Turkey Israel Germany Korea Algeria andTunisia [2ndash4 9 14ndash23]

In our study we surveyed POLHmutations in 16 Tunisianpatients with late onset of XP in order to assess the causativemutations of this disorder and to develop a rapid moleculardiagnostic test

2 Patients and Methods

21 Patients Sixteen suspected XP-V patients belonging toten consanguineous Tunisian families originated from differ-ent regions of Tunisia were investigated (Table 2) Their agewas ranging from 4 to 50 years

22 Methods Written informed consent was obtained fromall available family members or from parents of minorchildren Families were interviewed using a structured ques-tionnaire to collect information about family history consan-guinity affected members and associated diseases DNA wasisolated from peripheral blood leukocyte using salting-outmethod [24] or Qiagen kit DNA extraction

221 Genetic Analysis To confirm linkage to POLH geneavailable family members were genotyped using two poly-morphic microsatellite markers spanning a 04Mb inter-val near to POLH locus (cen-D6S207 and D6S1582(POLH-)tel) as previously described [4]Microsatellitemark-ers were selected from the genetic maps available on NCBIbrowsers and the CEPH genotype database (httpwwwcephbfrencephdb) on the basis of their heterozygositypercentage and closeness to the POLH gene Genotyping wasperformed as described elsewhere [25]

222 PCR Long-Range On absence of amplification ofPOLH exon 10 long PCR was performed using the ExpandLong Template PCR System Kit (Expand Long Range dNT-Pack 700 units120583L Roche) PCR was performed using dif-ferent primers (Table 3) The PCR program included 92∘Cfor 2min 10 cycles of 92∘C for 10 sec 60∘C for 15 sec 68∘C

for 10min and 20 cycles of the same program except thatthe extension step was extended by 20 sec per cycle PCRproducts were run on 1 agarose gel with the DNA ladder1 kb molecular size marker (GeneRulerTM)

223 Bioinformatic Analysis As several genomic rearrange-ments are commonly caused by recombination eventsinduced by repetitive elements present in the human genomethe genomic sequence of the POLH gene (NG 0092521) wasobtained and analyzed from chr6 43578281 to 43581387 posi-tion corresponding to exon 9 to exon 11 region Screening forrepetitive elements was performed using the RepeatMaskersoftware available at httpwwwrepeatmaskerorg (Table 1)

224 Sequencing and Mutation Analysis Long range PCRproducts were directly sequenced using the ABI 3130 GeneticAnalyzer by two pairs of primers (Table 3) Mutation analysisand breakpoints of the deletion were determined as thelast nucleotide showing sequence identity between wild andmutated sequences (Figure 3)

225 Mutation Nomenclature The genomic reference of thePOLH gene NG 0117631 was used to annotate the deletionaccording to the HGVS version 20 (Mutalyzer 20beta-26)

3 Results

31 Clinical Findings In this study we investigated sixteenpatients with late onset of XP features These patients belongto ten consanguineous families from different Tunisian geo-graphic areas (Table 2) For all patients skin hyperphotosen-sitivity to UVR began at a mean age of 4 years The mean ageat onset of the first skin cancer was 24 years Nonmelanomaskin cancer (NMSC) occurred in only 10 patients At least 3among them developed only basal cell carcinoma (BCC) and5 developed squamous cell carcinoma (SCC) combined withBCC (Table 2)

32 Genetic Analysis The genetic examination of XP-Vpatients was initially assessed through routine procedureswhich involved genotyping of all consanguineous XP-Vpatients and available related individuals Haplotype analysisshowed homozygosity for the closest two markers to POLHgene D6S1582 and D6S271 with a founder haplotype (129ndash188) in all investigated patients (Figure 1)

33 PCR Long-Range DNA samples from these patientsrepeatedly failed to yield PCR amplification products forexon 10 Therefore we assumed the presence of a genomicdeletion spanning exon 10 (del exon 10) As del exon 10was previously described in Italian patient at the genomicDNA level with 27 Kb deletion [2] we first screen for thisdeletion Therefore screening of this deletion by PCR didnot yield any amplification product confirming that thereare different breakpoints involved in our XP-V patients Inorder to identify the deletion size we amplified the sequencebetween exon 9 and exon 11 using primers POLH10ΔF andPOLH10ΔR showed in Table 3 Long range PCR revealed


