Heterogeneous clinical features in Cockayne syndrome-A patients with the same mutation and in siblings

Cockayne syndrome (CS) is a rare autosomal recessive disorder caused by mutations in ERCC6/CSB or ERCC8/CSA that participate in transcription-coupled nucleotide excision repair (TC-NER) of UV-induced DNA damage. CS patients display a large heterogeneity of clinical symptoms and severities, the reason of which is not fully understood, and little data is available for affected siblings. CS is largely undiagnosed in North Africa. We report here the clinical description as well as genetic and functional characterization of eight North African CS patients, including siblings. These patients, who belonged to six unrelated families, underwent complete clinical examination and biochemical analyses. Sanger sequencing was performed for the recurrent mutation in ve families, and targeted gene sequencing for one patient of the other family. We also performed RRS (Recovery RNA Synthesis) to conrm the functional impairment of DNA repair in the identied mutations. in another type of in regulation (11). in subtype. North two siblings siblings private > G) ERCC8 and four more CS patients clinically biochemically but the mutations have identied six our Cohort, Sanger sequencing identied a recurrent ERCC8 mutation, the homozygous c. identied in two Tunisian siblings Indels are the second most common class of mutation in the human genome involve domains with repetitive sequences ERCC8 encodes a 44 kDa protein, CSA that contains 7 WD40 domains each of is constituted by several WD repeats (Trp, W), aspartic acid (Asp, D)]. The c. 598_600delinsAA variation in ERCC8 patients could lead to a nonsense-mediated mRNA decay (NMD). detail, the alteration of the fourth evolutionarily conserved amino-acid residue in the WD4 repeated motif result in a premature stop codon after 12 aminoacids (AA). The WD motifs are required for the construction of the beta-propeller structure, formation of with transcription and repair DDB1, RNA polymerase II, TFIIH Responses to UV irradiation in primary broblasts were evaluated through unscheduled DNA synthesis (UDS) and recovery of RNA synthesis (RRS) after DNA damage, as described (44–46). Briey, cells were plated on coverslips in 6-well plates and exposed to UV-C doses at 0, 5, 10 and 15 J/m2. De novo DNA synthesis was measured via incorporation of 5-ethynyl-2'-deoxyuridine (EdU) after UV irradiation in 6 CS patient broblasts (CS1EA1, CS1EA2, CS2, CS6EA1, CS6EA2, CS7), one healthy donor control, one Xeroderma pigmentosum and one Cockayne syndrome (affected DNA repair) controls. RNA detection was performed by irradiating primary culture of broblasts with UV-C doses (0, 6, 12, and 20 J/m2). 5-ethynyl uridine (5-EU) incorporation was assessed after 24 hours of recovery from the UV exposure. The images were processed and analyzed with Image J for 50 randomly selected cells, originating from three independent experiments, and the average nuclear uorescence intensity was calculated.

Clinical, imaging, and genetic characterization of the eight patients are summarized in Table 1. This cohort includes 6 males and 2 females from six unrelated Tunisian families (CS1, CS2, CS6, CS7, CS11 and CS16). Consanguinity, examined by genealogical data, was found in 4 families (CS2, CS6, CS11, and CS16) and endogamy was reported for two other families (CS1 and CS7). All patients originated from the North West of Tunisia except CS2 that originated from South Tunisia. The mean age of patients at the time of examination was 3.4 years ranging from 1.5 year to 7 years. At the time of the study, all patients were alive (Fig. 1).   born in the eighth month of pregnancy. No perinatal asphyxia was reported. Birth weight was within the low normal range for all patients (mean birth weight: 2575 g, ranging from 1450 g (as for CS16) to 3400 g). Based on the head circumference at birth, all patients except CS16 (N = 7) were normocephalic (mean birth head circumference: 33.28 cm). Postnatally, all patients developed progressive growth failure and microcephaly (mean head circumference − 3SD: standard deviation de ned according to growth curve) (Table 1), (Supplementary Fig. 1).
Behavioral abnormalities, muscular neurological and neurosensory problems All patients arrived in our department for examination displayed psychomotor delay. Five patients were able to sit independently at a mean age of 13 months whereas two patients (CS11 and CS 16) were incapable to do so. Walking without support was acquired in two cases (CS6EA1et CS7) at 2 and 3 years, respectively, and then lost as the syndrome progressed (mainly due to contractures). Another patient (CS1EA1) was capable of walking with support at 30 months without reported abnormalities. The ve other patients were unable to walk alone.
As a general observation, none of the patients had language skills at the time of examination. However, they were outgoing and interactive. Behavioral disturbance with irritability and sleep disorder were not reported except for patient CS1EA1. Neurological examination showed a spasticity of limbs with predominance in lower limbs in all patients leading to a progressive exion retraction in 6 patients, associated with ataxia and tremor in three cases (CS1EA1, CS7, CS11), and neurological signs in one case (CS11, age 4

