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Uncommon nucleotide excision repair phenotypes revealed by targeted high-throughput sequencing

December 2016
Orphanet Journal of Rare Diseases 11(1)

DOI:10.1186/s13023-016-0408-0

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CC BY 4.0

Authors:

Cathy Obringer

University of Strasbourg

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Background Deficient nucleotide excision repair (NER) activity causes a variety of autosomal recessive diseases including xeroderma pigmentosum (XP) a disorder which pre-disposes to skin cancer, and the severe multisystem condition known as Cockayne syndrome (CS). In view of the clinical overlap between NER-related disorders, as well as the existence of multiple phenotypes and the numerous genes involved, we developed a new diagnostic approach based on the enrichment of 16 NER-related genes by multiplex amplification coupled with next-generation sequencing (NGS). Methods Our test cohort consisted of 11 DNA samples, all with known mutations and/or non pathogenic SNPs in two of the tested genes. We then used the same technique to analyse samples from a prospective cohort of 40 patients. Multiplex amplification and sequencing were performed using AmpliSeq protocol on the Ion Torrent PGM (Life Technologies). Results We identified causative mutations in 17 out of the 40 patients (43 %). Four patients showed biallelic mutations in the ERCC6(CSB) gene, five in the ERCC8(CSA) gene: most of them had classical CS features but some had very mild and incomplete phenotypes. A small cohort of 4 unrelated classic XP patients from the Basque country (Northern Spain) revealed a common splicing mutation in POLH (XP-variant), demonstrating a new founder effect in this population. Interestingly, our results also found ERCC2(XPD), ERCC3(XPB) or ERCC5(XPG) mutations in two cases of UV-sensitive syndrome and in two cases with mixed XP/CS phenotypes. Conclusions Our study confirms that NGS is an efficient technique for the analysis of NER-related disorders on a molecular level. It is particularly useful for phenotypes with combined features or unusually mild symptoms. Targeted NGS used in conjunction with DNA repair functional tests and precise clinical evaluation permits rapid and cost-effective diagnosis in patients with NER-defects.

Clinical symptoms of NER-related disorders and more frequently involved genes

…

Genes included in the targeted next generation sequencing strategy

…

Clinical description, molecular and cellular results of the 40 NER-defective patients studied by NGS

…

Clinical description, molecular and cellular results of the 40 NER-defective patients studied by NGS (Continued)

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R E S E A R C H Open Access

Uncommon nucleotide excision repair

phenotypes revealed by targeted high-

throughput sequencing

Nadège Calmels

, Géraldine Greff

, Cathy Obringer

, Nadine Kempf

, Claire Gasnier

, Julien Tarabeux

Marguerite Miguet

, Geneviève Baujat

, Didier Bessis

, Patricia Bretones

, Anne Cavau

, Béatrice Digeon

Martine Doco-Fenzy

, Bérénice Doray

, François Feillet

, Jesus Gardeazabal

, Blanca Gener

, Sophie Julia

Isabel Llano-Rivas

, Artur Mazur

, Caroline Michot

, Florence Renaldo-Robin

, Massimiliano Rossi

17,18

Pascal Sabouraud

, Boris Keren

20,21

, Christel Depienne

20,21

, Jean Muller

1,2

, Jean-Louis Mandel

and

Vincent Laugel

2,22

Abstract

Background: Deficient nucleotide excision repair (NER) activity causes a variety of autosomal recessive diseases

including xeroderma pigmentosum (XP) a disorder which pre-disposes to skin cancer, and the severe multisystem

condition known as Cockayne syndrome (CS). In view of the clinical overlap between NER-related disorders, as well as

the existence of multiple phenotypes and the numerous genes involved, we developed a new diagnostic approach

based on the enrichment of 16 NER-related genes by multiplex amplification coupled with next-generation sequencing

(NGS).

Methods: Our test cohort consisted of 11 DNA samples, all with known mutations and/or non pathogenic SNPs in

two of the tested genes. We then used the same technique to analyse samples from a prospective cohort of 40

patients. Multiplex amplification and sequencing were performed using AmpliSeq protocol on the Ion Torrent PGM

(Life Technologies).

Results: We identified causative mutations in 17 out of the 40 patients (43 %). Four patients showed biallelic mutations

in the ERCC6(CSB) gene, five in the ERCC8(CSA) gene: most of them had classical CS features but some had very mild and

incomplete phenotypes. A small cohort of 4 unrelated classic XP patients from the Basque country (Northern Spain)

revealed a common splicing mutation in POLH (XP-variant), demonstrating a new founder effect in this population.

Interestingly, our results also found ERCC2(XPD), ERCC3(XPB) or ERCC5(XPG) mutations in two cases of UV-sensitive

syndrome and in two cases with mixed XP/CS phenotypes.

Conclusions: Our study confirms that NGS is an efficient technique for the analysis of NER-related disorders on a

molecular level. It is particularly useful for phenotypes with combined features or unusually mild symptoms. Targeted

NGS used in conjunction with DNA repair functional tests and precise clinical evaluation permits rapid and cost-effective

diagnosis in patients with NER-defects.

Keywords: NER, NGS, Cockayne syndrome, xeroderma pigmentosum, ERCC2,ERCC3,ERCC5,ERCC6,ERCC8,POLH

* Correspondence: nadege.calmels@chru-strasbourg.fr

Laboratoire de Diagnostic Génétique, Institut de Génétique Médicale

d’Alsace (IGMA), Hôpitaux Universitaires de Strasbourg, 1 place de l’hôpital,

Strasbourg, France

Full list of author information is available at the end of the article

International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and

reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to

the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver

(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Calmels et al. Orphanet Journal of Rare Diseases (2016) 11:26

DOI 10.1186/s13023-016-0408-0

Background

Nucleotide excision repair (NER) is a multistep DNA repair

process in which a broad spectrum of DNA lesions are re-

moved in two stages - one acting genome-wide (GG-NER)

and the other on actively transcribed strands (TC-NER) [1].

Despiteacommonautosomalrecessivepatternofinherit-

ance, genetic defects in the NER pathway lead to a broad

variety of clinical conditions, namely: xeroderma pigmento-

sum (XP), trichothiodystrophy (TTD), Cockayne syndrome

(CS) and UV-sensitive syndrome (UVSS) (Table 1). Xero-

derma pigmentosum (Orpha number 910) is a genoder-

matosis characterized by extreme sensitivity to ultraviolet

(UV)-induced changes in the skin and eyes, and multiple

skin cancers. XP patients present sunlight-induced pigmen-

tation changes, a predisposition to skin cancer and in about

30 % of cases neurological degeneration with progressive

cognitive impairment. Classical XP results from defects in

one of seven genes, XPA through to G. XP-variant, charac-

terized by the absence of DNA repair deficits, results from

a deficiency in the translesion synthesis DNA polymerase,

polη[2]. Trichothiodystrophy (Orpha number 33364) is a

very rare heterogeneous group of disorders characterized

by brittle hair with low-sulfur content associated with

neurodevelopmental impairment. The spectrum of symp-

toms observed in TTD patients ranges from mild forms of

the disease, characterized by normal development with brit-

tle hair and scaling skin only, to very severe cases, charac-

terized by high mortality at a young age combined with

severe neurodevelopmental defects. Approximately 50 % of

patients with TTD exhibit marked photosensitivity. These

photosensitive patients are found to be carriers of biallelic

mutations, usually in the ERCC2(XPD) gene, or more rarely

in ERCC3(XPB) or TTDA [3]. All encode for subunits of

the dual function repair/transcription factor II H (TFIIH)

and result in a NER defect, which explains the photosensi-

tivity observed. Amongst the remaining 50 % of non-

photosensitive TTD cases, a minority (about 10 %) carry

biallelic mutations in TTDN1, a ubiquitously expressed

gene thought to play a role in the maintenance of the cell

cycle [4–6]. Cockayne syndrome (Orpha number 191) is a

multisystem disorder characterized by intellectual disability,

microcephaly, severe growth failure, sensory impairment,

peripheral neuropathy and cutaneous photosensitivity [7].

Various degrees of severity have been described, including:

a prenatal-onset form known as cerebro-oculo-facio-skel-

etal (COFS) syndrome (Orpha number 1466), an early-

onset or severe form (CS type II), a “classical”or moderate

form (CS type I) and a mild or late-onset form (CS type

III). The two major genes responsible for the disorder,

ERCC6(CSB) and ERCC8(CSA) were identified in the mid

1990s [8, 9]. More recently, defects in either XPF endo-

nuclease or in its partner ERCC1 have also been associated

with CS [10]. COFS patients show mutations mainly in

ERCC6(CSB) [11, 12], but also in ERCC2(XPD) [13],

ERCC5(XPG) [14–16] and ERCC1 [17]. A few rare cases

show combined features of CS and XP.Someofthese

patients present in the neonatal period with severe features

such as in COFS, which leads to early mortality. Others

Table 1 Clinical symptoms of NER-related disorders and more frequently involved genes

Cockayne syndrome COFS syndrome Trichothiodystrophy Xeroderma pigmentosum UV-sensitive syndrome

Growth failure ++ ++ + - -

Microcephaly ++ ++ +/−+/−-

Intellectual disability ++ ++ +/−+/−-

Retinal degeneration + +/−-- -

Cataracts + + + - -

Deafness + +/−+/−--

Photosensitivity + +/−+/−++ ++

Brittle hair - - ++ - -

Cancer - - - ++ -

Known involved genes ERCC6(CSB) ERCC6(CSB) ERCC2(XPD) XPC ERCC6(CSB)

ERCC8(CSA) ERCC2(XPD) ERCC3(XPB) ERCC2(XPD) ERCC8(CSA)

ERCC5(XPG) TTD-A XPA UVSSA(KIAA1530)

ERCC1 TTDN1 POLH

ERCC5(XPG)

DDB2(XPE)

ERCC3(XPB)

ERCC1

ERCC4(XPF)

COFS: cerebro-oculo-facio-skeletal syndrome

Calmels et al. Orphanet Journal of Rare Diseases (2016) 11:26 Page 2 of 16

have later onset and milder neurological/developmental

abnormalities with XP-like skin lesions which develop in

later life [18–20]. These combined presentations are mainly

linked to mutations in ERCC2(XPD), ERCC3(XPB) or

ERCC5(XPG) [16, 18, 19], but defects in ERCC4 have also

recently been observed [10]. Finally, UV-sensitive syn-

drome (Orpha number 178338) is characterized by isolated

cutaneous photosensitivity without any of the other features

associated with CS, and without the pre-disposition to skin

malignancy as in xeroderma pigmentosum. It has been

linked to mutations in ERCC6(CSB),ERCC8(CSA) and in

therecentlyidentifiedgeneencoding for UV-stimulated

scaffold protein A (UVSSA)[21–23]. Distinct functions of

the CS proteins aside from their role in TC-NER, may

account for the various clinical symptoms of CS and for the

differences between CS and UVSS [24]. Intriguingly, neither

thesitenorthenatureofthemutationinCSA or CSB

seems to correlate with the clinical differences observed

amongst patients with CS and UVSS [25].

Clinical recognition of NER defects remains challenging,

due to the remarkable heterogeneity and overlap of clinical

symptoms which exists between these conditions (Table 1).

Moreover, the existence of combined forms, such as XP/

CS, further complicates the diagnosis. Historically, the diag-

nosis was confirmed by DNA repair activity testing on pri-

mary fibroblasts, using ‘unscheduled DNA synthesis (UDS)’

to test GG-NER, and ‘recovery of RNA synthesis (RRS)’to

test TC-NER [26]. More recently, gene identification has

permitted molecular diagnosis of NER related disorders,

using Sanger sequencing of candidate genes. The clinical

presentations, as well as the results of UDS and RRS cellu-

lar tests, are used to guide the investigations towards the

gene involved. However, this gene-by-gene sequential ap-

proach is expensive and time consuming. Our goal was to

develop a single and efficient mutation-screening strategy

for all NER related disorders, and to improve our under-

standing of the clinical spectrum observed. In this study,

we describe our findings, based on the enrichment of 16

genes of the NER pathway by multiplex amplification

coupled with next-generation sequencing (NGS). Our co-

hort included 40 patients referred for suspicion of NER

defects.

Methods

Patients and samples

Initially, the NGS procedure was tested and validated on a

small cohort of 11 patients who had already been screened

for the entire coding sequence of ERCC6(CSB) and/or

ERCC8(CSA) genes. Next, a prospective cohort of 40 con-

secutive patients referred to our lab with suspicion of NER

defects was studied. For all patients, written consent for

genetic testing was obtained, either from adult probands or

from a legal representative in the case of minors.

DNA sample and quality control

Genomic DNA was extracted either from peripheral blood

or fibroblast cultures following standard procedures (QIA-

GEN). DNA concentration and quality were assessed at

each quality-control step using either Nanodrop® 8000,

Qubit® 2.0 fluorometer (Life technologies), or LabChip® GX

(Caliper).

Multiplex amplification

Targeted genomic regions covered exons from the major

isoform of the 16 genes involved in the NER pathway

(Table 2). Exon coordinates were extended to an additional

50 bp in flanking intronic sequences. Entire 5’and 3’un-

translated regions were included for ERCC6(CSB) and

ERCC8(CSA) genes only. Primer design for multiplex amp-

lification was performed with Ion AmpliSeq Designer

version 2.2 (reference IAD43922_95, Life Technologies).

The overall targeted regions span 62 kb, amplified in 452

amplicons (length between 125 and 175 bp), and are di-

vided into two pools.

NGS sequencing

NGS library preparation for sequencing of the targeted

genes was performed by multiplex amplification using the

Ion AmpliSeq Library Kit 2.0 (Life Technologies). Individ-

ual samples were then barcoded using Ion Xpress™Barcode

Adapters 1–16 Kits. The amplified librairies were purified

using Agencourt AMPure XP Beads (Beckman Coulter).

