Cockayne syndrome: The expanding clinical and mutational spectrum

Mechanisms of Ageing and Development 134 (2013) 161–170

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Cockayne syndrome: The expanding clinical and mutational spectrum Vincent Laugel * Department of Pediatrics, Strasbourg-Hautepierre University Hospital, Avenue Moliere, F-67098 Strasbourg, France

A R T I C L E I N F O

A B S T R A C T

Article history: Available online 18 February 2013

Cockayne syndrome is a progressive multisystem disorder characterized by a specific cellular defect in transcription-coupled repair. Typical features include developmental delay, failure to thrive, microcephaly, cutaneous photosensitivity, dental anomalies, progressive hearing loss, pigmentary retinopathy, cataracts and enophthalmia. Various levels of severity have been described including the ‘‘classical’’ or moderate type I CS, the early-onset or severe type II and the mild or late-onset type III. Adult-onset cases with prolonged survival and normal initial development have also been identified. At the opposite end of the scale, the most severely affected patients, showing a prenatal onset of the symptoms, are overlapping with the cerebro-oculo-facio-skeletal (COFS) syndrome. These overlapping subtypes build a continuous spectrum without clear thresholds. Revised diagnostic criteria are proposed to improve the recognition of the disease. Two thirds of the patients are linked to mutations in the CSB (ERCC6) gene, one third to mutations in the CSA (ERCC8) gene. At least 78 different mutations are known in the CSB gene and 30 in the CSA gene to date, in more than 120 genetically confirmed patients. Large clinical and molecular databases are needed to unravel genotype-phenotype correlations and to gain more insight into the underlying molecular mechanisms. ß 2013 Elsevier Ireland Ltd. All rights reserved.

Keywords: Cockayne syndrome Diagnostic criteria Clinical subtypes CSA CSB

1. Introduction Cockayne syndrome (CS) is an autosomal recessive multisystem disorder characterized by mental retardation, microcephaly, severe growth failure, sensorial impairment, cutaneous photosensitivity, dental decay, recognizable facial appearance with deep sunken eyes. This progressive and devastating condition is related to defective DNA transcription and/or repair and belongs to the family of nucleotide excision repair disorders together with xeroderma pigmentosum (XP) and trichothiodystrophy (TTD). The first two patients were reported by Sir Edward A. Cockayne in 1936 and the disease was described as a cachectic dwarfism with retinal atrophy and deafness (Cockayne, 1936). In this original report and in clinically similar patients reported later on, the first symptoms typically occurred in early childhood. Most clinical features were already described in the first reports of the condition. It is only from the late 1970s and early 1980s that the clinical diagnosis of CS could be confirmed by cellular testing, taking advantage of a particular sensitivity to UV light (Mayne and Lehmann, 1982; Schmickel et al., 1977). This defect in the transcription-coupled nucleotide excision repair then served as a definite hallmark to ascertain the diagnosis in all CS patients.

* Tel.: +33 3 88 12 84 98; fax: +33 3 88 12 83 30. E-mail address: [email protected] 0047-6374/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mad.2013.02.006

Genetic confirmation only became available in the mid-1990s after the identification of the two major genes responsible for the disorder, ERCC6 (CSB) and ERCC8 (CSA) (Henning et al., 1995; Troelstra et al., 1992). These cellular and molecular findings provided the basis to expand the clinical spectrum of the disease beyond the framework of the initial descriptions. Early-onset cases (Lowry, 1982; Moyer et al., 1982) and late-onset cases (Kennedy et al., 1980; Rapin et al., 2006) were then identified and proved to share the same cellular defect than the classical patients. Earlyonset cases and late-onset cases were named type II and type III respectively as compared to the classical (type I) patients described in the first place. Early-onset patients show congenital signs of the disease and late-onset cases may only be affected in late childhood or even adulthood. A comprehensive review of previously published cases led to the establishment of clinical diagnostic criteria in 1992 (Nance and Berry, 1992). CS is a progressive disorder and most symptoms appear and worsen with time. Actually, all CS patients show very similar features but the time of onset and the rate of progression vary widely among the subgroups. With several hundreds of CS patients have been identified and clinically characterized, it has also become clearer and clearer that CS has a continuous spectrum of severity and that there is no clear threshold between the largely overlapping subgroups. The limits of this constantly expanding clinical spectrum have been pushed even farther with the inclusion of very severely affected patients and very mildly affected patients who had been