Laboratory investigations
Mild serum aminotransferase elevation (2N) was noted in all patients before the age of three except CS11 and CS16. The biochemical analysis of AST/ALT was done longitudinally for patient CS1EA1, who showed cytolysis (964/1780) at the age of one year, but these values normalized progressively at the age of 3 years (47/53) ( Table 1).
CSF (Cerebrospinal Fluid) analysis was performed in the patient CS1EA1 and showed a mild increase of CSF lactate level (2.13 mmol/l; normal value < 2); this patient also displayed mild elevation of creatinine kinase (824 UI/l; normal value < 145).

Neuroimaging analysis
Computed tomography was performed for 7 patients (except CS16) and showed lenticular calci cations in all of them. Magnetic resonance imaging was performed in these 7 patients as well, showing hypomyelination in four cases (CS2, CS7, CS11, CS16) and cerebellar atrophy in other four cases (CS1EA1, CS2, CS7, CS16) ( Table 1). Cerebral MRI images of CS7 illustrate the white matter anomalies (Fig. 2).

Neurophysiological studies
Nerve conduction velocities were studied in 7 patients, and values were reduced (< 45m/s) in 4 patients (Table 1). Electroretinogram was performed once and was normal in this case.

Genetic analysis reveals the same mutation in six patients
We rst screened the eight patients by Sanger sequencing of ERCC8 exon 7, which revealed that six (CS2, both CS6, CS7, CS11, and CS16) out of eight CS patients were homozygous carriers for the variant NM_000082.3: c. 598_600delinsAA p.Tyr200Lysfs*12. This variant that introduces a stop codon and therefore a truncated protein has been previously described in several unrelated CS patients from North Africa (12, 13) (and unpublished data). We also con rmed parental segregation of the mutation in all cases (Fig. 3). The recurrent variation was absent in the affected siblings from (CS1 family).
Genetic analysis of the CS1 family -Identi cation of an intronic variant via targeted gene sequencing In one patient of the CS1A family, screening of 17 genes involved in NER pathway using NGS revealed a homozygous transversion in ERCC8 gene at the position NM_000082.3:c.843 + 1G > C (Fig. 3). This variant represents a transversion from guanine to cytosine located at the donor splice site of intron 9. This variation was reported previously in a Lebanese CS patient (14). Sanger sequencing con rmed that this mutation was homozygous in the two affected patients (siblings) from this family, and heterozygous in their parents, as expected.
-In silico effect of the variation on splicing site The variant modi ed the consensus donor splice site region in intron 9 of the gene ERCC8, from the conserved GT motif to CT. This mutation is expected to alter the mRNA splicing by affecting the donor site signal, according to the prediction tools Human Splicing Finder (HSF) and MaxEntScan. In particular using HSF, the potential impact of the variation was assessed through attribution of consensus value (CV) according to the matrices from Shapiro and Senepathy (15). The difference between the wild type and the mutant had a CV of (-32. 71%), and was predicted by the program to abolish the donor site, thereby affecting the splicing process.
-cDNA analysis of the splice site mutation To con rm and functionally validate the mutation at the splice site, mRNA extracted from primary broblasts derived from the CS1EA1 patient and a healthy control were compared. PCR ampli cation of cDNA from exon 8 to exon 11, using appropriate primers, resulted in a shorter fragment in CS1EA1 compared to the control. Sequence analysis of the amplicon revealed that exon 9 is missing in the transcript of the CS1EA1 patient. At the protein level, skipping of exon 9 is predicted to shift the reading frame leading to the emergence of a premature stop codon, eight amino acid after (p.Ala240Glyfs*8), and therefore a protein of only 246aa in length (instead of 396aa) (Fig. 4).
-Cellular response to UV in CS patients UV irradiation tests showed that 6 cell lines derived from CS patients, exhibited reduced response to UV compared to healthy controls. Response to increasing doses (0-15 J/m 2 ) of UV radiation was rst assessed by RNA recovery synthesis (RRS) that displayed strongly reduced RNA synthesis in all CS samples compared to the healthy control, with a better response for CS6EA1. As expected, cells derived from CS patients displayed unscheduled DNA synthesis (UDS) comparable to healthy controls (whereas the XP positive control patient had low UDS levels). Altogether these results indicate defective capacity to repair UV induced DNA damage on the transcribed strand in CS patients, including those that do not display abnormal sensitivity to sunlight (Fig. 4), in agreement with previous ndings (5, 16) .

Mutations in eight CS patients
Essentially two genes have been associated with CS, namely ERCC6 in 68% and ERCC8 in 32% of patients (6). The situation is possibly reversed in Tunisia and Arab countries, where ERCC8 mutations seem to be more frequent (5,13,14,17,18). The present study expands the clinical spectrum and increases the relevance of two mutations in the CSA subtype. These genetic defects seem to be speci c to the Tunisian and North African population, as they have not been reported elsewhere, at least to date. Indeed, since the rst description of CS by Dr. Cockayne in 1936, only eight patients have been reported in the Tunisian population: two siblings have one of the mutation described here (c.598_600delinsAA) (12) (13), two other siblings have a private mutation (c.400-2A > G) in ERCC8 (18), and four more CS patients have been clinically and biochemically characterized but the mutations have not been identi ed (19,20) .
In six patients of our Cohort, Sanger sequencing identi ed a recurrent ERCC8 mutation, namely the homozygous mutation c. 598_600delinsAA p.Tyr200Lysfs*12, which was previously identi ed in two Tunisian siblings (12,13). Indels are the second most common class of mutation in the human genome (21), and often involve domains with repetitive sequences (22). ERCC8 encodes a 44 kDa protein, CSA that contains 7 WD40 domains each of which is constituted by several WD repeats [tryptophan (Trp, W), aspartic acid (Asp, D)]. The c. 598_600delinsAA variation in ERCC8 patients could lead to a nonsense-mediated mRNA decay (NMD). In detail, the alteration of the fourth evolutionarily conserved amino-acid residue in the WD4 repeated motif may result in a premature stop codon after 12 aminoacids (AA). The WD motifs are required for the construction of the beta-propeller structure, which is important for protein complex formation and interactions of CSA with transcription and repair factors DDB1, RNA polymerase II, TFIIH (10, 23).
The relatively larger proportion of ERCC8 defects, and in particular the c.598_600delinsAA mutation, in Tunisian patients can be attributed to a founder effect. Further investigations including haplotype analysis are required to verify whether this is the case. Interestingly, one of the six patients has Algerian ancestries suggesting that this variation is a founder mutation in North Africa.
Furthermore, via targeted gene sequencing, we detected in two patients (CSEA1 and CS1EA2) a variation that has not been previously reported in the Tunisian population, i.e. c.843 + 1G > C. This homozygous mutation leads to the abolition of the consensus donor splice site in intron 9, generating a novel splice event, and leading to exon 9 skipping in the ERCC8 gene and the emergence of a premature stop codon. The donor splice mutation detected in patients from the CS1 family leads to a shorter protein lacking the last two WD40 domains, which may affect the function of this protein. This variant co-segregated in the CS1 family members, an additional argument indicating that this variant is causal of the CS disorder in these patients.
clinical manifestations as microcephaly and ataxia at birth are not unique for CS and have been described also in mitochondria-associated diseases, which makes the CS diagnosis more di cult at early stages.
Therefore, cutaneous photosensitivity was classi ed as a minor criterion in the diagnosis of CS that appears only in about 75% of patients and was not correlated with the type of genetic defect in the TCR-NER pathway. Our data, with the two siblings from the CS1 family (mutation c.843 + 1G > C), as well as the CS11 patient mutation c.598_600delinsAA not displaying clinical photosensitivity con rm that this defect is not an essential criterium for CS. The absence of clinical photosensitivity required assessing the repair of UV-induced DNA damage by TC-NER in primary broblasts from these patients. Indeed, since 1977 it has been shown that broblasts from CS patients had increased sensitivity to UV irradiation (29). Conventional methods to assess TC-NER include RRS following UV damage that is impaired in CS (30), and unscheduled DNA synthesis that is not affected in these patients whereas it is in XP patients (31). When clinical photosensitivity is identi ed in CS, it remains very moderate compared to other forms of genodermatosis related to defects of the NER system.
Comparison between conventional mild phenotype CS patients and CS patients who did not show photosensitivity displayed no difference in RRS, which was low in both groups as compared to controls. This indicates that photosensitivity, even if not clinically visible, is present at the cellular level in CS. RRS following UV damage remains useful to con rm the diagnostic and is complementary to genetic investigations.
This further substantiate that Cockayne syndrome may not be accounted to the defective NER system alone. Indeed, variations in ERCC6 and ERCC8 genes were also associated with the UV sensitive syndrome (UVSS), a milder form clinically characterized by mild cutaneous symptoms. In UVSS patients, reduced RRS after UV radiations was also observed, indicating that despite TC-NER was impaired this defect did not lead to neurodegeneration or premature ageing typical of CS.
Lack of association between CS and clinical photosensitivity in some patients suggests that other or additional mechanisms than the DNA repair defect are involved in the etiology of CS. In this context, CS exhibit altered mitochondrial metabolism and an accumulation of oxidative stress at the cellular level (32,33). CSA and CSB are indeed multifunctional proteins that are involved in several processes in addition to DNA repair (34,35).