Before library pooling of up to 8 patients, amplified libraries

were validated using the 2100 BioAnalyzer System (Agi-

lent). Emulsion PCR of the pooled library was performed

using the OneTouch™2 system (Life Technologies). Enrich-

ment of the template-positive ion sphere particles (ISPs,

containing clonally amplified DNA) was performed using

the Ion OneTouch ES (Life Technologies), according to the

manufacturer’s instructions. The percentage of templated

ISPs compared to total ISPs was estimated using an Ion

Sphere™Quality Control kit on a Qubit® 2.0 Fluorometer

(Life Technologies). The enriched ISPs were loaded on

either Ion 314 (validation phase only) or Ion 316 chips and

sequenced with an Ion Personal Genome Machine (PGM,

Life Technologies).

NGS bioinformatic analysis: read mapping, variant calling

and filtering

Sequencing data was collected and analyzed by the

Torrent Suite v4.4 (Life Technologies), including base

calling, barcode sorting, alignement to the reference

genome (GRCh37), variant calling and coverage ana-

lysis. Low stringency default parameters for germline

mutations were used on the Variant Caller plugin of the

Ion Torrent Browser with the exception of indel and

multiple nucleotide polymorphism maximum strand

bias that was set to 1.

Calmels et al. Orphanet Journal of Rare Diseases (2016) 11:26 Page 3 of 16

DNA sequences were visualized using Alamut Visual

2.7.1 (Interactive Biosoftware). Initial variant filtering

included the removal of frequent variants (minor allele

frequency ≥0.01) present either in dbSNP142 [27], in the

Exome Variant Server [28] or in the Exome Aggregation

Consortium (ExAC) [29]. Recurrent variations found in

more than 10 % of the patients in our cohort were also

filtered. Subsequent variant annotation and ranking was

performed using VaRank v1.4.0 [30] configured with Ala-

mut Batch (Interactive biosoftware).

Gap filling and variant confirmation using Sanger

sequencing

Gaps in NGS coverage of the targeted regions (i. e. cover-

age <30X, the threshold recommended in the literature

[31, 32]), were filled with Sanger sequencing for coding

sequences of the ERCC6(CSB) and ERCC8(CSA) genes

only. Variants identified by the NGS approach were

confirmed by Sanger DNA sequencing and familial segre-

gation analyses were performed as extensively as possible.

Sequences were obtained either on 3130xl or 3500 Genetic

Analyzer (Applied Biosystems), aligned with the Sequen-

cing Pilot software (JSI) and compared with the corre-

sponding genomic DNA reference sequence (GRCh37).

Splicing variants were validated by Sanger sequencing on

cDNA obtained by reverse transcription of RNA ex-

tracted from patient’s fibroblasts when available. All

primer sequences are available upon request.

Illumina OminiExpress-24 chips

Samples were processed on Illumina OmniExpress-24 v1

arrays using the Infinium assay as previously described [33],

and results were analyzed using Illumina GenomeStudio

software. Reference standards were set with the Illumina

Genetrain 2.0 algorithm using the 96 samples processed

during the same run. Regions of homozygosity were called

with the Illumina cnvPartition 3.1.6 algorithm, with a min-

imal region size of 1 Mb.

Cell lines and culture conditions

All cells used in our study were human primary fibroblasts

derived from patients. Cells were cultured in Dulbecco’s

Modified Eagle Medium (DMEM) (Ref. 21885–025,

GIBCO) supplemented with 10 % FBS (Ref. 10500–064,

GIBCO) and 1 % Antiseptic-Antimycotic (Ref. 15240–062,

GIBCO) at 37 °C in a humidified atmosphere containing

5%CO

until RRS and UDS was initiated.

RRS assay

Cells were plated on coverslips in 6-well plates at a con-

fluency of 7x10

cells per well. After 2 days of growing, cells

were washed with PBS, followed by irradiation with a range

of UV-C doses (6-12-20 J/m

). The non-irradiated plate(s)

acted as references. After UV-irradiation, cells were incu-

bated for 23 h for RNA Synthesis recovery in DMEM

supplemented with FBS. Then, after washing with PBS, cells

were labelled with 5-ethynyl-uridine (EU; Invitrogen) for

2 h. Cells were then washed again with PBS, followed by

Table 2 Genes included in the targeted next generation sequencing strategy

Official gene symbol Legacy name Ref Seq NM # # Exons Size of coding exons (bp) Targeted region size (bp) Theoretical coverage

DDB1 NM_001923.4 27 3423 6,150 94.67 %

DDB2 XPE NM_000107.2 10 1284 2,268 99.03 %

ERCC1 NM_001983.3 10 894 2,102 99.57 %

ERCC2 XPD NM_000400.3 23 2283 4,894 98.12 %

ERCC3 XPB NM_000122.1 15 2349 3,864 99.48 %

ERCC4 XPF NM_005236.2 11 2751 3,862 100.00 %

ERCC5 XPG NM_000123.3 15 3561 5,076 100.00 %

ERCC6 CSB NM_000124.3 21 4482 9,127 99.99 %

ERCC8 CSA NM_000082.3 12 1191 3,256 98.65 %

GTF2H5 TTD-A NM_207118.2 3 216 418 100.00 %

MPLKIP TTDN1 NM_138701.3 2 540 742 100.00 %

POLH NM_006502.2 11 2142 3,152 100.00 %

USP7 NM_003470.2 31 3309 6,440 93.88 %

UVSSA KIAA1530 NM_020894.2 14 2130 3,443 99.07 %

XPA NM_000380.3 6 822 1,428 100.00 %

XPC NM_004628.4 16 2823 4,767 100.00 %

Total 227 34200 58,721

Targeted genomic regions covered coding exons with their intron boundaries (50 pb) from the major isoform of 16 genes involved in NER pathway. 5’and 3’

untranslated regions (UTR) were included for ERCC6(CSB) and ERCC8(CSA) genes only. The total panel size was 62 kb considering the 3 genes located on sex

chromosomes (SRY, AMELX and AMELY) added as gender internal quality control

Calmels et al. Orphanet Journal of Rare Diseases (2016) 11:26 Page 4 of 16

fixationandpermeabilization.Thelaststepinvolvedan

azide-coupling reaction and DAPI staining (Click-iT RNA

HCS Assay, Invitrogen). Finally coverslips were washed in

PBS, and mounted on glass slides with Ibidi Mounting

Medium (Biovalley).

Photographs of the cells were taken with a fluorescent

microscope (Imager.Z2) equipped with a CCD camera

(AxioCam, Zeiss). The images were processed and analyzed

with the ImageJ software. At least 50 cells were randomly

selected, and the average nuclear fluorescent intensity was

calculated.

UDS assay

Cells were plated on coverslips in 6-well plates at a con-

fluency of 7x10

cells per well. After 2 days of growing,

cells were washed with PBS, followed by irradiation with a

range of UV-C doses (5-10-15 J/m

), the non-irradiated

plate(s) being the reference. After UV-irradiation, cells

were incubated for 3 h with 5-ethynyl-2’-deoxyuridine

(EdU; Invitrogen) contained in F 10 medium without thy-

midine supplemented with dialyzed FBS and 5-fluoro-2-

deoxyuridine (Fudr; Sigma). After washing with PBS,

cells were then incubated for 15 min in full normal

medium (F 10 + antibiotics + 15 % FBS) complemented

with cold thymidine (thymidine 5’triphosphate; Sigma).

Cells were then washed again with PBS, followed by

fixation and permeabilization, azide-coupling reaction

and DAPI staining. At the end, coverslips were washed

in PBS, and mounted on glass slides with Ibidi Mount-

ing Medium (Ref. 50001, Biovalley).

Photographs of the cells were taken and analyzed as for

the RRS assay. At least 50 non-S-phase cells were randomly

selected, and the average nuclear fluorescent intensity was

calculated.

Results

Targeted regions: design strategy

Our goal was to develop an efficient mutation-screening

process for the diagnosis of patients with phenotypes

suggestive of NER defects. We chose a multiplex amplifica-

tion approach coupled with NGS to focus on genomic

sequencing of all NER genes known to be involved in

human diseases at the time the study was designed, as well

as on their direct interactors (Table 2). Three genes located

on sex chromosomes (SRY, AMELX and AMELY)werein-

cluded for quality control purposes.

Analysis and evaluation

The procedure was first performed on a cohort of 11

samples, previously tested by Sanger sequencing of

ERCC6(CSB) and/or ERCC8(CSA) genes. Pathogenic

mutations were detected in 5 samples, as well as non-

pathogenic variants which were found in all.

Initially, we tested several combinations using between

one and four patient DNA samples, pooled together on

314 or 316 arrays. Based on these preliminary results,

we then pooled 8 patient DNA samples on a 316 array

for diagnostic testing.

We assessed the sensitivity and specificity of single

nucleotide variation (SNV) detection by comparing NGS

results with allelic states of SNVs detected by Sanger

sequencing of ERCC6(CSB) and ERCC8(CSA) in the 11

samples. Several variation types (missense, nonsense,

splice mutations and small deletion of one base) at dif-

ferent allelic states were tested (Additional file 1). All 63

previously identified single base variations were detected in

their correct heterozygous/homozygous state. A further

94,321 normal ERCC6(CSB) and/or ERCC8(CSA) bases

were also correctly sequenced with no false positives. In

our sample (N= 63 variations, with 100 % correct detection

rate), the probability of a false-negative event can be esti-

mated at 3/N (=5 % with N= 64). This corresponds to an

overall sensitivity of ≥95 %, calculated using the ‘rule of 3’

estimate of power, with respect to sample size [34]. The

overall specificity, calculated by the same method using

data from the 94,321 normal bases, can be estimated at

≥99.99 %.

Finally, analysis of variations in copy number was

assessed using an amplicon-based enrichment NGS tech-

nique, a quantitative method used to compare the number

of reads for each amplicon in each sample of the run [35].

A heterozygous 4.6 Mb deletion of chromosome 10q11,

encompassing the whole ERCC6(CSB) gene, was clearly

detected by this process, as well as by direct observation

of the coverage data using Alamut Visual. In contrast, we

were unable to detect an ERCC6(CSB) deletion limited to

the first two exons of the gene, with this technique (data

not shown).

Results from the 40 patient cohort

We analyzed data obtained from a cohort of 40 consecu-

tive patients referred for suspected Cockayne syndrome

(n= 30), xeroderma pigmentosum (n= 5), UV sensitive

syndrome (n= 2) or COFS syndrome (n= 3). Our tech-

nique allowed us to generate a high-quality sequencing

dataset, with a mean depth of coverage of 944X. On

average, 95 ± 3 % of targeted regions were covered more

than 30X for each patient. Depth of coverage was highly

variable (944 ± 493 X) but always over 172X. Few regions,

(n= 14 out of 452 amplicons, or 3 % of the amplicons)

appeared to be consistently or frequently (>50 % of the

patients) poorly covered (mean coverage < 30X) (Additional

file 2). Of these, the first group are mainly highly GC-rich

regions which are difficult to amplify, and also difficult to

study by capture enrichment processes as demonstrated by

the limited coverage of these regions on the ExAC database.

Other poorly covered regions are low GC-content regions

Calmels et al. Orphanet Journal of Rare Diseases (2016) 11:26 Page 5 of 16

with numerous small polyA or polyT tracts. Such homo-

polymer stretches are well known to be prone to high

error rates with Ion Torrent PGM sequencing technology

[36, 37]. An average of 67 ± 12 unfiltered variations per

patient was observed.

We detected clear pathogenic biallelic mutations in 17

of the 40 cases (43 %). No potentially pathogenic variant

could be identified in any of the 16 targeted genes in the

remaining 23 patients (57 %).

Diagnosis of patients with classical phenotypes

Amongst the confirmed mutation group (17 patients), two

thirds (n= 11) displayed a molecular diagnosis correspond-

ing to the classical clinical presentation. Concerning CS,

four patients (# 1 to 4) showed biallelic mutations in the

ERCC6(CSB) gene and three patients (# 5 to 7) in the

ERCC8(CSA) gene. They all display a classical Cockayne

phenotype with growth failure, microcephaly, intellectual

disability, pyramidal and extra-pyramidal signs and neuro-

sensorial impairment (Table 3). Several novel mutations

were found in these two genes including 2 frameshift, 3

splicing and 1 missense mutation (Table 4). None of these

variants were present in the EVS or ExAC databases. The

novel ERCC8(CSA) missense variant, c.356C > T p.Ser119-

Leu, involved an amino-acid located in a WD40 repeat

domain. It is predicted to be deleterious by SIFT, probably

damaging by PolyPhen2 and disease causing by Mutation-

Taster. Whilst the parents were not available for segregation

analysis (patient #6), this missense mutation is located in a

region in which many other clearly pathogenic missenses

have been reported. The second mutation in our patient is

a splice consensus mutation with a high pathogenicity

potential.

Concerning XP, analysis of a group of six patients

from four families coming from Northern Spain (Can-

tabria, n=1,Basque Country, n= 5) allowed us to iden-

tify a founder mutation in the POLH gene (Additional

file 2). All patients except for one have developed one

or several skin cancers (melanomas, basocellular car-

cinomas and epidermoid carcinomas) with an onset of

between 15 and 41 years of age. Four of the patients experi-

ence photophobia. Increased number of lentigines (n=2/6)

and exaggerated and prolonged sunburn response (n=2/6)

are also described. One patient suffers from hearing prob-

lems. No other symptoms have been reported. All patients

exhibited a normal NER level after UV irradiation, even on

UDS or RRS assay. This is a hallmark of XP variant (XP-V),

a disease linked to the POLH gene. NGS was performed on

a single patient from each family (patients # 10 to 13). In 3

of these 4 patients, we identified a homozygous POLH

variation at the canonical donor site of intron 6, c.764 +

1G > A. Parental segregation confirmed the heterozygous

carrier status of the parents in one family. The last patient

(patient # 10), was compound heterozygous for the same

POLH splicing mutation and for a novel POLH stop

mutation: c.1445C > A p.Ser482*. cDNA studies on the

homozygous patients revealed two truncated transcripts

(Additional file 3): the first splice form, with the greatest

expression, uses a cryptic splice donor site, 42 bp upstream

from the end of exon 6. The resulting in-frame deletion

(r.723_764del42; p.Ser242_Ile255del14) leads to shortening

of the polymerase eta protein by 14 amino acids. The

second splice variant has a deletion of the entire exon 6

(104 bp) (r.661_764del104) expected to produce a 221-

amino-acid truncated protein (p.Val221Profs*2). Whereas

the c.764 + 1G > A mutation is largely unknown (described

once in the ExAC database, in one out of 11,564 alleles

from a Latino population), the c.764 + 1G > C mutation has

been described in patient XP31BE [38] with exactly the

same abnormal transcripts. As expected in XP-V cell lines,

fibroblasts collected from our Spanish patient exhibited a

specific reduction in post-UV survival in the presence of

caffeine (data not shown). Haplotype analysis using the

Illumina HumanOmniExpress-24 SNP chip showed a com-

mon homozygous haplotype of 975 kb encompassing the

POLH gene in the patients of families # 11, 12 and 13

(Additional file 4). The patient with compound heterozy-

gosity presented a genotype over the 257 SNPs in the inter-

val completely compatible with the founder haplotype.

Uncommon diagnoses established by targeted NGS

The remaining third of cases from our confirmed mutation

group (n= 6 out of 17 patients) displayed atypical findings

such as mild and incomplete symptoms, combined pheno-

types or involvement of extremely rarely implicated genes.

Of these cases, we identified two close ERCC8(CSA)

missense mutations in two CS patients showing incomplete

or mild symptoms. The younger patient (#8) is a 16 year

old boy presenting with an incomplete clinical picture with

normal growth (25th centile), slight intellectual deficiency,

behavioral difficulties, congenital unilateral deafness, pro-

gressive enophthalmia and cutaneous photosensitivity

(Additional file 5). The older patient (#9), a 26-year-old

man, was referred for small stature, microcephaly, congeni-

tal deafness, learning difficulties and photosensitivity associ-

ated with truncal cutaneous spots and cerebellar atrophy

(Additional file 5). Both patients, born to consanguineous

parents, are homozygous carriers of a missense variant,

respectively c.730C > T p.His244Tyr for patient #8 and

c.793A > C p.Thr265Pro for patient #9. These variations,

which are absent from the EVS and ExAC databases,

involve conserved amino-acids localized in the same func-

tional WD40 domain and are predicted as deleterious by

Sift and PolyPhen2. Segregation studies have confirmed the

carrier status of the parents in both families. The two

healthy siblings of patient #8 and four healthy siblings of

patient #9 were either heterozygous carriers of the familial

missense variant or homozygous for the wild type allele.

Calmels et al. Orphanet Journal of Rare Diseases (2016) 11:26 Page 6 of 16

Table 3 Clinical description, molecular and cellular results of the 40 NER-defective patients studied by NGS

Patient

Gender Clinical

indication

Intellectual

disability/

psychomotor

delay

Growth

failure

Micro-

cephaly

Photo-

sensitivity

Neuro-

sensorial

impairement

Cancer RRS UDS NGS

diagnosis

Mutated

gene

Mutations

(c.)

Mutations

(p.)

Parentation

segregation

1FCS + +++ + -↓NA + ERCC6(CSB) c.[(?_-164)_(−15+44

+1_136-1)];[2047C>T]

p.[?];[Arg683*] + (only

mother)

2MCS + +++ + -↓NA + ERCC6(CSB) c.[2599-

26A>G];[4115delG]

p.[Met867Thrfs*14];

[Gly1372Glufs*22]

3MCS + ++NA+ -NANA+ ERCC6(CSB) c.[2060C>T];[3862C>T] p.[Ser687Leu];[Arg1288*] +

4 M CS + + NA + - - ↓N+ ERCC6(CSB) c.[543G>T];[543

+4delA]

p.[Lys181Asn];[?] +

5 F CS + + + + + - NA NA + ERCC8(CSA) c.[927delT];[1041

+1G>T]

p.[Phe309Leufs*19];[?] +

6 F CS + + + ++ + - ↓N+ ERCC8(CSA) c.[356C>T(;)618-1G>A] p.[Ser119Leu(;)?] NA

7MCS + +++ - -↓NA + ERCC8(CSA) c.[611C>A];[1122

+1delG]

p.[Thr604Lys];[?] + (only

mother)

8 M CS + - - + + - I N + ERCC8(CSA) c.[730C>T];[730C>T] p.[His244Tyr];[His244Tyr] +

9MCS + +++ + -INA+ ERCC8(CSA) c.[793A>C];[793A>C] p.[Thr265Pro];

[Thr265Pro]

10 F XP - NA - + - + N N + POLH c.[764

+1G>A(;)1445C>A]

p.[?(;)Ser482*] NA

11 F XP - NA NA + - + N N + POLH c.[764+1G>A(;)764

+1G>A]

p.[?(;)?] NA

12 F XP - NA - + - + N N + POLH c.[764+1G>A(;)764

+1G>A]

p.[?(;)?] NA

13 M XP - NA NA + NA + N N + POLH c.[764+1G>A];[764

+1G>A]

p.[?];[?] +

14 M CS + - + ++ + - NA ↓+ERCC2(XPD) c.[1847G>C];[2047C>T] p.[Arg616Pro];[Arg683Trp] + (only

mother)

15 F UVSS - - - ++ - - NA ↓+ERCC2(XPD) c.[2047C>T];[2047C>T] p.[Arg683Trp];

[Arg683Trp]

16 F CS + + + + - - ↓↓ +ERCC5(XPG) c.[2200-

10C>G];[2200-

10C>G]

p.[?];[?] +

17 M UVSS - - - ++ + - ↓↓ +ERCC3(XPB) c.[296T>C(;)325C>T] p.[Phe99Ser(;)

Arg109*]

18 M CS + + + - + - NA NA -

19 F CS + + + NA + - N ↓-

20 F XP + + NA ++ + - NA NA -

Calmels et al. Orphanet Journal of Rare Diseases (2016) 11:26 Page 7 of 16

Table 3 Clinical description, molecular and cellular results of the 40 NER-defective patients studied by NGS (Continued)

21 M CS + + + NA - - N N -

22 F CS + + + - + - I N -

23 M CS + + + NA + - I NA -

24 F CS + + - - - - N N -

25 M CS + + + - - - N NA -

26 F CS + + + - - - N N -

27 M CS - + + - + - NA NA -

28 M COFS NA + + NA + - N N -

29 M CS + + + + + - NA NA -

30 F CS + + + + + - N ↓-

31 M CS + + + - + - NA NA -

32 F CS + + + + - - ↓N-

33 M CS + + + - - - N N -

34 F CS + + + - - - NA NA -

35 F CS + + + - + - NA NA -

36 F CS + + + - + - NA NA -

37 M CS + + + - + - NA NA -

38 F CS + + + NA + - NA NA -

39 F COFS NA + + NA NA - NA NA -

40 M COFS NA + + NA NA - N I -

Undescribed variations are in bold. Variations are described according to the latest nomenclature conventions described in HGVS. I: inconclusive results, N: normal; NA: not available; RRS: recovery of RNA synthesis;

UDS: unscheduled DNA synthesis

Calmels et al. Orphanet Journal of Rare Diseases (2016) 11:26 Page 8 of 16

Table 4 Novel variations identified in ERCC6(CSB), ERCC8(CSA), ERCC2(XPD), ERCC3(XPB) ERCC5(XPG) and POLH genes

Gene DNA

variant

cDNA variant Protein Type (DNA) Localization Variant

predicted

effect

Functionnal

domain

Frequency

in EVSdb

Frequency

in ExACdb

Prediction among SIFT

and PolyPhen 2 and

Mutation Taster

Patient

ERCC6(CSB) c.543G>T r.423_543del (exon 3

deletion)

p.Lys181Asn Substitution exon 3 Missense

+ splicing

- NF NF D, PD, DC 4

c.4115delG - p.Gly1372Glufs*22 Deletion exon 21 Frameshift - NF NF - 2

ERCC8(CSA) c.356C>T - p.Ser119Leu Substitution exon 4 Missense WD40 NF NF D, PD, DC 6

c.730C>T - p.His244Tyr Substitution exon 9 Missense WD40 NF NF D, PD, DC 8

c.793A>C - p.Thr265Pro Substitution exon 9 Missense WD40 NF NF D, PD, DC 9

c.927delT - p.Phe309Leufs*19 Deletion exon 10 Frameshift - NF NF - 5

c.1041

+1G>T

Not tested p.? Substitution intron 10 Splicing - NF NF - 5

c.1122

+1delG

Not tested p.? Deletion intron 11 Splicing - NF NF - 7

ERCC3(XPB) c.325C>T - p.Arg109* Substitution exon 3 Nonsense - 0.000462 0.000478 - 17

ERCC3(XPB) c.2200-

10C>G

r.2199_2200ins2200-

9_2200-1 (partial

insertion of intron 9)

p.Glu734_Thr1186delinsIleLeu* Substitution intron 9 Splicing - NF NF - 16

POLH c.764

+1G>A

r.723_764del42 (partial

del exon 6)

p.Ser242_Ile255del14 Substitution intron 6 Splicing - NF 8.241e-06 - 10, 11,

12, 13

p.Val221Profs*2

r.661_764del104 (del

exon 6)

POLH c.1445C>A - p.Ser482* Substitution exon 11 Nonsense - NF NF - 10

Ddeleterious, DC disease causing, EVSdb Exome Variant Server database, ExACdb Exome Aggregation Consortium database, NF no frequency data available, PD probably damaging

Calmels et al. Orphanet Journal of Rare Diseases (2016) 11:26 Page 9 of 16

Studies of ERCC8(CSA) mRNA in patient #9 found a

normal-sized transcript with the homozygous p.Thr265Pro

missense mutation. RRS functional assay was inconclusive

in these two cases: repeated experiments showed variable

results which prevented us from drawing any formal con-

clusions about the efficiency of TC-NER in these patients

under experimental conditions (data not shown).

One patient referred for CS and one for UVSS were

linked to the ERCC2(XPD) helicase gene, with a final diag-

nosis of XP/CS and XP respectively. Patient #14 was the

first child of young unrelated healthy parents. His birth

weight was 2760 g (10

centile), length was 47.5 cm (5

centile) and his head circumference was 33 cm (5th centile).

He was referred for early and severe photosensitivity, learn-

ing difficulties, peripheral neuropathy and deafness. At the

age of 14, he presented microcephaly (head circumference:

50.5 cm (below the 3

centile)) without growth failure

(height: 162 cm (mean) and weight: 44.4 kg (25

centile)).

No evidence of calcification or leucodystrophy was revealed

on brain imaging. No cutaneous malignancies were de-

scribed at the age of 16. The patient has a younger sister

(15 year old) who presents a less severe phenotype without

microcephaly or neuropathy, but with learning disabilities

and mild photosensitivity. Surprisingly, we identified two

compound heterozygous missense variations (already de-

scribed) in ERCC2(XPD): c.1847G > C and c.2047C > T.

These lead respectively to the protein changes p.Arg616Pro

and p.Arg683Trp. The p.Arg683Trp mutation was inher-

ited from the mother, the father was not tested. The af-

fected sister shares the same genotype as patient #14. Both

variations, involving highly conserved amino-acids and

located in a helicase domain, have been previously reported

in either XP or in TTD patients [39, 40]. Our patient

shared the same genotype as the XP17PV patient present-

ing with XP and mild neurological abnormalities [40]. His

first tumor appeared at 22 years of age. The second

ERCC2(XPD) patientofthecohort(#15)isthefirstchildof

consanguineous healthy parents, referred by the dermatolo-

gist for UVSS. She presents severe photosensitivity with

normal growth and psychomotor development. No cutane-

ous cancer was described at the age of 4. She was found to

be homozygous for the common ERCC2(XPD) missense

mutation, c. 2047C > T p. Arg683Trp, as was her affected

younger sister. The parents are heterozygous carriers of the

mutation. Both patients display a severely decreased UDS.

A further CS patient was diagnosed with combined

XP/CS, linked to the rarely involved ERCC5(XPG) gene.

Patient #16 was the first child of young healthy parents.

No consanguinity was known but the parents came from

very close villages. The patient was born at term. Her

birth weight was 2700 g (25

centile), length was 51 cm

(mean) and her head circumference was 31 cm (below

centile). Apgar score was 10. The patient was evalu-

ated at the age of 4 years and 4 months. At that time,

she was 81 cm tall, weighed 7 kg, and had a head cir-

cumference of 35 cm (all well below the 1

centile). She

displayed delayed developmental milestones (she sat at the

age of one year, walked when she was 2 years old and spoke

single words at the age of 3.5). She exhibited an abnormal

ataxic gait. She also had deep set eyes and signs of retinal

pigmentary degeneration. Auditory assessment was normal.