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first considered as belonging to distinct entities. Cerebro-oculofacio-skeletal syndrome (COFS) and UV-sensitive syndrome (UVSS) were described independently from CS in the 1970s and early 1980s (Fujiwara et al., 1981; Lowry et al., 1971; Pena and Shokeir, 1974), but eventually proved to share the same cellular defect as the canonical Cockayne patients (Graham et al., 1998; Itoh et al., 1994). COFS syndrome was first reported within the Manitoba aboriginal population as an autosomal recessive disorder defined by arthrogryposis, microcephaly, cataracts and microphthalmia. These features demonstrate a very early onset of the disease in the fetus. COFS patients show mutations mainly in CSB but also in XPD, XPG and ERCC1 (Laugel et al., 2008; Meira et al., 2000). UVSS is only characterized by cutaneous photosensitivity without any of the other features of the CS picture and without cancer proneness. It has been linked to mutations in CSB, CSA and a novel gene UVSSA (Horibata et al., 2004; Miyauchi-Hashimoto et al., 1998; Nakazawa et al., 2012; Nardo et al., 2009; Schwertman et al., 2012; Zhang et al., 2012). These entities are now considered as additional CS variants and appear to be the opposite ends of the same continuous spectrum, but they do not fully meet the canonical diagnostic criteria for CS anymore. Finally, some rare patients show combined features of CS and XP, and are linked to mutations in XPD, XPB or XPG. Most XP–CS patients show a very severe phenotype, close to type II CS with severe skin photosensitivity (Lindenbaum et al., 2001; Vermeulen et al., 1993). XPB–CS and XPG–CS complexes have been associated with survival into adulthood with inconstant development of multiple malignancies (Rapin et al., 2006). The minimal incidence of CS has been evaluated in Western Europe at 2.7 cases per million births (Kleijer et al., 2008). Higher incidences in recent years and in countries where diagnostic tests are routinely available suggest that CS is still likely to be underdiagnosed in many circumstances. 2. Disease characteristics 2.1. Facial appearance The characteristic facial appearance of CS patients is mainly due to the loss of subcutaneous and orbital fat (Suppl. Fig. 1). The resulting enophthalmia is one of the most specific hallmarks for clinical diagnosis. However, it must be kept in mind that this facial appearance only develops with time (Suppl. Fig. 2) and may not be easily recognizable in the early stages of the diseases. It is also noticeable that COFS patients usually do not show the classical cachectic appearance. Appropriate tube feeding may also deeply modify this cachectic facial appearance but the enophthalmia usually persists. 2.2. Growth Growth failure is a prominent and constant feature of CS and is considered as a major diagnostic criterion. It is also often one of the earliest signs of the disease. All patients show progressive growth retardation. Age of onset and growth rate vary among CS subgroups and parallels the severity of the other key symptoms. Usually, in types I and II, weight is affected slightly earlier and more severely than length/height. Conversely, short stature is predominant in mildly affected cases. It should be underlined that length or height measurements can be very inaccurate and often underestimated in CS patients due to joint contractures and that weight partly depends on nutritional management and neurological limitation of oral intake. Intra-uterine growth retardation is inconstantly observed in early-onset cases and is always followed by progressive growth failure in the early postnatal period. Body weight and length are typically normal at birth in type I and type