Heterogeneous clinical features in patients with the same mutation and siblings
CS is a clinically heterogeneous disease and is caused by a large number of distinct mutations in ERCC6 or ERCC8 (5,36). For comparison, other monogenic diseases, for instance the Hutchinson-Guilford progeria syndrome (HGPS) is due to a single point mutation that blocks the physiological processing of the Laminin A protein (37). Conversely, 38 pathogenic variants have been described just for ERCC8/CSA and which concern totally 84 CS patients (36). Since genotype/phenotype correlation remains elusive, relevant information may originate from the study and the assessment of their clinical symptoms in multiple patients and, when available, siblings carrying the same mutation. However, this situation is rather infrequent and only three other cases of siblings as well as patients carrying the same mutations (13, 38) have been described in CS. The present study that reports a detailed clinical characterization of six patients, including two siblings that carry the same mutation as well as two siblings carrying a second mutation, represents a powerful data set to address this question.
The six patients carrying the c.598_600delinsAA mutation shared common characteristics: early age symptoms [0-24 months], prenatal abnormalities as microcephaly, cerebellar hypoplasia, olighydramnios, and lower post-natal weight and height. They also displayed different combinations (presence/absence) of other defects like normal or low birth weight and height, ataxia, cataracts, dental abnormalities, hypomyelination, cerebellar atrophy, etc. Importantly, within this group the two CS6 siblings displayed remarkable phenotypic differences, like post-natal height, independent walking, dental abnormalities, and cryptorchidism.
Two siblings from the CS1 family (mutation c.843 + 1G > C) presented high levels of transaminase which are commonly observed in other CS patients re ecting a possible mild liver damage (4,39). Moreover, the younger of the two patients displayed severe symptoms like the emergence of cataracts at an early age. Indeed, the presence of cataracts is normally associated with a worst probability of survival and death before the age of 7 for CS patients (40). Only one of the two siblings (CS1EA1, a male) showed prenatal microcephaly olighydramnios and cataracts. Conversely, only the other sibling (CS1EA2, a female) showed bird-like nose dysmorphism, limb spasticity, ataxia, hair and dental abnormalities, cerebellar atrophy. These clinical differences in the context of the same mutation and, in the case of siblings also of comparable genetic background, underscore the large heterogeneity of CS clinical symptoms that is di cult to reconcile with a simple genotype/phenotype alteration, and the reason of which remains obscure.
It is important to note that the clinical heterogeneity of patients that share the same recurrent mutation increases the di culty for clinicians to con rm the clinical diagnosis of this disease, and may generate confusion with pathologies that display related symptoms like those linked to mitochondrial etiopathology such as mitochondrial cytopathies. Moreover, the clinical heterogeneity in CS may represent a further challenge for treatments, which have not been developed for CS to date.