She had cutaneous photosensitivity associated with dry skin

and numerous pigmented naevi. Her brain MRI showed

de-myelination of the white matter (Additional file 6). Liver

enzymes were slightly elevated (SGOT = 43 U/l; SGPT =

99U/l). Both RRS and UDS were severely decreased. We

identified a novel homozygous ERCC5(XPG) variation at

the acceptor site of intron 9, c.2200-10C > G, the first spli-

cing mutation described in this gene. Both parents were

found to be heterozygous carriers of the variant. cDNA

studies revealed a significant biological impact with no

normal transcript but with several aberrant species leading

to early protein termination. The major transcript included

the last 9 bp of intron 9 between exon 9 and 10, leading to

a premature stop codon (p.Glu734_Thr1186delinsIleLeu*).

As expected in ERCC5(XPG) XP/CS cell lines [41], the

patient’s fibroblasts exhibited a specific reduction of post-

UV survival in the presence of methylene blue (data not

shown).

The last atypical result involved a teenager referred

for UVSS who was found to carry mutations in the

ERCC3(XPB) gene.To date, eight different mutations in

nine patients from six families have been reported. Patient

#17 displayed severe and early-onset photosensitivity with

erythema and progressive lentigines on exposed areas

(Additional file 7). No skin cancer was described at the age

of 13. General health was good, with normal growth and

psychomotor development. Moderate unilateral hypoacou-

sia was described. The patient has a younger brother who

suffers from less severe photosensitivity. As expected, both

UDS and RRS were severely decreased. We identified two

close heterozygous substitutions in ERCC3(XPB): c.296 T >

C and c.325C > T, leading to the p.Phe99Ser missense

mutation and to the p.Arg109* nonsense mutation respect-

ively. As both mutations were never observed on the same

NGS read (Additional file 7), we can consider that they are

located in trans, even without analyzing parental samples

(which are unavailable). The p.Phe99Ser missense mutation

has been previously reported in association with the c.471

+ 1G > A p.K157insTSDS* splicing mutation in two mild

XP/CS siblings [42] and with the c.1273C > T p.Arg425*

nonsense mutation in two XP siblings [43]. The p.Arg109*

nonsense mutation has never been described in NER

patients but is present in the EVS and ExAC database at a

very low frequency (0.08 % in Europeans). This appar-

ent discrepancy between the rarity of reported cases of

ERCC3(XPB) mutations and the relatively high fre-

quency of the p.Arg109* mutation in Europeans raises

Calmels et al. Orphanet Journal of Rare Diseases (2016) 11:26 Page 10 of 16

the question of possible fetal lethality when truncated

mutations in this gene are present on both alleles.

This hypothesis is consistent with the absence of such

cases in the litterature.

Discussion

Our aim was to develop a single and efficient mutation-

screening process for NER defects and to improve our

understanding of the clinical spectrum of these overlap-

ping and sometimes combined disorders. We performed

targeted next generation sequencing of 16 NER genes in

a cohort of 40 consecutive patients presenting with

phenotypes suggestive of DNA repair defects. Our find-

ings confirm the efficiency of this process, which is able

to simultaneously analyze 95 ± 3 % of targeted regions in

8 patients in a single run. Coverage is sufficiently sensi-

tive (overall sensitivity ≥95 % in the preliminary cohort)

to guarantee reliable detection of heterozygous variants

and small indels. Whole gene deletion is also detectable,

but the method is currently not sensitive enough (in our

hands), to detect smaller heterozygous deletions (for

example, a heterozygous deletion of the first two exons

of the ERCC6(CSB) gene was not detected). This could

be explained by the heterogeneity of the samples tested

in one run (various sample types analyzed such as blood

or fibroblast cultures, various DNA extraction methods

used), as well as by the small number of patients per run,

preventing normalization of depth of coverage on a large

cohort. These limitations are unfortunately difficult to

improve upon. Nevertheless, missing heterozygous CNV

is less harmful in the context of exclusive autosomal

recessive disorders than in dominant diseases. Presence of

a CNV could indeed be considered when a single point

mutation is found, then checked by a quantitative assay.

We identified causative mutations in 43 % of the cohort

(17 out of 40 patients). This approach permits rapid con-

firmation of the molecular diagnosis for classical presenta-

tions of CS and XP. It has established the diagnosis in

seven typical CS cases (patients # 1 to 7) with pathogenic

mutations in either ERCC6(CSB) or ERCC8(CSA), and in

four XP cases (patients # 10 to 13) with mutations in

POLH. The technique is also reliable in cases with uncom-

mon phenotypes (combined features or unusually mild

symptoms or very rarely involved genes). For example, two

of our patients initially referred for CS have a final diagnosis

of XP-CS complex (patient #14 mutated in ERCC2(XPD)

and patient #16 mutated in ERCC5(XPG)). Two further

cases referred for UVSS had mutations in XP genes (patient

#15 in ERCC2(XPD) and patient #17 in ERCC3(XPB)).

These results clearly illustrate the challenge that clinical

diagnosis of NER defects presents, and entirely justifies the

multi-gene approach in molecular diagnosis. The diag-

nosis of NER diseases usually relies on established clin-

ical criteria [7, 44, 45] but these criteria are often only

fulfilled in patients at a relatively advanced stage of the

disease. Clinical diagnosis is therefore difficult in young

or late-onset cases that do not entirely fit the criteria.

NGS is a particularly good alternative for confirming

such cases at an early stage. The benefits of early diag-

nosisarewellknowninNERdefects,forexample,in

order to prevent cutaneous cancer in XP and to delay

sensory impairments and manage feeding difficulties in

CS. Appropriate genetic counseling is beneficial for

family members in all cases.

As expected, the majority of causative mutations iden-

tified in the CS patients were found to be located in the

two main CS genes, ERCC6(CSB) (patients #1 to 4) and

ERCC8(CSA) (patients #5 to 9). We detected eight novel

mutations in these genes, four missenses predicted to be

pathogenic, two frameshift mutations by single nucleotide

deletions, and two mutations in splicing donor consensus

sequences (Table 4). Including the last review [44] as well

as recent case reports [46–51], the number of known muta-

tions now rises to 86 for ERCC6(CSB) and 38 for

ERCC8(CSA).

We also describe the first splicing mutation in

ERCC5(XPG) in an XP-CS patient (patient #16, homo-

zygous for the c.2200-10C > G mutation). Biallelic mu-

tations in the ERCC5(XPG) gene have previously been

associated with XP (group G), CS, XP-CS and COFS

[14, 16]. In the NER pathway, the XPG endonuclease

performs a 3’incision of the abnormal strand and stabi-

lizes the basal transcription factor TFIIH. Patients have

either truncating or point mutations that abolish TFIIH

interaction, with truncating mutations being associated

withthemoreseverephenotypes.Sofar,around20

patients with a defect in the ERCC5(XPG) gene and 30

different disease-causing mutations have been described

worldwide [14, 16, 52, 53]: 20 nucleotide substitutions (9

nonsenses and 11 missenses), 9 deletions of only 1 to 4

bases, 1 duplication and 1 large deletion [16]. We identified

a homozygous splicing mutation without any expression of

the normal transcript in cutaneous fibroblasts, leading to a

severe XP-CS phenotype. The patient, initially referred for

suspected CS, has a more complex diagnosis. This molecu-

lar finding radically changes the clinical management,

which will include cutaneous cancer prevention and genetic

counseling for the family.

The ERCC3(XPB) gene is even more rarely involved in

human diseases. Coding a helicase, it is mutated in only

nine reported patients from six families, with remarkable

phenotypic heterogeneity: XP/CS (5 patients from 4

families), relatively mild XP without CS (2 siblings) and

TTD (2 siblings) [43]. To date only eight different muta-

tions have been identified in ERCC3(XPB):2missense

mutations, 4 truncating mutations (nonsense mutations,

deletion or insertion of one base pair) and 2 splicing

mutations [43]. Genotype/phenotype correlation studies

Calmels et al. Orphanet Journal of Rare Diseases (2016) 11:26 Page 11 of 16

of the short ERCC3(XPB) patient cohort have suggested

that partially active missense mutations may explain symp-

toms in milder patients, whilst severe XP/CS complex

patients have nonsense or consensus splice mutations with

low levels of altered XPB proteins. Patient #17, a teenager

referred for UVSS due to severe and early onset photosensi-

tivity (without skin malignancy or neurological impairment)

(Table 3), was found to be compound heterozygous for the

known p.Phe99Ser mutation and for a novel nonsense

p.Arg109* mutation. A similar genotype was found in two

XP siblings, XP33BR and XP1SA, which carry p.Phe99Ser

and p.Arg425* mutations [43]. ERCC3(XPB) mutations

have been reported in XP/CS, XP alone and TTD patients,

but never in UVSS patients as of yet. As our patient is only

13 years old, skin malignancy may still develop. Patients

XP33BR and XP1SA which carry a similar genotype, both

had multiple basal cell carcinomas, the first of which was

diagnosed at 28 and 29 years old [43]. Long term follow up

of our patient is necessary in order to test the hypothesis

that the clinical spectrum of ERCC3(XPB) mutations ex-

tends to UVSS. As described previously, once again the

molecular diagnosis leads to appropriate clinical follow-up.

Our cohort also included six XP patients from 4 unre-

lated families coming from the Basque country and

surrounding area in Northern Spain. Surprisingly, all cases

had mutations in the same gene, POLH,encodingthe

translesion DNA synthesis polymerase eta. Biallelic muta-

tions in this gene have previously been associated with

Xeroderma-Pigmentosum Variant (XP-V), which is distinct

from classical XP due to the absence of insufficient DNA

repair measured either by UDS or RRS. XP-V patients also

have a relatively milder phenotype with late onset symp-

toms and delayed progression [54]. More than 60 muta-

tions have been identified in the POLH gene, in cell lines

derived from XP-V patients from different geographic

locations [54]. The four patients from our cohort and their

two siblings shared the same previously undescribed

splicing mutation (NM_006502.2:c.764 + 1G > A). This

was either in the homozygous form (5 out of 6 patients)

or in combination with a novel nonsense mutation

(NM_006502.2:c.1445C > A, p.Ser482*) (in one patient).

Haplotype analysis showed a founder haplotype of 975 kb

encompassing the POLH gene in the 4 families that were

investigated (3 homozygous, 1 compound heterozygous).

This suggests that a common ancestor carrying the muta-

tion lived approximately 500–1000 years ago (using com-

parison calculations for a similar sized founder haplotype)

[55]. Similar founder mutations in the POLH gene have

been reported in other populations such as in Japan,

Korea [38, 56] and Tunisia [57]. Such founder effects have

previously been described in patients from the Basque

country for several other diseases such as Parkinson disease

[58], fatal familial insomnia [59] and limb-girdle muscular

dystrophy type 2A [60]. These findings are consistent with

the fact that this population has been relatively isolated in

terms of genetic mixing in the past. Consequently, we

suggest systematic screening for this mutation in all mild

XP patients coming from the Basque country and its

surrounding area.

Under our experimental conditions, classical functional

assays failed to clearly identify two mild CSA patients (# 8

and 9), who have proven pathogenic mutations in the same

WD40 proteic domain. We suspect that fluorescent RRS

assay may overlook mild CS cases, especially when associ-

ated with missense mutations. A different patient carrying a

homozygous missense mutation in the same domain was

reported to have low RRS, but this was associated with a

more severe type I clinical phenotype (patient 08STR2

[25]). In the few previously reported cases of mild late-

onset CS, decreased RRS has always been an associated

finding [25, 45, 61–69]. However, the classical diagnostic

procedure for CS usually recommends performing molecu-

lar sequencing only after identification of a functional

defect in the RRS, and thus atypical CS patients with

normal or inconclusive RRS may be missed. We believe

that these results should be included in the decision making

tree for diagnosis of NER-diseases. In the flowchart pro-

posed by Jia N et al., the authors suggest performing UDS

and RRS assays prior to molecular sequencing (either by

the Sanger method or NGS) [70]. Our experience suggests

that strong clinical suspicion should prompt molecular

investigations. We advocate performing cellular and mo-

lecular tests in parallel, as in most cases cellular tests still

remain important for correct interpretation of nucleotide

variants found by NGS.

Finally, no potentially pathogenic variants were identified

in 23 patients included in the cohort (57 %). All except for

one was referred for CS or COFS syndrome. Gap filling by

Sanger sequencing of the two most commonly involved

genes ERCC6(CSB) and ERCC8(CSA) did not reveal any

anomalies.This relatively high rate of undiagnosed patients

may be explained in part by the fact that 15 % of the cohort

(6 out of 40 patients) had already been explored by Sanger

sequencing of both ERCC6(CSB) and ERCC8(CSA) genes.

Such “prescreening”artificially decreases the rate of positive

diagnoses. Moreover, the 40 patients included in the cohort

were not selected to fulfill the criteria of NER diseases

precisely. Rather, they represent the day to day cases

referred to our laboratory, with many patients presenting

non-specific symptoms such as microcephaly or intellectual

disability. Widening the inclusion criteria allows for diagno-

sis in patients who do not present all the classic diagnostic

features, but it also leads to a lower rate of positive diag-

noses overall. Most of these negative results occur in

patients who are probably not true CS patients, and

further molecular investigations are needed in order to

establish a differential diagnosis. In one case (patient #

32), the phenotype was evocative of a DNA repair defect

Calmels et al. Orphanet Journal of Rare Diseases (2016) 11:26 Page 12 of 16

measured by cellular tests. This case highlights the limits

of our targeted approach, as pathogenic mutations could

be localized in non-targeted regions such as promoters or

deep intronic regions. Such mutations are indeed known

in the ERCC8(CSA) gene, with the example of the deep

intronic c.173 + 1046A > G mutation [25]. While cDNA

sequencing of ERC6(CSB) and ERCC8(CSA) genes did not

retrieve any abnormality in patient #32, other NER tran-

scripts have not been studied. Another explanation could

be the presence of a small copy number variation (CNV),

barely detectable by multiplex-based NGS strategies.

Finally, a non-targeted gene could be involved in this

patient. At least two new genes have been documented in

NER disorders since our study was designed. A missense

mutation in the proliferating cell nuclear antigen (PCNA),

an essential DNA replication accessory protein, was iden-

tified in a single family with four patients sharing pheno-

typic similarities to XP, CS and also ataxia telangiectasia

(AT) [71]. In cells from affected individuals, both UDS

and RRS were reproducibly decreased. A family with a

novel X-linked form of non-photosensitive TTD was also

recently described, with a nonsense mutation in a gene of

unknown function, RNF113A [72]. Whereas the targeted

sequencing approach is the simplest tool for analyzing the

genetic variants of a focused panel of genes via NGS, it

will miss newly identified genes and prevents the re-

analysis of data. Exome or full-genome strategies are more

exhaustive alternatives, and are recommended in these

patients. Such approaches have been succesfully used or

suggested for the diagnosis of XP [73, 74].

Conclusions

Targeted NGS of 16 genes involved in the NER pathway

has been demonstrated to be an efficient alternative to

the sequential Sanger approach for the molecular diag-

nosis of patients with a suspected NER defect, with a

positive diagnosis rate of 43 %. We believe that this

novel approach is the most appropriate solution to the

diagnostic challenge presented by patients with com-

bined specific neurological, dermatological and growth

characteristics. This approach enables rapid confirm-

ation of the molecular diagnosis in classical presenta-

tions of CS and XP. It can also be used to determine

atypical phenotypes with combined features or very mild

symptoms. Targeted NGS should be used in addition to

DNA repair functional tests and precise clinical assess-

ment in order to allow rapid, thorough and cost-

effective diagnosis in patients with NER-defects.

Additional files

Additional file 1: Spectrum of previously identified variations within

the validation cohort of 11 patients. 11 patients already tested by Sanger

sequencing of ERCC6(CSB) and/or ERCC8(CSA) genes were explored by

targeted NGS strategy. All 63 previously identified single base variations

were identified in their correct heterozygous/homozygous state. A

heterozygous 4.6 Mb deletion of chromosome 10q11, encompassing the

whole ERCC6(CSB) gene, was clearly detected but the method was not

sensitive enough to detect an ERCC6(CSB) deletion limited to the first two

exons of the gene. (XLSX 14 kb)

Additional file 2: Poorly covered amplicons (mean coverage < 30X).

Some amplicons appeared to be consistently poorly covered (in all

patients of the cohort); others appeared frequently poorly covered

(in more than 50 % of the patients) (XLSX 13 kb)

Additional file 3: Novel POLH splice mutation in non-related XP-variant

patients. A: Pedigrees of the 4 XP-variant families coming from Northern

Spain and mutated in the POLH gene. The arrows indicate the four patients

studied by NGS. Patients #11, 12 and 13 were homozygous for the splice

mutation c.764 + 1G> A whereas patient #10 was compound heterozygous

for mutations c.764 + 1G > A and c.1445C > A (p.Ser482*). B: RT-PCR using

RNA from patients #10 and 11, with forward primer in exon 5 and reverse

primer in exon 8, showed a single 365 bp band in normal cells and two

bands at323 bp and 261 bp in the homozygous patient #11. The diagram

indicates partial deletion (42 bp) of exon 6 in the type I splice variant and

deletion of the entire 104 bp of exon 6 in the type-II splice variant. The

normal transcript was the major transcript in the heterozygous patient #10,

with a fain band corresponding to the type I splice variant. (PPTX 92 kb)

Additional file 4: POLH founder mutation in Northern Spain. Haplotype

analysis was performed on the 6 XP-variant patients coming from Northern

Spain with mutations in the POLH gene (chr6:43,543,878-43,588,260[hg19]).

Despite a genome wide study using the Illumina HumanOmniExpress-24

SNP chip, the table focuses on the SNP localized in the vicinity of the POLH

gene on chromosome 6 (between 42,000,000 and 44,000,000). Patient #11,

patient #12 and her sister and patient #13 and his sister were homozygous

for the c.764 + 1G > A mutation. Patient #10 was compound heterozygous

c.[764 + 1G > A(;)1445C > A]. Parents of patient #13 were heterozygous

carriers of the splice mutation. The five homozygous patients share a region

of homozygosity (975 kb) including the POLH gene (from rs3800291 at

position 42,751,457 to rs866236 at position 43,726,956). The compound

heterozygous patient #10 and the carrier parents have at least one allele in

common with the founder haplotype all along the 975 kb region.

(XLSX 46 kb)

Additional file 5: Clinical pictures of patients #8 (A) and #9 (B) (mutated in

ERCC8(CSA)). (PPTX 403 kb)

Additional file 6: Clinical pictures and brain MRI of patients #16

(mutated in ERCC5(XPG)). Sagittal T1 and axial T2-Flair (PPTX 970 kb)

Additional file 7: Clinical picture and molecular results of patients #17

(mutated in ERCC3(XPB)). A: clinical picture of patient #17. B: Patient #17 reads

alignment (thanks to Alamut Visual) showing that both mutations c.296 T > C

and c.325C >T were never observed on the same read. Forward reads are in

green and reverse reads are in blue. (PPTX 477 kb)

Competing interests

The authors declare that they have no competing interests.

Authors’contributions

NCandVLdesignedthestudy.GB,DB,PB,AC,BDi,MDF,BD,FF,JG,BG,SJ,ILR,VL,

AM, CM, FRR, MR and PS provided DNA samples and clinical information. GG, NK

and MM carried out the molecular genetic studies. CO carried out cellular studies.

BK and CD carried out SNP arrays studies. JM performed bioinformatics studies.

NC, GG, CO and VL collected and analyzed the data. CG and JT provided technical

support and conceptual advice. NC, VL and JLM provided project coordination

and wrote the manuscript. All authors read and approved the final manuscript.

Acknowledgements

We wish to thank all patients and families included in this study, and our

clinician colleagues who referred patients and provided clinical information.

We warmly thank Dr Helen Fothergill for reviewing the manuscript. This

work was partially supported by a grant from Agence de la Biomédecine.

Calmels et al. Orphanet Journal of Rare Diseases (2016) 11:26 Page 13 of 16

Author details

Laboratoire de Diagnostic Génétique, Institut de Génétique Médicale

d’Alsace (IGMA), Hôpitaux Universitaires de Strasbourg, 1 place de l’hôpital,

Strasbourg, France.

Laboratoire de Génétique Médicale –INSERM U1112,

Institut de Génétique Médicale d’Alsace (IGMA), Faculté de médecine de

Strasbourg, 11 rue Humann, Strasbourg, France.

Centre de Référence

Maladies Osseuses Constitutionnelles, Département de Génétique, Hôpital

Necker-Enfants malades, Paris, France.

Département de Dermatologie,

Hôpital Saint-Éloi, 80 avenue Augustin-Fliche, 34295 Montpellier, France.

Service d’Endocrinologie Pédiatrique, diabète et maladies héréditaires du

métabolisme, Hôpital Femme Mère enfant, GH Est, 59 boulevard Pinel, Bron,

France.

Service de Pédiatrie Générale, Hôpital Necker-Enfants malades, Paris,

France.

Service de Pédiatrie, CHU de Reims, Hôpital Maison Blanche, 45 rue

Cognacq-Jay, Reims, France.

Service de Génétique et Biologie de la

Reproduction CHU de Reims, Hôpital Maison Blanche, 45 rue Cognacq-Jay,

Reims, France.

Service de Génétique, CHU La Réunion, Hôpital Félix Guyon,

Allée des Topazes, Saint-Denis, France.

Centre de Référence des Maladies

Héréditaires du Métabolisme, Service de Médecine Infantile, INSERM NGERE

954, CHU Brabois Enfants, Allée du Morvan, Vandœuvre les Nancy, France.

Servicio de Dermatología, Cruces University Hospital, BioCruces Health

Research Institute, Baracaldo Vizcaya, Spain.

Servicio de Genética, Cruces

University Hospital, BioCruces Health Research Institute, Baracaldo Vizcaya,

Spain.

Service de Génétique Médicale, CHU de Toulouse - Hôpital Purpan,

Place du Docteur Baylac, Toulouse, France.

Department of Pediatrics,

Pediatric Endocrinology and Diabetes, Faculty of Medicine, University of

Rzeszów, Rzeszów, Poland.

Service de Génétique Médicale, Hôpital Necker

Enfants-Malades, 24 Bd du Montparnasse, Paris, France.

Service de

Neurologie, Hôpital Robert Debré, 48 boulevard Serurier, Paris, France.

Centre de Référence des Anomalies du Développement, Service de

Génétique, Hospices Civils de Lyon, Lyon, France.

INSERM U1028; CNRS

UMR5292; CNRL TIGER Team, Lyon, France.

Service de Pédiatrie A -

Neurologie pédiatrique, CHU de Reims - American Memorial Hospital, 47 rue

Cognacq Jay, Reims, France.

AP-HP, Hôpital de la Pitié-Salpêtrière,

Département de Génétique, F-75013 Paris, France.

Sorbonne Universités,

UPMC Univ Paris 06, Inserm, CNRS, UM 75, U 1127, UMR 7225, ICM, F-75013,

Paris, France.

Service de Pédiatrie, Hôpitaux Universitaires de Strasbourg, 1

avenue Molière, Strasbourg, France.

Received: 28 November 2015 Accepted: 16 March 2016

References

1. Marteijn JA, Lans H, Vermeulen W, Hoeijmakers JH. Understanding

nucleotide excision repair and its roles in cancer and ageing. Nat Rev Mol

Cell Biol. 2014;15(7):465–81.

2. Masutani C, Kusumoto R, Yamada A, Dohmae N, Yokoi M, Yuasa M, Araki M,

Iwai S, Takio K, Hanaoka F. The XPV (xeroderma pigmentosum variant) gene

encodes human DNA polymerase eta. Nature. 1999;399(6737):700–4.

3. Stefanini M, Botta E, Lanzafame M, Orioli D. Trichothiodystrophy: from basic

mechanisms to clinical implications. DNA Repair (Amst). 2010;9(1):2–10.

4. Botta E, Offman J, Nardo T, Ricotti R, Zambruno G, Sansone D, Balestri P,

Raams A, Kleijer WJ, Jaspers NG, Sarasin A, Lehmann AR, Stefanini M.

Mutations in the C7orf11 (TTDN1) gene in six nonphotosensitive

trichothiodystrophy patients: no obvious genotype-phenotype relationships.

Hum Mutat. 2007;28(1):92–6.

5. Nakabayashi K, Amann D, Ren Y, Saarialho-Kere U, Avidan N, Gentles S,

MacDonald JR, Puffenberger EG, Christiano AM, Martinez-Mir A, Salas-Alanis

JC, Rizzo R, Vamos E, Raams A, Les C, Seboun E, Jaspers NG, Beckmann JS,

Jackson CE, Scherer SW. Identification of C7orf11 (TTDN1) gene mutations

and genetic heterogeneity in nonphotosensitive trichothiodystrophy. Am J

Hum Genet. 2005;76(3):510–6.

6. Zhang Y, Tian Y, Chen Q, Chen D, Zhai Z, Shu HB. TTDN1 is a Plk1-

interacting protein involved in maintenance of cell cycle integrity. Cell Mol

Life Sci. 2007;64(5):632–40.

7. Wilson BT, Stark Z, Sutton RE, Danda S, Ekbote AV, Elsayed SM, Gibson

L, Goodship JA, Jackson AP, Keng WT, King MD, McCann E, Motojima T,

MurrayJE,OmataT,PilzD,PopeK,SugitaK,WhiteSM,WilsonIJ.The

Cockayne Syndrome Natural History (CoSyNH) study: clinical findings in

102 individuals and recommendations for care. Genet Med. 2015. doi:

10.1038/gim.2015.

8. Henning KA, Li L, Iyer N, McDaniel LD, Reagan MS, Legerski R, Schultz RA,

Stefanini M, Lehmann AR, Mayne LV, Friedberg EC. The Cockayne syndrome

group A gene encodes a WD repeat protein that interacts with CSB protein

and a subunit of RNA polymerase II TFIIH. Cell. 1995;82(4):555–64.

9. Troelstra C, van Gool A, de Wit J, Vermeulen W, Bootsma D, Hoeijmakers JH.

ERCC6, a member of a subfamily of putative helicases, is involved in Cockayne’s

syndrome and preferential repair of active genes. Cell. 1992;71(6):939–53.

10. Kashiyama K, Nakazawa Y, Pilz DT, Guo C, Shimada M, Sasaki K, Fawcett H,

Wing JF, Lewin SO, Carr L, Li TS, Yoshiura K, Utani A, Hirano A, Yamashita S,

Greenblatt D, Nardo T, Stefanini M, McGibbon D, Sarkany R, Fassihi H,

Takahashi Y, Nagayama Y, Mitsutake N, Lehmann AR, Ogi T. Malfunction of

nuclease ERCC1-XPF results in diverse clinical manifestations and causes

Cockayne syndrome, xeroderma pigmentosum, and Fanconi anemia. Am J

Hum Genet. 2013;92(5):807–19.

11. Laugel V, Dalloz C, Tobias ES, Tolmie JL, Martin-Coignard D, Drouin-Garraud

V, Valayannopoulos V, Sarasin A, Dollfus H. Cerebro-oculo-facio-skeletal

syndrome: three additional cases with CSB mutations, new diagnostic

criteria and an approach to investigation. J Med Genet. 2008;45(9):564–71.