III. Slowing of the growth rate is usually observed in the second year of life in type I patients and after 2 years of life in type III patients. Weight and height can reach an early plateau in the most severely affected patients. Moderate weight loss can be observed in the late stages of the disease. Even if the age of onset and growth rate vary among the subgroups, weight and height eventually reach a similar and very severe level in all groups, well below - 3 standard deviations, during infancy for type II patients, during childhood for type I patients and in their late teens for type III patients (Laugel, personal communication; Nance and Berry, 1992; Natale, 2011). Body mass index is usually low in CS type I and II and in the normal inferior range in type III patients. The specific loss of subcutaneous fat is responsible for the ‘‘cachectic’’ appearance of CS patients and the BMI is often not as low as it appears to be, at least until the late stages of the disease. Nutrition is a key issue for CS patients and their caregivers. Oral intake is often limited by swallowing difficulties particularly in the most severely neurologically impaired patients. Gastroesophageal reflux and recurrent vomiting are common in young CS patients and often require that they be given repeated small quantities of food. Gastrostomy tube feeding is routinely required in type II CS patients. 2.3. Nervous system Developmental delay is another major diagnostic criterion for CS, leading to mild to profound intellectual disability. Secondary neurological deterioration and cognitive decline is common in later stages of the disease in all groups. The severity of the developmental delay is usually correlated with the overall severity of the disease. COFS and CS type II patients show very limited development. Poor feeding and weak cry are often present in the neonatal period, together with axial hypotonia and peripheral hypertonia. These patients are usually unable to sit or stand unaided and have no language or only very few words. CS type I patients show normal developmental milestones in the first months of life. Motor and speech delay can be observed at the end of the first year or during the second year of life. CS type I patients usually learn to walk but often lose this ability as the disease progresses. These patients can understand and make simple sentences. In spite of this developmental delay, CS patients are unanimously considered as outgoing and interactive in all reports, including the original paper by E. Cockayne (1936). CS type III patients may only have mild intellectual disability and learning difficulties in primary school. Some patients have even been reported to have normal intellectual capacities and should probably be considered as yet another subgroup of severity. Cognitive decline and early dementia in adulthood are often reported in the oldest patients of these categories, typically after 30 or 40 years of age. CS patients also show many other symptoms of progressive neurological dysfunction. Most CS patients show a unique combination of pyramidal, extra-pyramidal, cerebellar and peripheral signs. Limb hypertonia and spasticity is an early symptom in the most severely affected patients. Briskness of tendon reflexes decreases rapidly as the peripheral nerve dysfunction progresses. Cerebellar signs such as gait ataxia, action tremor and dysarthric speech are almost constant in all CS patients, with various degrees of severity. Cerebellar ataxia can be an inaugural sign in the lateonset subgroup. Seizures are usually not a major issue in CS patients even if seizures are probably slightly more common in CS patients than in the general population. No specific clinical or EEG pattern has been described. Progressive microcephaly is a constant feature in CS patients. The absence of microcephaly at 3 years of age has been recognized

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as an exclusion criteria for type I CS (Nance and Berry, 1992) but it should probably be more convenient to consider the presence of microcephaly as a third major criteria. Head circumference can already be low at birth in COFS and CS type II patients but the microcephaly always becomes more severe with age in all groups. The severity of the microcephaly can reach very low levels, constantly below 3 SD. Neuroimaging studies have shown that cerebral and cerebellar atrophy, brain calcifications and white matter anomalies are cardinal features of CS which progresses with time (Koob et al., 2010). White matter loss and ventricle enlargement are probably the earliest detectable signs of CS on brain imaging and are present at the onset of neurological symptoms in all clinical subtypes. White matter signal anomalies on MRI are mainly related to primary hypomyelination (Fig. 1). Secondary demyelination and astrogliosis are present in some cases and have been previously

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demonstrated in neuropathological reports of CS cases (see specific review). Calcifications can be located in the basal ganglia, in the dentate nuclei and the subcortical white matter (Fig. 2). They can be missing in the first year of life even in severe cases. Dense or punctuate, symmetrical calcifications in the putamina are usually predominant in type I and III patients and can be associated with calcifications in the dentate nuclei in later stages. Diffuse subcortical calcifications are predominant in CS II and COFS patients. Meningeal vessels calcifications have been described in neuroimaging and pathological reports. Progressive cerebellar atrophy is also an early and common feature, with global volume loss, shrunken folia, and cisterna magna enlargement. CSF analysis can show elevated protein level. Signs of diffuse demyelinating peripheral neuropathy can be evidenced by nerve conduction studies from the early stages of the disease in the majority of CS patients.

Fig. 1. Axial T1 (a), Flair (b) and T2-weighted (c) brain MRI pictures in a CS type I patient showing hypomyelinated white matter at 8 years. (d) Sagittal T1-weighted MRI picture showing severe cerebellar and brainstem atrophy in a CS type III patient at 28 years.

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Fig. 2. Brain CT-scan images showing typical diffuse subcortical calcifications in a CS type II patient (a, b) and putaminal calcifications (arrow) in two different CS type I patients (c, d).