Characteristics of the Tunisian cohort
We reported six patients with the same homozygous variation, including one of Algerian origin. This mutation was previously observed in two other Tunisian patients (41), which suggests that it is a founder mutation in the region. The CS6 siblings were born from a consanguineous marriage. Although the CS1 siblings were born from a non-consanguineous marriage, the emergence of the homozygous mutation, and thereby of CS, is likely due to the high rate of endogamy in this region. In Tunisia, the high rate of endogamy contributes to the increased risk (96.64%) of recessive diseases in isolated communities even without consanguinity (42).
The two siblings of the CS1 family harbor the same genetic variation as in a previously reported Lebanese patient, who also displayed a severe CS phenotype (14). North Africa's abundant prehistoric and historic cultural heritage has added to the diversity of the genetic pool of its population nowadays (43). This pool originates from a combination of Middle Eastern, Sub-Saharan Africa and Western European genetic components. For instance, the two Tunisian CS1 patients described here share a variant with the Lebanese patient born from Druze parents, possibly dating back to a common ancestry.

Conclusions
In the present work we report the largest cohort of patients with Cockayne syndrome-A in Tunisia to date, and enlarged the description of ERCC8/CSA variants globally. This study provides genetic, biochemical, and clinical data on siblings and multiple patients carrying the same ERCC8/CSA variant, underscoring the large heterogeneity of CS beyond the mutation. Although all CS-derived cells explored in this work had a DNA repair defect following UV exposure, some patients including those with a severe phenotype, did not show clinical photosensitivity. This nding con rms the notion that photosensitivity is not an essential clinical feature of this pathology, and further questions the mechanistic link between some clinical manifestations and the de cit of the DNA repair system.
A thorough clinical characterization in CS patients, in whom the deleterious effect of the identi ed mutations has been con rmed, should facilitate the early management of other patients in the future, and the establishment of a prenatal diagnosis. Moreover, thanks to the awareness of the families studied, an antenatal investigation was carried out three times for two consanguineous families at risk (two times for CS1 family and one time for CS6 family). Our study shows that collaboration between clinicians and researchers has an important impact on the follow-up of patients' families, mainly for the pathology of CS, a very severe disease which has no treatment to date.
All broblasts primary cultures were assessed at comparable passage number (passage number 3 to 4).

Analysis of ERCC8 cDNA from primary dermal broblasts cultures
Total RNA from 10 6 of dermal broblasts was isolated using Trizol reagent (Sigma-Aldrich) according to the manufacture's instruction. To avoid contamination with genomic DNA, samples were treated with DNase (invitrogen). The cDNA was synthesized from 1µg of RNA using oligo dT primers with the Superscript Reverse transcriptase II (Invitrogen), according to the manufacturer's instructions. For the analysis of the region of interest, polymerase chain reaction was used to amplify the cDNA spanning exon 8 to exon 12 (F:5' GTGAGAAGAGCATCAGGATG3' R:5' CCAGAATGTTGCAGTCTCTG3'). Which was assessed in agarose gel and compared to healthy for amplicon's length and further analyzed via Sanger sequencing.
Responses to UV irradiation in primary broblasts were evaluated through unscheduled DNA synthesis (UDS) and recovery of RNA synthesis (RRS) after DNA damage, as described (44)(45)(46). Brie y, cells were plated on coverslips in 6-well plates and exposed to UV-C doses at 0, 5, 10 and 15 J/m2. De novo DNA synthesis was measured via incorporation of 5-ethynyl-2'-deoxyuridine (EdU) after UV irradiation in 6 CS patient broblasts (CS1EA1, CS1EA2, CS2, CS6EA1, CS6EA2, CS7), one healthy donor control, one Xeroderma pigmentosum and one Cockayne syndrome (affected DNA repair) controls. RNA detection was performed by irradiating primary culture of broblasts with UV-C doses (0, 6, 12, and 20 J/m2). 5-ethynyl uridine (5-EU) incorporation was assessed after 24 hours of recovery from the UV exposure. The images were processed and analyzed with Image J for 50 randomly selected cells, originating from three independent experiments, and the average nuclear uorescence intensity was calculated.

Declarations
Ethic approval and consent for publication The study was approved by Institute Pasteur Ethics Committee in Tunisia (reference 2017/31/I/LR16IPT05/V2), in accordance with the Declaration of Helsinki Principles. Written informed consent was obtained from the legal tutor of all patients for genetic investigation and publication.

Figure 1
Pedigree of the six unrelated Tunisian families. The studied proband is indicated with an arrow.  Lower panel, sequencing of the mutant transcript (MT), which con rmed the aberrant splicing event compared to the wild type (WT); the stop codon in the mutant is indicated by an asterisk. Figure 5