Table1Sequ

ence

analysisof

repetitivee

lementsof

the9

358b

psequ

ence

ofPO


usingrepeatmaskerS

oftware

scorediv

del

ins

Query

sequ

ence

Positionin

query-

C +Matchingrepeat

Repeatcla

ssfa

mily

-Position

inrepeat(le

ft)endbegin

linkage

begin

end

(left)

+repeat

classfa

mily

begin

end

(left)

idgraph

ic1692

135

04

04

chr6POLH

43572521+43581878

657

893

(8465)

CAluJr

SINEAlu

(2)

310

741

2266

104

03

43

chr6POLH

43572521+43581878

925

1240

(8118)

CAluSq

SINEAlu

(9)

304

12

658

263

86

46

chr6POLH

43572521+43581878

1267

1581

(7777)

CL1MA8

LINEL1

(25)

6266

5940

32432

120

00

00

chr6POLH

43572521+43581878

1644

1944

(7414)

CAluSx1

SINEAlu

(10)

302

24

3529

149

69

42

chr6POLH

43572521+43581878

1958

2302

(7056)

CL1MB8

LINEL1

(0)

6178

5821

52464

108

00

00

chr6POLH

43572521+43581878

2303

2609

(6749)

CAluSc5

SINEAlu

(2)

307

16

3529

161

7044

chr6POLH

43572521+43581878

2610

3080

(6278

CL1MB8

LINEL1

(342)

5820

5323

52287

9255

00

chr6POLH

43572521+43581878

3081

3372

(5986)

+AluSq2

SINEAlu

1308

(5)

71739

178

107

18chr6POLH

43572521+43581878

3373

3510

(5848)

CL1MB8

LINEL1

(829)

5333

5170

5810

187

1270

chr6POLH

43572521+43581878

3744

3909

(544

9)C

AluJo

SINEAlu

(19)

293

137

8535

296

9714

chr6POLH

43572521+43581878

3990

4509

(4849)

+L2

aLINEL2

(2804)

3365

(61)

92528

100

00

10chr6POLH

43572521+43581878

5271

5581

(3777)

CAluSx1

SINEAlu

(4)

308

110

13100

59

28

chr6POLH

43572521+43581878

5582

5615

(3743)

+(TCT

TTA)n

Simplerepeat

136

(0)

11684

56

00

00

chr6POLH

43572521+43581878

6162

6233

(3125)

+AluSq10

SINEAlu

172

(241)

122510

103

00

00

chr6POLH

43572521+43581878

6991

7300

(2058)

+AluSq2

SINEAlu

1310

(3)

132679

7000

00

chr6POLH

43572521+43581878

7564

7863

(1495)

CAluSq

SINEAlu

(13)

300

114

2503

84

07

00

chr6POLH

43572521+43581878

8018

8313

(1045)

CAluSg

SINEAlu

(11)

299

215

214

247

186

10chr6POLH

43572521+43581878

8598

8683

(675)

+MER

5ADNAhAT

-Charlie

2102

(87)

16196

161

00

00

chr6POLH

43572521+43581878

8684

8714

(644

)+

MER

5ADNAhAT

-Charlie

(159)

189

(0)

17



ofTu

nisia

nXP

-Vpatie

nts

Patie

nts

Affected

patie

nts

Sex

Age

(years)

Age

aton

seto

fthe

1stX

Pmacules

erythema(

years)

Geographic

Orig

in

Age

aton

seto

f1stTu

mor

inyears(Num

bero

ftum

ors)

Photop

hobia

Radiotherapy

Tumor

post

Radiotherapy

BCC

SCC

Other

tumors

XPV6K

E1

M47

(diedat

50)

4Ke

f22

(6)

20(12)

mdash+minus

+++

+++

XPV15GA

1F

184

Gafsa

15(2)

16(2)

+minus

XPV17-1B

3F

174

Bizerte

00

0mdash

XPV17-2

BF

114

00

0mdash

XPV17-3

BF

45

00

0mdash

XP18G

1F

434

Gafsa

16(8)

21(3)

KA+minus

+++

+++

XPV20G

3F

315

Gafsa

ND(gt10)

ND(gt2)

+++

+

XPV43-1

7M

ND

5Za

ghou

an38

(8)

23(4)

KAand

Actin

icKe

ratosis

+minus

+(lo

cal)

mdash

XPV43-2

F45

541

(1)

+minus

XPV48G

1F

466

Gafsa

047

(1)

+minus

XPV53Z

1M

507

Fahs

Zagh

ouan

37(4)

+(lo

cal)

+minus

XPV79-1

3(1died)

F13

3To

zeurG

afsa

10(3)

mdashKA

+++minus

XPV79-2

F18

3KA

mdash

XPV91-1

3F

296

Tozeur

Actin

icKe

ratosis

mdash

XPV91-2

F21

6mdash

XPV91-3

F24

6mdash

SCC

spinocellcarcinom

aBC

CbasalcellC

arcino

maKA

keratho

acantum(+minus)m

oderatep

heno

type(mdash)a

bsence(++

+)several













4 Discussion




43 2 2

Family XPV17B

129188

129188

153168

149194

129188

129188

129188

129188

12918

129188

129188

129188

139186

139184

3

149155

3

3 3 1 3 4 1 2

145190

139186

145190

Family XPV15GA

129188

129188

Family XPV6KE

8 129188

129188

129188

129188


D6S1582

D6S451

04Mb

036Mb

0051Mb


10000 bp6000 bp

1kbXP-V17-1

XP-V91-1

XP-V(P) Control





150 160 170 180 190 200 210

(a)

Alu-Sq2 intron 9

Alu-Sq2 intron 10

Alu-Sq2 intron 9

Alu-Sq2 intron 10

Alu-Sq2 intron 9

Alu-Sq2 intron 10

Alu-Sq2 intron 9

Alu-Sq2 intron 10









(b)


Alu-Sq2 Alu-Sq2

Alu-Sq2

Exon 9 Exon 11


3925bp

1

2

(c)




5 Conclusion





Acknowledgments




References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table1Sequ

ence

analysisof

repetitivee

lementsof

the9

358b

psequ

ence

ofPO


usingrepeatmaskerS

oftware

scorediv

del

ins

Query

sequ

ence

Positionin

query-

C +Matchingrepeat

Repeatcla

ssfa

mily

-Position

inrepeat(le

ft)endbegin

linkage

begin

end

(left)

+repeat

classfa

mily

begin

end

(left)

idgraph

ic1692

135

04

04

chr6POLH

43572521+43581878

657

893

(8465)

CAluJr

SINEAlu

(2)

310

741

2266

104

03

43

chr6POLH

43572521+43581878

925

1240

(8118)

CAluSq

SINEAlu

(9)

304

12

658

263

86

46

chr6POLH

43572521+43581878

1267

1581

(7777)

CL1MA8

LINEL1

(25)

6266

5940

32432

120

00

00

chr6POLH

43572521+43581878

1644

1944

(7414)

CAluSx1

SINEAlu

(10)

302

24

3529

149

69

42

chr6POLH

43572521+43581878

1958

2302

(7056)

CL1MB8

LINEL1

(0)

6178

5821

52464

108

00

00

chr6POLH

43572521+43581878

2303

2609

(6749)

CAluSc5

SINEAlu

(2)

307

16

3529

161

7044

chr6POLH

43572521+43581878

2610

3080

(6278

CL1MB8

LINEL1

(342)

5820

5323

52287

9255

00

chr6POLH

43572521+43581878

3081

3372

(5986)

+AluSq2

SINEAlu

1308

(5)

71739

178

107

18chr6POLH

43572521+43581878

3373

3510

(5848)

CL1MB8

LINEL1

(829)

5333

5170

5810

187

1270

chr6POLH

43572521+43581878

3744

3909

(544

9)C

AluJo

SINEAlu

(19)

293

137

8535

296

9714

chr6POLH

43572521+43581878

3990

4509

(4849)

+L2

aLINEL2

(2804)

3365

(61)

92528

100

00

10chr6POLH

43572521+43581878

5271

5581

(3777)

CAluSx1

SINEAlu

(4)

308

110

13100

59

28

chr6POLH

43572521+43581878

5582

5615

(3743)

+(TCT

TTA)n

Simplerepeat

136

(0)

11684

56

00

00

chr6POLH

43572521+43581878

6162

6233

(3125)

+AluSq10

SINEAlu

172

(241)

122510

103

00

00

chr6POLH

43572521+43581878

6991

7300

(2058)

+AluSq2

SINEAlu

1310

(3)

132679

7000

00

chr6POLH

43572521+43581878

7564

7863

(1495)

CAluSq

SINEAlu

(13)

300

114

2503

84

07

00

chr6POLH

43572521+43581878

8018

8313

(1045)

CAluSg

SINEAlu

(11)

299

215

214

247

186

10chr6POLH

43572521+43581878

8598

8683

(675)

+MER

5ADNAhAT

-Charlie

2102

(87)

16196

161

00

00

chr6POLH

43572521+43581878

8684

8714

(644

)+

MER

5ADNAhAT

-Charlie

(159)

189

(0)

17



ofTu

nisia

nXP

-Vpatie

nts

Patie

nts

Affected

patie

nts

Sex

Age

(years)

Age

aton

seto

fthe

1stX

Pmacules

erythema(

years)

Geographic

Orig

in

Age

aton

seto

f1stTu

mor

inyears(Num

bero

ftum

ors)

Photop

hobia

Radiotherapy

Tumor

post

Radiotherapy

BCC

SCC

Other

tumors

XPV6K

E1

M47

(diedat

50)

4Ke

f22

(6)

20(12)

mdash+minus

+++

+++

XPV15GA

1F

184

Gafsa

15(2)

16(2)

+minus

XPV17-1B

3F

174

Bizerte

00

0mdash

XPV17-2

BF

114

00

0mdash

XPV17-3

BF

45

00

0mdash

XP18G

1F

434

Gafsa

16(8)

21(3)

KA+minus

+++

+++

XPV20G

3F

315

Gafsa

ND(gt10)

ND(gt2)

+++

+

XPV43-1

7M

ND

5Za

ghou

an38

(8)

23(4)

KAand

Actin

icKe

ratosis

+minus

+(lo

cal)

mdash

XPV43-2

F45

541

(1)

+minus

XPV48G

1F

466

Gafsa

047

(1)

+minus

XPV53Z

1M

507

Fahs

Zagh

ouan

37(4)

+(lo

cal)

+minus

XPV79-1

3(1died)

F13

3To

zeurG

afsa

10(3)

mdashKA

+++minus

XPV79-2

F18

3KA

mdash

XPV91-1

3F

296

Tozeur

Actin

icKe

ratosis

mdash

XPV91-2

F21

6mdash

XPV91-3

F24

6mdash

SCC

spinocellcarcinom

aBC

CbasalcellC

arcino

maKA

keratho

acantum(+minus)m

oderatep

heno

type(mdash)a

bsence(++

+)several













4 Discussion




43 2 2

Family XPV17B

129188

129188

153168

149194

129188

129188

129188

129188

12918

129188

129188

129188

139186

139184

3

149155

3

3 3 1 3 4 1 2

145190

139186

145190

Family XPV15GA

129188

129188

Family XPV6KE

8 129188

129188

129188

129188


D6S1582

D6S451

04Mb

036Mb

0051Mb


10000 bp6000 bp

1kbXP-V17-1

XP-V91-1

XP-V(P) Control





150 160 170 180 190 200 210

(a)

Alu-Sq2 intron 9

Alu-Sq2 intron 10

Alu-Sq2 intron 9

Alu-Sq2 intron 10

Alu-Sq2 intron 9

Alu-Sq2 intron 10

Alu-Sq2 intron 9

Alu-Sq2 intron 10









(b)


Alu-Sq2 Alu-Sq2

Alu-Sq2

Exon 9 Exon 11


3925bp

1

2

(c)




5 Conclusion





Acknowledgments




References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















ofTu

nisia

nXP

-Vpatie

nts

Patie

nts

Affected

patie

nts

Sex

Age

(years)

Age

aton

seto

fthe

1stX

Pmacules

erythema(

years)

Geographic

Orig

in

Age

aton

seto

f1stTu

mor

inyears(Num

bero

ftum

ors)

Photop

hobia

Radiotherapy

Tumor

post

Radiotherapy

BCC

SCC

Other

tumors

XPV6K

E1

M47

(diedat

50)

4Ke

f22

(6)

20(12)

mdash+minus

+++

+++

XPV15GA

1F

184

Gafsa

15(2)

16(2)

+minus

XPV17-1B

3F

174

Bizerte

00

0mdash

XPV17-2

BF

114

00

0mdash

XPV17-3

BF

45

00

0mdash

XP18G

1F

434

Gafsa

16(8)

21(3)

KA+minus

+++

+++

XPV20G

3F

315

Gafsa

ND(gt10)

ND(gt2)

+++

+

XPV43-1

7M

ND

5Za

ghou

an38

(8)

23(4)

KAand

Actin

icKe

ratosis

+minus

+(lo

cal)

mdash

XPV43-2

F45

541

(1)

+minus

XPV48G

1F

466

Gafsa

047

(1)

+minus

XPV53Z

1M

507

Fahs

Zagh

ouan

37(4)

+(lo

cal)

+minus

XPV79-1

3(1died)

F13

3To

zeurG

afsa

10(3)

mdashKA

+++minus

XPV79-2

F18

3KA

mdash

XPV91-1

3F

296

Tozeur

Actin

icKe

ratosis

mdash

XPV91-2

F21

6mdash

XPV91-3

F24

6mdash

SCC

spinocellcarcinom

aBC

CbasalcellC

arcino

maKA

keratho

acantum(+minus)m

oderatep

heno

type(mdash)a

bsence(++

+)several













4 Discussion




43 2 2

Family XPV17B

129188

129188

153168

149194

129188

129188

129188

129188

12918

129188

129188

129188

139186

139184

3

149155

3

3 3 1 3 4 1 2

145190

139186

145190

Family XPV15GA

129188

129188

Family XPV6KE

8 129188

129188

129188

129188


D6S1582

D6S451

04Mb

036Mb

0051Mb


10000 bp6000 bp

1kbXP-V17-1

XP-V91-1

XP-V(P) Control





150 160 170 180 190 200 210

(a)

Alu-Sq2 intron 9

Alu-Sq2 intron 10

Alu-Sq2 intron 9

Alu-Sq2 intron 10

Alu-Sq2 intron 9

Alu-Sq2 intron 10

Alu-Sq2 intron 9

Alu-Sq2 intron 10









(b)


Alu-Sq2 Alu-Sq2

Alu-Sq2

Exon 9 Exon 11


3925bp

1

2

(c)




5 Conclusion





Acknowledgments




References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























4 Discussion




43 2 2

Family XPV17B

129188

129188

153168

149194

129188

129188

129188

129188

12918

129188

129188

129188

139186

139184

3

149155

3

3 3 1 3 4 1 2

145190

139186

145190

Family XPV15GA

129188

129188

Family XPV6KE

8 129188

129188

129188

129188


D6S1582

D6S451

04Mb

036Mb

0051Mb


10000 bp6000 bp

1kbXP-V17-1

XP-V91-1

XP-V(P) Control





150 160 170 180 190 200 210

(a)

Alu-Sq2 intron 9

Alu-Sq2 intron 10

Alu-Sq2 intron 9

Alu-Sq2 intron 10

Alu-Sq2 intron 9

Alu-Sq2 intron 10

Alu-Sq2 intron 9

Alu-Sq2 intron 10









(b)


Alu-Sq2 Alu-Sq2

Alu-Sq2

Exon 9 Exon 11


3925bp

1

2

(c)




5 Conclusion





Acknowledgments




References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















43 2 2

Family XPV17B

129188

129188

153168

149194

129188

129188

129188

129188

12918

129188

129188

129188

139186

139184

3

149155

3

3 3 1 3 4 1 2

145190

139186

145190

Family XPV15GA

129188

129188

Family XPV6KE

8 129188

129188

129188

129188


D6S1582

D6S451

04Mb

036Mb

0051Mb


10000 bp6000 bp

1kbXP-V17-1

XP-V91-1

XP-V(P) Control





150 160 170 180 190 200 210

(a)

Alu-Sq2 intron 9

Alu-Sq2 intron 10

Alu-Sq2 intron 9

Alu-Sq2 intron 10

Alu-Sq2 intron 9

Alu-Sq2 intron 10

Alu-Sq2 intron 9

Alu-Sq2 intron 10









(b)


Alu-Sq2 Alu-Sq2

Alu-Sq2

Exon 9 Exon 11


3925bp

1

2

(c)




5 Conclusion





Acknowledgments




References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















150 160 170 180 190 200 210

(a)

Alu-Sq2 intron 9

Alu-Sq2 intron 10

Alu-Sq2 intron 9

Alu-Sq2 intron 10

Alu-Sq2 intron 9

Alu-Sq2 intron 10

Alu-Sq2 intron 9

Alu-Sq2 intron 10









(b)


Alu-Sq2 Alu-Sq2

Alu-Sq2

Exon 9 Exon 11


3925bp

1

2

(c)




5 Conclusion





Acknowledgments




References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















A Founder Large Deletion Mutation in Xeroderma Pigmentosum-Variant Form in Tunisia: Implication for Molecular Diagnosis and Therapy

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