12. Meira LB, Graham Jr JM, Greenberg CR, Busch DB, Doughty AT, Ziffer DW,

Coleman DM, Savre-Train I, Friedberg EC. Manitoba aboriginal kindred with

original cerebro-oculo- facio-skeletal syndrome has a mutation in the

Cockayne syndrome group B (CSB) gene. Am J Hum Genet. 2000;66(4):1221–8.

13. Graham Jr JM, Anyane-Yeboa K, Raams A, Appeldoorn E, Kleijer WJ,

Garritsen VH, Busch D, Edersheim TG, Jaspers NG. Cerebro-oculo-facio-

skeletal syndrome with a nucleotide excision-repair defect and a mutated

XPD gene, with prenatal diagnosis in a triplet pregnancy. Am J Hum Genet.

2001;69(2):291–300.

14. Drury S, Boustred C, Tekman M, Stanescu H, Kleta R, Lench N, Chitty LS,

Scott RH. A novel homozygous ERCC5 truncating mutation in a family with

prenatal arthrogryposis–further evidence of genotype-phenotype

correlation. Am J Med Genet A. 2014;164A(7):1777–83.

15. Hamel BC, Raams A, Schuitema-Dijkstra AR, Simons P, van der Burgt I,

Jaspers NG, Kleijer WJ. Xeroderma pigmentosum–Cockayne syndrome

complex: a further case. J Med Genet. 1996;33(7):607–10.

16. Lehmann J, Schubert S, Schafer A, Apel A, Laspe P, Schiller S, Ohlenbusch A,

Gratchev A, Emmert S. An unusual mutation in the XPG gene leads to an

internal in-frame deletion and a XP/CS complex phenotype. Br J Dermatol.

2014;171(4):903–5.

17. Jaspers NG, Raams A, Silengo MC, Wijgers N, Niedernhofer LJ, Robinson AR,

Giglia-Mari G, Hoogstraten D, Kleijer WJ, Hoeijmakers JH, Vermeulen W. First

reported patient with human ERCC1 deficiency has cerebro-oculo-facio-

skeletal syndrome with a mild defect in nucleotide excision repair and severe

developmental failure. Am J Hum Genet. 2007;80(3):457–66.

18. Horibata K, Kono S, Ishigami C, Zhang X, Aizawa M, Kako Y, Ishii T, Kosaki R,

Saijo M, Tanaka K. Constructive rescue of TFIIH instability by an alternative

isoform of XPD derived from a mutated XPD allele in mild but not severe

XP-D/CS. J Hum Genet. 2015;60(5):259–65.

19. Lindenbaum Y, Dickson D, Rosenbaum P, Kraemer K, Robbins I, Rapin I.

Xeroderma pigmentosum/cockayne syndrome complex: first

neuropathological study and review of eight other cases. Eur J Paediatr

Neurol. 2001;5(6):225–42.

20. Vermeulen W, Jaeken J, Jaspers NG, Bootsma D, Hoeijmakers JH. Xeroderma

pigmentosum complementation group G associated with Cockayne

syndrome. Am J Hum Genet. 1993;53(1):185–92.

21. Nakazawa Y, Sasaki K, Mitsutake N, Matsuse M, Shimada M, Nardo T, Takahashi Y,

Ohyama K, Ito K, Mishima H, Nomura M, Kinoshita A, Ono S, Takenaka K,

Masuyama R, Kudo T, Slor H, Utani A, Tateishi S, Yamashita S, Stefanini M,

Lehmann AR, Yoshiura K, Ogi T . Mutations in UVSSA cause UV-sensitive

syndrome and impair RNA polymerase IIo processing in transcription-coupled

nucleotide-excision repair. Nat Genet. 2012;44(5):586–92.

22. Schwertman P, Lagarou A, Dekkers DH, Raams A, van der Hoek AC, Laffeber

C, Hoeijmakers JH, Demmers JA, Fousteri M, Vermeulen W, Marteijn JA . UV-

sensitive syndrome protein UVSSA recruits USP7 to regulate transcription-

coupled repair. Nat Genet. 2012;44(5):598–602.

23. Zhang X, Horibata K, Saijo M, Ishigami C, Ukai A, Kanno S, Tahara H, Neilan

EG, Honma M, Nohmi T, Yasui A, Tanaka K . Mutations in UVSSA cause UV-

sensitive syndrome and destabilize ERCC6 in transcription-coupled DNA

repair. Nat Genet. 2012;44(5):593–7.

24. Spivak G, Hanawalt PC. Host cell reactivation of plasmids containing

oxidative DNA lesions is defective in Cockayne syndrome but normal in

UV-sensitive syndrome fibroblasts. DNA Repair (Amst). 2006;5(1):13–22.

Calmels et al. Orphanet Journal of Rare Diseases (2016) 11:26 Page 14 of 16

25. Laugel V, Dalloz C, Durand M, Sauvanaud F, Kristensen U, Vincent MC,

Pasquier L, Odent S, Cormier-Daire V, Gener B, Tobias ES, Tolmie JL,

Martin-Coignard D, Drouin-Garraud V, Heron D, Journel H, Raffo E, Vigneron

J, Lyonnet S, Murday V, Gubser-Mercati D, Funalot B, Brueton L, Sanchez Del

Pozo J, Munoz E, Gennery AR, Salih M, Noruzinia M, Prescott K, Ramos L et

al.. Mutation update for the CSB/ERCC6 and CSA/ERCC8 genes involved in

Cockayne syndrome. Hum Mutat. 2010;31(2):113–26.

26. Mayne LV, Lehmann AR. Failure of RNA synthesis to recover after UV

irradiation: an early defect in cells from individuals with Cockayne’s

syndrome and xeroderma pigmentosum. Cancer Res. 1982;42(4):1473–8.

27. The Database of Single Nucleotide Polymorphisms [http://www.ncbi.nlm.

nih.gov/SNP/]. Accessed: Build ID: 142.

28. The Exome Variant Server [http://evs.gs.washington.edu/EVS/]. Accessed:

Sept 2015.

29. The Exome Aggregation Consortium [http://exac.broadinstitute.org].

Accessed: Sept 2015.

30. Geoffroy V, Pizot C, Redin C, Piton A, Vasli N, Stoetzel C, Blavier A, Laporte J,

Muller J. VaRank: a simple and powerful tool for ranking genetic variants.

Peer J. 2015;3:e796.

31. Singh RR, Patel KP, Routbort MJ, Reddy NG, Barkoh BA, Handal B, Kanagal-

Shamanna R, Greaves WO, Medeiros LJ, Aldape KD, Luthra R. Clinical

validation of a next-generation sequencing screen for mutational hotspots

in 46 cancer-related genes. J Mol Diagn. 2013;15(5):607–22.

32. Weiss MM, Van der Zwaag B, Jongbloed JD, Vogel MJ, Bruggenwirth HT, Lekanne

Deprez RH, Mook O, Ruivenkamp CA, van Slegtenhorst MA, van den Wijngaard

A, Waisfisz Q, Nelen MR, van der Stoep N. Best practice guidelines for the use of

next-generation sequencing applications in genome diagnostics: a national

collaborative study of Dutch genome diagnostic laboratories. Hum Mutat. 2013;

34(10):1313–21.

33. Steemers FJ, Chang W, Lee G, Barker DL, Shen R, Gunderson KL. Whole-genome

genotyping with the single-base extension assay. Nat Methods. 2006;3(1):31–3.

34. Mattocks CJ, Morris MA, Matthijs G, Swinnen E, Corveleyn A, Dequeker

E, Muller CR, Pratt V, Wallace A. A standardized framework for the

validation and verification of clinical molecular genetic tests. Eur J Hum

Genet. 2010;18(12):1276–88.

35. Pasmant E, Parfait B, Luscan A, Goussard P, Briand-Suleau A, Laurendeau I,

Fouveaut C, Leroy C, Montadert A, Wolkenstein P, Vidaud M, Vidaud D.

Neurofibromatosis type 1 molecular diagnosis: what can NGS do for you

when you have a large gene with loss of function mutations? Eur J Hum

Genet. 2014;23(5):596–601.

36. Loman NJ, Misra RV, Dallman TJ, Constantinidou C, Gharbia SE, Wain J,

Pallen MJ. Performance comparison of benchtop high-throughput

sequencing platforms. Nat Biotechnol. 2012;30(5):434–9.

37. Tarabeux J, Zeitouni B, Moncoutier V, Tenreiro H, Abidallah K, Lair S, Legoix-Ne P,

Leroy Q, Rouleau E, Golmard L, Barillot E, Stern MH, Rio-Frio T, Stoppa-Lyonnet D,

Houdayer C. Streamlined ion torrent PGM-based diagnostics: BRCA1 and BRCA2

genes as a model. Eur J Hum Genet. 2014;22(4):535–41.

38. Inui H, Oh KS, Nadem C, Ueda T, Khan SG, Metin A, Gozukara E, Emmert S,

Slor H, Busch DB, Baker CC, DiGiovanna JJ, Tamura D, Seitz CS, Gratchev A,

Wu WH, Chung KY, Chung HJ, Azizi E, Woodgate R, Schneider TD, Kraemer

KH. Xeroderma pigmentosum-variant patients from America, Europe, and

Asia. J Invest Dermatol. 2008;128(8):2055–68.

39. Broughton BC, Steingrimsdottir H, Weber CA, Lehmann AR. Mutations in the

xeroderma pigmentosum group D DNA repair/transcription gene in

patients with trichothiodystrophy. Nat Genet. 1994;7(2):189–94.

40. Takayama K, Salazar EP, Lehmann A, Stefanini M, Thompson LH, Weber CA.

Defects in the DNA repair and transcription gene ERCC2 in the cancer-prone

disorder xeroderma pigmentosum group D. Cancer Res. 1995;55(23):5656–63.

41. Soltys DT, Rocha CR, Lerner LK, de Souza TA, Munford V, Cabral F, Nardo T,

Stefanini M, Sarasin A, Cabral-Neto JB, Menck CF. Novel XPG (ERCC5)

mutations affect DNA repair and cell survival after ultraviolet but not

oxidative stress. Hum Mutat. 2013;34(3):481–9.

42. Vermeulen W, Scott RJ, Rodgers S, Muller HJ, Cole J, Arlett CF, Kleijer WJ,

Bootsma D, Hoeijmakers JH, Weeda G. Clinical heterogeneity within

xeroderma pigmentosum associated with mutations in the DNA repair and

transcription gene ERCC3. Am J Hum Genet. 1994;54(2):191–200.

43. Oh KS, Khan SG, Jaspers NG, Raams A, Ueda T, Lehmann A, Friedmann PS,

Emmert S, Gratchev A, Lachlan K, Lucassan A, Baker CC, Kraemer KH.

Phenotypic heterogeneity in the XPB DNA helicase gene (ERCC3):

xeroderma pigmentosum without and with Cockayne syndrome. Hum

Mutat. 2006;27(11):1092–103.

44. Laugel V. Cockayne syndrome: the expanding clinical and mutational

spectrum. Mech Ageing Dev. 2013;134(5–6):161–70.

45. Nance MA, Berry SA. Cockayne syndrome: review of 140 cases. Am J Med

Genet. 1992;42(1):68–84.

46. Cui YP, Chen YY, Wang XM, Wang XL, Nan X, Zhao H. Two Novel

Heterozygous Mutations in ERCC8 Cause Cockayne Syndrome in a Chinese

Patient. Pediatr Neurol. 2015;53(3):262–5.

47. Luo Y, Ling Y, Chen J, Xu X, Chen C, Leng F, Cheng J, Chen M, Lu Z. A new

mutation in the CSB gene in a Chinese patient with mild Cockayne

syndrome. Clin Case Rep. 2014;2(2):33–6.

48. Shehata L, Simeonov DR, Raams A, Wolfe L, Vanderver A, Li X, Huang Y,

Garner S, Boerkoel CF, Thurm A, Herman GE, Tifft CJ, He M, Jaspers NG, Gahl

WA. ERCC6 dysfunction presenting as progressive neurological decline with

brain hypomyelination. Am J Med Genet A. 2014;164A(11):2892–900.

49. Swartz JM, Akinci A, Andrew SF, Sigirci A, Hirschhorn JN, Rosenfeld RG,

Dauber A, Hwa V. A novel ERCC6 splicing variant associated with a mild

Cockayne syndrome phenotype. Horm Res Paediatr. 2014;82(5):344–52.

50. Xin B, Wang H. Identification of Two Novel ERCC6 Mutations in Old Order

Amish with Cockayne Syndrome. Mol Syndromol. 2012;3(6):288–90.

51. Yu S, Chen L, Ye L, Fei L, Tang W, Tian Y, Geng Q, Yi X, Xie J. Identification

of two missense mutations of ERCC6 in three Chinese sisters with Cockayne

syndrome by whole exome sequencing. PLoS One. 2014;9(12):e113914.

52. Schafer A, Schubert S, Gratchev A, Seebode C, Apel A, Laspe P, Hofmann L,

Ohlenbusch A, Mori T, Kobayashi N, Schurer A, Schon MP, Emmert S.

Characterization of three XPG-defective patients identifies three missense

mutations that impair repair and transcription. J Invest Dermatol. 2013;

133(7):1841–9.

53. Stenson PD, Ball EV, Mort M, Phillips AD, Shiel JA, Thomas NS, Hofmann

L, Ohlenbusch A, Mori T, Kobayashi N, Schurer A, Schon MP, Emmert S.

Human Gene Mutation Database (HGMD): 2003 update. Hum Mutat.

2003;21(6):577–81.

54. Opletalova K, Bourillon A, Yang W, Pouvelle C, Armier J, Despras E, Ludovic

M, Mateus C, Robert C, Kannouche P, Soufir N, Sarasin A. Correlation of

phenotype/genotype in a cohort of 23 xeroderma pigmentosum-variant

patients reveals 12 new disease-causing POLH mutations. Hum Mutat. 2014;

35(1):117–28.

55. Hamadouche T, Poitelon Y, Genin E, Chaouch M, Tazir M, Kassouri N,

Nouioua S, Chaouch A, Boccaccio I, Benhassine T, De Sandre-Giovannoli A,

Grid D, Levy N, Delague V. Founder effect and estimation of the age of the

c.892C > T (p.Arg298Cys) mutation in LMNA associated to Charcot-Marie-

Tooth subtype CMT2B1 in families from North Western Africa. Ann Hum

Genet. 2008;72(Pt 5):590–7.

56. Masaki T, Ono R, Tanioka M, Funasaka Y, Nagano T, Moriwaki S, Nishigori C.

Four types of possible founder mutations are responsible for 87 % of

Japanese patients with Xeroderma pigmentosum variant type. J Dermatol

Sci. 2008;52(2):144–8.

57. Ben Rekaya M, Laroussi N, Messaoud O, Jones M, Jerbi M, Naouali C,

Bouyacoub Y, Chargui M, Kefi R, Fazaa B, Boubaker MS, Boussen H, Mokni M,

Abdelhak S, Zghal M, Khaled A, Yacoub-Youssef H. A founder large deletion

mutation in Xeroderma pigmentosum-Variant form in Tunisia: implication

for molecular diagnosis and therapy. Biomed Res Int. 2014;2014:256245.

58. Mata IF, Hutter CM, Gonzalez-Fernandez MC, de Pancorbo MM, Lezcano E,

Huerta C, Blazquez M, Ribacoba R, Guisasola LM, Salvador C, Gomez-Esteban

JC, Zarranz JJ, Infante J, Jankovic J, Deng H, Edwards KL, Alvarez V, Zabetian

CP. Lrrk2 R1441G-related Parkinson’s disease: evidence of a common

founding event in the seventh century in Northern Spain. Neurogenetics.

2009;10(4):347–53.

59. Rodriguez-Martinez AB, Alfonso-Sanchez MA, Pena JA, Sanchez-Valle R, Zerr

I, Capellari S, Calero M, Zarranz JJ, de Pancorbo MM. Molecular evidence of

founder effects of fatal familial insomnia through SNP haplotypes around

the D178N mutation. Neurogenetics. 2008;9(2):109–18.

60. Cobo AM, Saenz A, Poza JJ, Urtasun M, Indakoetxea B, Urtizberea JA, Lopez

de Munain A, Calafell F. A common haplotype associated with the Basque

2362AG –> TCATCT mutation in the muscular calpain-3 gene. Hum Biol.

2004;76(5):731–41.

61. Brumback RA, Yoder FW, Andrews AD, Peck GL, Robbins JH. Normal

pressure hydrocephalus. Recognition and relationship to neurological

abnormalities in Cockayne’s syndrome. Arch Neurol. 1978;35(6):337–45.

62. Fryns JP, Bulcke J, Verdu P, Carton H, Kleczkowska A, Van den Berghe

H. Apparent late-onset Cockayne syndrome and interstitial deletion of

Calmels et al. Orphanet Journal of Rare Diseases (2016) 11:26 Page 15 of 16

the long arm of chromosome 10 (del(10)(q11.23q21.2)). Am J Med

Genet. 1991;40(3):343–4.

63. Hashimoto S, Suga T, Kudo E, Ihn H, Uchino M, Tateishi S. Adult-onset

neurological degeneration in a patient with Cockayne syndrome and a null

mutation in the CSB gene. J Invest Dermatol. 2008;128(6):1597–9.

64. Inoue T, Sano N, Ito Y, Matsuzaki Y, Okauchi Y, Kondo H, Horiuchi N, Nakao

K, Iwata M. An adult case of Cockayne syndrome without sclerotic

angiopathy. Intern Med. 1997;36(8):565–70.

65. Kennedy RM, Rowe VD, Kepes JJ. Cockayne syndrome: an atypical case.

Neurology. 1980;30(12):1268–72.

66. Komatsu A, Suzuki S, Inagaki T, Yamashita K, Hashizume K. A kindred with

Cockayne syndrome caused by multiple splicing variants of the CSA gene.

Am J Med Genet A. 2004;128A(1):67–71.

67. McDaniel LD, Legerski R, Lehmann AR, Friedberg EC, Schultz RA.

Confirmation of homozygosity for a single nucleotide substitution mutation

in a Cockayne syndrome patient using monoallelic mutation analysis in

somatic cell hybrids. Hum Mutat. 1997;10(4):317–21.

68. Miyauchi H, Horio T, Akaeda T, Asada Y, Chang HR, Ishizaki K, Ikenaga

M. Cockayne syndrome in two adult siblings. J Am Acad Dermatol.

1994;30(2 Pt 2):329–35.

69. Rapin I, Weidenheim K, Lindenbaum Y, Rosenbaum P, Merchant SN, Krishna

S, Dickson DW. Cockayne syndrome in adults: review with clinical and

pathologic study of a new case. J Child Neurol. 2006;21(11):991–1006.

70. JiaN,NakazawaY,GuoC,ShimadaM,SethiM,TakahashiY,UedaH,

Nagayama Y, Ogi T. A rapid, comprehensive system for assaying DNA

repair activity and cytotoxic effects of DNA-damaging reagents. Nat

Protoc. 2015;10(1):12–24.

71. Baple EL, Chambers H, Cross HE, Fawcett H, Nakazawa Y, Chioza BA, Harlalka

GV, Mansour S, Sreekantan-Nair A, Patton MA, Muggenthaler M, Rich P,

Wagner K, Coblentz R, Stein CK, Last JI, Taylor AM, Jackson AP, Ogi T,

Lehmann AR, Green CM, Crosby AH. Hypomorphic PCNA mutation

underlies a human DNA repair disorder. J Clin Invest. 2014;124(7):3137–46.

72. Corbett MA, Dudding-Byth T, Crock PA, Botta E, Christie LM, Nardo T,

Caligiuri G, Hobson L, Boyle J, Mansour A, Friend KL, Crawford J, Jackson G,

Vandeleur L, Hackett A, Tarpey P, Stratton MR, Turner G, Gecz J, Field M. A

novel X-linked trichothiodystrophy associated with a nonsense mutation in

RNF113A. J Med Genet. 2015;52(4):269–74.

73. Menck CF, Munford V. DNA repair diseases: What do they tell us about

cancer and aging? Genet Mol Biol. 2014;37(1 Suppl):220–33.

74. Ortega-Recalde O, Vergara JI, Fonseca DJ, Rios X, Mosquera H, Bermudez

OM, Medina CL, Vargas CI, Pallares AE, Restrepo CM, Laissue P. Whole-

exome sequencing enables rapid determination of xeroderma

pigmentosum molecular etiology. PLoS One. 2013;8(6):e64692.

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Calmels et al. Orphanet Journal of Rare Diseases (2016) 11:26 Page 16 of 16

Atypical features in two adult patients with Cockayne syndrome and analysis of genotype-phenotype correlation

Article

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Apr 2023
CHINESE MED J-PEKING

Current state of knowledge of human DNA polymerase eta protein structure and disease-causing mutations

Article

Jul 2022
MUTAT RES-REV MUTAT

POLη, encoded by the POLH gene, is a crucial protein for replicating damaged DNA and the most studied specialized translesion synthesis polymerases. Mutations in POLη are associated with cancer and the human syndrome xeroderma pigmentosum variant, which is characterized by extreme photosensitivity and an increased likelihood of developing skin cancers. The myriad of structural information about POLη is vast, covering dozens of different mutants, numerous crucial residues, domains, and posttranslational modifications that are essential for protein function within cells. Since POLη is key vital enzyme for cell survival, and mutations in this protein are related to aggressive diseases, understanding its structure is crucial for biomedical sciences, primarily due to its similarities with other Y-family polymerases and its potential as a targeted therapy-drug for tumors. This work provides an up-to-date review on structural aspects of the human POLη: from basic knowledge about critical residues and protein domains to its mutant variants, posttranslational modifications, and our current understanding of therapeutic molecules that target POLη. Thus, this review provides lessons about POLη's structure and gathers critical discussions and hypotheses that may contribute to understanding this protein's vital roles within the cells.

A mild case of Cockayne syndrome with a novel start-loss variant of ERCC8

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Full-text available

Nov 2024

Cockayne syndrome (CS) is a progressive multisystem disorder characterized by growth failure, microcephaly, developmental delay, and photosensitivity. The characteristic symptoms appear during early childhood in most patients with CS. Herein, we report a mild case of CS with a novel start-loss variant in ERCC8 that did not show the characteristic symptoms of CS during early childhood and exhibited sudden growth failure after the age of 10 years.

The clinical spectrum associated with ERCC5 mutations: Is there a relationship between phenotype and genotype?

Article

Full-text available

Jul 2024

Mutations in the ERCC5 gene can lead to different clinical phenotypes, few articles have reported the clinical phenotypes in detail and explained the relationship between genotype and phenotype. The clinical data of cases with ERCC5 gene mutations diagnosed in our center and reported in previous studies were collected. The cases were divided into three groups based on phenotype; the differences of clinical manifestation and genotype among groups were analyzed. Genetic tests showed a complex heterozygous mutation of the ERCC5 gene with paternal C.402_C.403 (exon 4) insA (p.T135Nfs*28) and maternal C.1096 (exon 8) C > T (p.R366X.821) in our case. The gene mutation has not been reported and was predicted to seriously affect the protein structure. According to a review of 59 cases of ERCC5 mutations, cerebrooculofacioskeletal syndrome (COFS) occurred in 16 cases, XP in 19 cases, and XP/CS in 24 cases. The incidence of physical retardation, mental retardation, peripheral neuropathy, magnetic resonance abnormalities and fundus/vision abnormalities in XP/CS patients was significantly higher than that in XP patients. In addition, patients with the XP/CS phenotype were more prone to appearance abnormalities, deafness, and epilepsy, and cheilitis and tumors were more common in patients with the XP phenotype, but the differences were not significant. XP/CS can cause abnormal liver function and even fatality, which should be given attention. ERCC5 mutation‐related diseases were characterized by mild to severe clinical phenotypes. In addition to tumors, liver function should be considered in ERCC5‐related diseases, and patients should be cautious with medication to avoid drug‐induced liver injury.

Cockayne syndrome without UV-sensitivity in Vietnamese siblings with novel ERCC8 variants

Article

Full-text available

Jun 2022

Cockayne syndrome (CS) is a rare progeroid disorder characterized by growth failure, microcephaly, photosensitivity, and premature aging, mainly arising from biallelic ERCC8 (CS-A) or ERCC6 (CS-B) variants. In this study we describe siblings suffering from classical Cockayne syndrome but without photosensitivity, which delayed a clinical diagnosis for 16 years. By whole-exome sequencing we identified the two novel compound heterozygous ERCC8 variants c.370_371del (p.L124Efs*15) and c.484G>C (p.G162R). The causality of the ERCC8 variants, of which one results in a frameshift and the other affects the WD3 domain, was tested and confirmed by a rescue experiment investigating DNA repair in H2O2 treated patient fibroblasts. Structural modeling of the p.G162R variant indicates effects on protein-protein interaction. This case shows the importance to test for ERCC6 and ERCC8 variants even if patients do not present with a complete CS phenotype.

Investigations and treatment of photodermatoses

Article

Apr 2024

Smitha Prabhu

The photodermatoses comprise of a miscellaneous set of photosensitive disorders with abnormal cutaneous response to sunlight. The diagnosis is mostly clinical and is augmented by methods such as phototesting, photoprovocation testing, and photopatch testing, which are discussed below. Treatment includes photoprotection, prophylactic phototherapy, as well as topical and systemic immunosuppression to varying extents. Various newer modalities for photoprotection as well as treatment are being explored.

Whole-exome sequencing revealed a novel ERCC8 variant in an Iranian large family with Cockayne syndrome

Article

Feb 2024

ClinGen guidance for use of the PP1/BS4 co-segregation and PP4 phenotype specificity criteria for sequence variant pathogenicity classification

Article

Dec 2023
AM J HUM GENET

The 2015 American College of Medical Genetics and Genomics and the Association for Molecular Pathology variant classification publication established a standard employed internationally to guide laboratories in variant assessment. Those recommendations included both pathogenic (PP1) and benign (BS4) criteria for evaluating the inheritance patterns of variants, but details of how to apply those criteria at appropriate evidence levels were sparse. Several publications have since attempted to provide additional guidance, but anecdotally, this issue is still challenging. Additionally, it is not clear that those prior efforts fully distinguished disease-gene identification considerations from variant pathogenicity considerations nor did they address autosomal-recessive and X-linked inheritance. Here, we have taken a mixed inductive and deductive approach to this problem using real diseases as examples. We have developed a practical heuristic for genetic co-segregation evidence and have also determined that the specific phenotype criterion (PP4) is inseparably coupled to the co-segregation criterion. We have also determined that negative evidence at one locus constitutes positive evidence for other loci for disorders with locus heterogeneity. Finally, we provide a points-based system for evaluating phenotype and co-segregation as evidence types to support or refute a locus and show how that can be integrated into the Bayesian framework now used for variant classification and consistent with the 2015 guidelines.