2.4. Bones and joints Arthrogryposis and congenital kyphosis is the specific hallmark of COFS syndrome and may require early specific orthopedic management. In other CS subtypes, the neurological deterioration induces progressive joint stiffness and deformations. Hip, knee, ankle contractures are very frequently observed and require intensive physiotherapy. Surgery is recommended only if a functional benefit can be expected, or to improve positioning and to relieve chronic pain. These joint retractions as well as the spine deformations can make it very difficult to obtain reliable height or length measurements in CS patients. Osteoporosis is often reported on standard X-rays but has never been thoroughly documented in patients. It is yet unclear if this osteoporosis is a specific feature of the disease or if it is a consequence of the decreased mobility of the patients. Bone fractures have not been reported particularly often in CS patients. 2.5. Teeth Dental anomalies are common in CS patients and the presence of dental caries is considered among the minor diagnostic criteria (Nance and Berry, 1992). However, caries are observed in only 50– 75% of the patients and is obviously not a specific feature of CS.

Numerous anomalies in tooth shape, size, number and structure have recently been documented (Arenas-Sordo et al., 2006; BlochZupan, personal communication). Enamel hypoplasia seems to be a particular hallmark of the disease and can be observed in almost all patients. Tooth malposition, hypodontia (missing permanent teeth), shovel-shaped upper central incisors are commonly observed. Caries are a consequence of preexisting enamel hypoplasia, decreased salivation, poor oral hygiene due to neurological impairment, gastro-oesophageal reflux and vomiting. 2.6. Skin Cutaneous photosensitivity is a cardinal feature of CS especially with regard to the underlying DNA repair defect and cellular sensitivity to UV light. It is only considered a minor diagnostic criterion as it is clinically prominent in only two thirds to three fourths of the CS patients. In these patients mild to severe sunburns are reported even after short exposure to sunlight. Pigmented macules on sun exposed areas are common in older CS patients. Anhidrosis, thin and dry hair, subtle nail dystrophy, skin atrophy, cyanotic livedo and edema of the extremities are consistently reported in CS patients. The mechanism of this livedo is unclear but may be related to the neurological involvement (Lipsker, personal communication).

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No skin cancer has been reported in CS patients mutated in the CSB or CSA genes. Skin malignancies have only been observed in patients presenting with the complex XP–CS phenotype. 2.7. Sensory systems Progressive sensorineural hearing loss is almost a constant feature in CS patients when appropriate hearing tests can be performed, but it is not always clinically significant in the early stages of the disease process. Neurological and cognitive impairment can prevent or delay the clinical diagnosis of hearing loss. The severity of the hearing loss seems to be well correlated with the overall severity of the disease. Hearing aids are very useful and very well tolerated in type I CS patients in spite of their intellectual disability. Cochlear implants have been used in several patients (Morris et al., 2007). Type III CS patients have usually a mild and late hearing impairment. Pigmentary degeneration of the retina has been considered as one of the cardinal hallmarks of CS since the very first reports. This retinal degeneration is progressive throughout the life of the patients and only becomes clinically significant in advanced stages of the disease. Fundus examination most often reveals the classic ‘‘salt and pepper’’ retinopathy. Electroretinogram reveals abnormal photopic and scotopic responses long before any clinical impairment. The severity of the retinal degeneration and specifically the ERG anomalies seem to parallel the global severity of the disease. Optic disk pallor is frequently if not systematically associated with the retinal degeneration. In some cases, optic atrophy clearly precedes the retinal anomalies, indicating that it cannot be a consequence of the retinopathy (Dollfus et al., 2003). Cataracts are present in approximately one half of the patients. Cataracts can be congenital, especially in the most severe subtypes, or progressive. Cataracts are more frequent in the most severe subtypes and the presence of congenital cataracts is classically considered as a poor prognosis factor, but this statement has been challenged in a recent survey (Natale, 2011). Cataracts may be of various types, including cortical, posterior, subcapsular, and nuclear types. Structural changes of the eye such as microphthalmia and iris hypoplasia have been reported in the most severely affected patients and specifically in COFS patients. Microphthalmia may sometimes be difficult to differentiate from the enophthalmia that is observed in all CS patients due to the loss of orbital fat tissue. Photophobia, decreased or absent lacrimation and miotic pupils with resistance to mydriatics are additional common features of CS. The poor response to mydriatics may cause surgical difficulties. 2.8. Kidney Renal complications have been reported in CS patients since 1966 (Ohno and Hirooka, 1966) but still remain probably largely overlooked even if they represent potentially lethal events. