Mutations in eight CS patients
Mutation of essentially two genes has been associated with CS, namely ERCC6 in 68% and ERCC8 in 32% of patients [5]. The situation is possibly reversed in Tunisia and Arab countries, where ERCC8 mutations seem to be more frequent [4, 15, 16, 19, 20]. The present study expands the clinical spectrum and increases the relevance of two CSA mutations. These genetic defects seem to be specific to the Tunisian and North African population, as they have not been reported elsewhere, at least to date. Indeed, since the first description of CS by Dr. Cockayne in 1936, only eleven patients have been reported in the Tunisian population: two siblings with one of the mutations described in the present study (c.598_600delinsAA) in ERCC8/CSA [14, 15], two other siblings with a private mutation (c.400-2A > G) in ERCC8/CSA [20], and three patients with the novel c3156dup mutation in ERCC6/CSB [10]. Four more CS patients have been clinically and biochemically characterized but their respective mutations have not been identified [21, 22].
In six patients of our cohort, Sanger sequencing identified a recurrent ERCC8 variant, namely the homozygous mutation c.598_600delinsAA p.(Tyr200Lysfs*12), which was previously identified in two Tunisian siblings [14, 15]. ERCC8 encodes a 44 kDa protein, CSA that contains 7 WD40 domains. Each of these domains is constituted by several WD [tryptophan (Trp, W), aspartic acid (Asp, D)] repeats. The c. 598_600delinsAA variant 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 is predicted to result in a premature stop codon after 12 aminoacids. 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 the transcription and repair factors DDB1, RNA polymerase II, TFIIH [13, 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 had Algerian ancestries suggesting that this variant is a possible founder mutation in North Africa (Fig. 4).
Furthermore, via targeted gene sequencing, we detected in two patients (CS1EA1 and CS1EA2) a variant 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 site, which leads to exon 9 skipping in the ERCC8 gene and the emergence of a premature stop codon. This donor splice mutation is predicted to generate 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, further supporting this variant as causal of the CS disorder in these patients.
The c.843 + 1G > C variant has been described in a CS patient from Lebanon [16], but the conclusions on the consequence of this variant on the transcript differ in our study. Indeed, Chelby et al. suggested that intron 9 (located between exons 9 and 10) was present in this variant because a PCR test with primers located in these two exons failed to amplify a fragment, indicating the presence of a long intron 9. However, one of the primers used in this PCR was located exactly in exon 9. In this case, the reason for lack of amplification was rather the absence of exon 9, in agreement with our findings. Moreover, the presence of intron 9 was not further demonstrated. Another possibility is that this transcript was not detected in the previous study because it is poorly expressed. In the absence of exon 9, the amplification obtained by Chelby et al. with a pair of primers englobing the region comprised between exon 9 to intron 9 could be due to contaminating DNA acting as a competitor in the PCR reaction [24], if samples were not treated with DNase before RT-PCR, as we did. According to our data, which are compatible with a splicing variant, this mutation has ultimately the same consequences as the c.843 + 2T > G and c.843 + 5G > C variants that have also been suggested to alter donor splice site and lead to a premature stop codon p.(Ala240Glyfs*8) [14, 25].
Remarkable clinical features and lack of clinical photosensitivity
Each of the reported cases in the present study displays distinct clinical features. It is worth to note that some patients (CS1 siblings, CS11, and CS16) suffered from intra-uterine growth retardation. This clinical feature is more frequently associated with the severe form of CS type II, which is usually linked to mutation in ERCC6. Conversely, all patients of this study were linked to the ERCC8 gene, which is normally associated with less severe forms [18, 26]. Other clinical manifestations as microcephaly and ataxia at birth are not specific to CS, and have been also described in mitochondria-associated diseases, which makes the CS diagnosis more difficult at early stages.
Previous studies reported CS patients that do not present clinical photosensitivity, as in Tunisian, Turkish, Italian, and Moroccan populations [4, 21, 27, 28]. Therefore, cutaneous photosensitivity was classified as a minor criterion in the diagnosis of CS as it appears in about 75% of patients, and was not correlated with the type of genetic defect in the TCR-NER pathway. Our data, with 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 confirm that this defect is not an essential criterium for CS. The absence of clinical photosensitivity required to assess whether the repair of UV-induced DNA damage by TC-NER in primary fibroblasts from these patients was affected. Indeed, fibroblasts from CS patients have increased sensitivity to UV irradiation [29], indipendently of the extent of clincal photosensitivity. Conventional methods to assess TC-NER include RRS following UV damage that is impaired in CS [30], and UDS that is not affected in these patients whereas it is in XP patients [31]. To be noticed, when clinical photosensitivity is identified in CS, it remains rather moderate compared to other forms of genodermatosis related to defects of the NER system.
In the present study, conventional mild phenotype CS patients as well as CS patients who did not show photosensitivity displayed similarly low RRS values compared to healthy controls. This result confirms that photosensitivity, although not clinically visible, is present at the cellular level in these patients.
Altogether these findings further substantiate that Cockayne syndrome may not be solely accounted for the defective NER system. Indeed, variants in ERCC6 and ERCC8 genes have been also associated with the UV sensitive syndrome (UVSS), a milder form clinically characterized by mild cutaneous symptoms [32]. In UVSS patients, reduced RRS after UV radiations was also observed, indicating that the TC-NER impairment did not lead to neurodegeneration or premature ageing as it is the case in 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 [33, 34]. CSA and CSB are indeed multifunctional proteins that are involved in several processes in addition to DNA repair [35, 36].
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 [4, 9]. For comparison, other monogenic diseases, for instance the Hutchinson-Guilford progeria syndrome (HGPS) is mostly due to a single point mutation that activates an alternative splicing site that produces an altered form of the lamin A protein [37]. Conversely, 38 pathogenic variants have been described just for ERCC8/CSA and which concern totally 84 CS patients [9]. Since genotype/phenotype correlation remains elusive in CS, relevant information may originate from the assessment of 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 [15, 20, 38], and a few cases of patients carrying the same mutations [10] 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 other siblings carrying another 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 concerning for instance post-natal height, independent walking, dental abnormalities, and cryptorchidism.
The two siblings from the CS1 family (mutation c.843 + 1G > C) presented high levels of transaminase which are commonly observed in other CS patients, possibly reflecting a mild liver damage [3, 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 backgrounds, underscore the large heterogeneity of CS clinical symptoms that is difficult 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 difficulty for clinicians to confirm 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 CS-A cohort
We reported six patients with the same homozygous variant, including one that appeared to have an Algerian ancestry (according to the genealogical questionnaire). 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 variant as in a previously reported Lebanese patient, who also displayed a severe CS phenotype [16]. North Africa's abundant prehistoric and historic cultural heritage has contributed 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. In fact, Druze was first reported under the Fatimid Dynasty, a dynasty that originated in Tunisia and spread to some region of Middle East [44]. Druze is a closed community with high rate of inbreeding (around 53%), which has increased the rate of autosomal recessive diseases [45].