TFIIH central activity in nucleotide excision repair to prevent disease

Article

Sep 2023
DNA REPAIR

Clinical and molecular diagnosis of genodermatoses: Review and perspectives

Article

Nov 2022

Genodermatoses are a complex and heterogeneous group of genetic skin disorders characterized by variable expression and clinical and genetic heterogeneity, rendering their diagnosis challenging. DNA‐based techniques, like whole‐exome sequencing, can establish a diagnosis in 50% of cases. RNA‐sequencing is emerging as an attractive tool that can obtain information regarding gene expression while integrating functional genomic data with regard to the interpretation of variants. This increases the diagnostic rate by an additional 10‐15%. In the present review, we detail the clinical steps involved in the diagnosis of genodermatoses, as well as the current DNA‐based technologies available to clinicians. Herein, the intention is to facilitate a better understanding of the possibilities and limitations of these diagnostic technologies. In addition, this review could guide dermatologists through new emerging techniques, such as RNA‐sequencing and its applications to familiarizing them with future techniques. Currently, this multi‐omics approach is likely the best strategy designed to promote the diagnosis of patients with genodermatoses, and discover new skin disease genes that could result in novel targeted therapies.

The Cockayne Syndrome Natural History (CoSyNH) study: Clinical findings in 102 individuals and recommendations for care

Article

Full-text available

Jul 2015
GENET MED

Cockayne syndrome (CS) is a rare, autosomal-recessive disorder characterized by microcephaly, impaired postnatal growth, and premature pathological aging. It has historically been considered a DNA repair disorder; fibroblasts from classic patients often exhibit impaired transcription-coupled nucleotide excision repair. Previous studies have largely been restricted to case reports and small series, and no guidelines for care have been established. One hundred two study participants were identified through a network of collaborating clinicians and the Amy and Friends CS support groups. Families with a diagnosis of CS could also self-recruit. Comprehensive clinical information for analysis was obtained directly from families and their clinicians. We present the most complete evaluation of Cockayne syndrome to date, including detailed information on the prevalence and onset of clinical features, achievement of neurodevelopmental milestones, and patient management. We confirm that the most valuable prognostic factor in CS is the presence of early cataracts. Using this evidence, we have created simple guidelines for the care of individuals with CS. We aim to assist clinicians in the recognition, diagnosis, and management of this condition and to enable families to understand what problems they may encounter as CS progresses.Genet Med advance online publication 23 July 2015Genetics in Medicine (2015); doi:10.1038/gim.2015.110.

VaRank: A simple and powerful tool for ranking genetic variants

Article

Full-text available

Mar 2015

Background. Most genetic disorders are caused by single nucleotide variations (SNVs) or small insertion/deletions (indels). High throughput sequencing has broadened the catalogue of human variation, including common polymorphisms, rare variations or disease causing mutations. However, identifying one variation among hundreds or thousands of others is still a complex task for biologists, geneticists and clinicians. Results. We have developed VaRank, a command-line tool for the ranking of genetic variants detected by high-throughput sequencing. VaRank scores and prioritizes variants annotated either by Alamut Batch or SnpEff. A barcode allows users to quickly view the presence/absence of variants (with homozygote/heterozygote status) in analyzed samples. VaRank supports the commonly used VCF input format for variants analysis thus allowing it to be easily integrated into NGS bioinformatics analysis pipelines. VaRank has been successfully applied to disease-gene identification as well as to molecular diagnostics setup for several hundred patients. Conclusions. VaRank is implemented in Tcl/Tk, a scripting language which is platform-independent but has been tested only on Unix environment. The source code is available under the GNU GPL, and together with sample data and detailed documentation can be downloaded from http://www.lbgi.fr/VaRank/.

A rapid, comprehensive system for assaying DNA repair activity and cytotoxic effects of DNA-damaging reagents

Article

Full-text available

Jan 2015

DNA repair systems protect cells from genomic instability and carcinogenesis. Therefore, assays for measuring DNA repair activity are valuable, not only for clinical diagnoses of DNA repair deficiency disorders but also for basic research and anticancer drug development. Two commonly used assays are UDS (unscheduled DNA synthesis, requiring a precise measurement of an extremely small amount of repair DNA synthesis) and RRS (recovery of RNA synthesis after DNA damage). Both UDS and RRS are major endpoints for assessing the activity of nucleotide excision repair (NER), the most versatile DNA repair process. Conventional UDS and RRS assays are laborious and time-consuming, as they measure the incorporation of radiolabeled nucleosides associated with NER. Here we describe a comprehensive protocol for monitoring nonradioactive UDS and RRS by studying the incorporation of alkyne-conjugated nucleoside analogs followed by a fluorescent azide-coupling click-chemistry reaction. The system is also suitable for quick measurement of cell sensitivity to DNA-damaging reagents and for lentivirus-based complementation assays, which can be used to systematically determine the pathogenic genes associated with DNA repair deficiency disorders. A typical UDS or RRS assay using primary fibroblasts, including a virus complementation test, takes 1 week to complete.

Identification of Two Missense Mutations of ERCC6 in Three Chinese Sisters with Cockayne Syndrome by Whole Exome Sequencing

Article

Full-text available

Dec 2014
PLOS ONE

Cockayne syndrome (CS) is a rare autosomal recessive disorder, the primary manifestations of which are poor growth and neurologic abnormality. Mutations of the ERCC6 and ERCC8 genes are the predominant cause of Cockayne syndrome, and the ERCC6 gene mutation is present in approximately 65% of cases. The present report describes a case of Cockayne syndrome in a Chinese family, with the patients carrying two missense mutations (c.1595A>G, p.Asp532Gly and c.1607T>G, p.Leu536Trp) in the ERCC6 gene in an apparently compound heterozygote status, especially, p.Asp532Gly has never been reported. The compound heterozygote mutation was found in three patients in the family using whole exome sequencing. The patients' father and mother carried a heterozygous allele at different locations of the ERCC6 gene, which was confirmed by Sanger DNA sequencing. The two mutations are both located in the highly conserved motif I of ATP-binding helicase and are considered "Damaging," "Probably Damaging," "Disease Causing," and "Conserved", indicating the role of DNA damage in the pathogenetic process of the disease. The results not only enrich the ERCC6 mutations database, but also indicate that whole exome sequencing will be a powerful tool for discovering the disease causing mutations in clinical diagnosis.

ERCC6 Dysfunction Presenting as Progressive Neurological Decline With Brain Hypomyelination

Article

Full-text available

Nov 2014
AM J MED GENET A

Mutations in ERCC6 are associated with growth failure, intellectual disability, neurological dysfunction and deterioration, premature aging, and photosensitivity. We describe siblings with biallelic ERCC6 mutations (NM_000124.2:c. [543 + 4delA];[2008C > T]) and brain hypomyelination, microcephaly, cognitive decline, and skill regression but without photosensitivity or progeria. DNA repair assays on cultured skin fibroblasts confirmed a defect of transcription-coupled nucleotide excision repair and increased ultraviolet light sensitivity. This report expands the disease spectrum associated with ERCC6 mutations. © 2014 Wiley Periodicals, Inc.

Two Novel Heterozygous Mutations in ERCC8 Cause Cockayne Syndrome in a Chinese Patient

Article

Jun 2015

Cockayne syndrome (MIM #133540, Cockayne syndrome B; 216400, Cockayne syndrome A) is a rare autosomal recessive inherited disease in which the characteristic symptoms are premature aging, cachectic dwarfism, lack of subcutaneous fat, neurological alterations, light sensitivity, and failure to thrive. The mutated gene responsible for this syndrome has been identified as usually either CSA (CKN1, ERCC8) or CSB (ERCC6). In this study, we describe the case of a 7-year-old Chinese boy with characteristic symptoms of Cockayne syndrome A and the conduction of mutation screening of the CSA gene. The patient was diagnosed with Cockayne syndrome in the pediatrics clinic for growth failure and developmental delay. We collected peripheral blood samples of the patient and his parents and then extracted the genomic DNA. DNA samples from control subjects and the patient were subjected to polymerase chain reaction amplification. All exons and the flanking intron-exon boundaries of CSA were amplified; then, the polymerase chain reaction products were directly sequenced for mutation screening. Two novel heterozygous CSA mutations, c.551-2A>C and c.394_398delTTACA, were identified in the patient. The c.551-2A>C mutation originates from his father and changed the splice acceptor site AG to CG, thus possibly causing alternative splicing. The c.394_398delTTACA from his mother caused a frameshift after the amino acid at position 132, thus introducing a premature stop codon in the gene sequence. These mutations extend the mutation spectrum of Cockayne syndrome in the context of Chinese race and provide possibilities of prenatal diagnosis for future offsprings in this family. Copyright © 2015 Elsevier Inc. All rights reserved.

Constructive rescue of TFIIH instability by an alternative isoform of XPD derived from a mutated XPD allele in mild but not severe XP-D/CS

Article

Feb 2015

Mutations in XPD cause xeroderma pigmentosum (XP), XP and Cockayne syndrome (CS) crossover syndrome (XP/CS), trichothiodystrophy and cerebro-oculo-facio-skeletal syndrome (COFS). COFS represents the most severe end of the CS spectrum. This study reports two Japanese patients, COFS-05-135 and COFS-Chiba1, who died at ages of <1 year and exhibited typical COFS manifestations caused by XPD mutations p.[I619del];[R666W] and p.[G47R];[I619del], respectively. Two other cases of severe XP-D/CS (XP group D/CS), XP1JI (p.[G47R];[0]) and XPCS1PV (p.[R666W];[0]), died at ages <2 years. On the other hand, two cases of mild XP-D/CS, XP1NE (p.[G47R];[L461V;V716_R730del]) and XPCS118LV (p.[L461V;V716_R730del];[R666W]), lived beyond 37 years of age. p.I619Del and p.[L461V;V716_R730del] are functionally null; therefore, despite the differences in clinical manifestations, the functional protein in all of these patients was either p.G47R or p.R666W. To resolve the discrepancies in these XPD genotype-phenotype relationships, the p.[L461V;V716_R730del] allele was analyzed and we found that p.[L461V;A717G] was expressed from the same allele as p.[L461V;V716_R730del] by authentic splicing. Additionally, p.[L461V;A717G] could partially rescue the loss of XPD function, resulting in the milder manifestations observed in XP1NE and XPCS118LV.Journal of Human Genetics advance online publication, 26 February 2015; doi:10.1038/jhg.2015.18.

A novel X-linked trichothiodystrophy associated with a nonsense mutation in RNF113A

Article

Jan 2015

Trichothiodystrophy (TTD) is a group of rare autosomal recessive disorders that variably affect a wide range of organs derived from the neuroectoderm. The key diagnostic feature is sparse, brittle, sulfur deficient hair that has a 'tiger-tail' banding pattern under polarising light microscopy. We describe two male cousins affected by TTD associated with microcephaly, profound intellectual disability, sparse brittle hair, aged appearance, short stature, facial dysmorphism, seizures, an immunoglobulin deficiency, multiple endocrine abnormalities, cerebellar hypoplasia and partial absence of the corpus callosum, in the absence of cellular photosensitivity and ichthyosis. Obligate female carriers showed 100% skewed X-chromosome inactivation. Linkage analysis and Sanger sequencing of 737 X-chromosome exons and whole exome sequencing was used to find the responsible gene and mutation. Linkage analysis localised the disease allele to a 7.75 Mb interval from Xq23-q25. We identified a nonsense mutation in the highly conserved RNF113A gene (c.901 C>T, p.Q301*). The mutation segregated with the disease in the family and was not observed in over 100 000 control X chromosomes. The mutation markedly reduced RNF113A protein expression in extracts from lymphoblastoid cell lines derived from the affected individuals. The association of RNF113A mutation with non-photosensitive TTD identifies a new locus for these disorders on the X chromosome. The extended phenotype within this family includes panhypopituitarism, cutis marmorata and congenital short oesophagus. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Normal Pressure Hydrocephalus

Article

Jun 1978
ARCH NEUROL-CHICAGO

Roger A. Brumback

• Normal pressure hydrocephalus (NPH) in adults is a well-known cause of dementia. We describe NPH in children having the recessively inherited Cockayne's syndrome (CS). Cockayne's syndrome is characterized by cachectic dwarfism, neurological dysfunction, and cutaneous sunlight sensitivity. We noted that the NPH-associated triad of dementia, gait disturbance, and incontinence developed in CS patients. Computerized tomography of the brain in our four CS patients showed hydrocephalic enlargement of the brain ventricles greatest in the older patients. There was no evidence of cortical atrophy except in the one patient who had CS with xeroderma pigmentosum. Lumbar puncture and radionuclide cisternography in the two patients tested showed normal CSF pressure, with complete blockade to flow of radionuclide above the tentorium cerebelli, ventricular reflux, and delayed absorption. Studies of NPH in CS may elucidate the pathophysiology of NPH and methods to alter its sequelae.

A Novel ERCC6 Splicing Variant Associated with a Mild Cockayne Syndrome Phenotype

Article

Nov 2014

Background: Cockayne syndrome is an autosomal recessive, heterogeneous syndrome with classical features, including short stature, microcephaly, developmental delay, neuropathy, and photosensitivity. New genomic approaches offer improved molecular diagnostic potential. Methods: Whole-exome sequencing was employed to study a consanguineous extended family with severe short stature and variable presentations of peripheral neuropathy, lipoatrophy, photosensitivity, webbed neck, and hirsutism. Results: We identified a novel homozygous ERCC6 variant at the donor splice site of intron 9 (c.1992 + 3A>G), which was predicted to only slightly perturb splicing efficiencies. Assessment of primary fibroblast-derived mRNAs, however, revealed a dominant splicing species that utilized an unsuspected putative donor splice site within exon 9, resulting in predicted early protein termination (p.Arg637Serfs*34). Conclusions: We describe a new splicing ERCC6 defect causal of Cockayne syndrome. The application of exome sequence analysis was integral to diagnosis, given the complexity of phenotypic presentation in the affected family members. The novel splicing defect, furthermore, illustrates how a seemingly minor change in the relative strength of a splice site can have significant biological consequences.

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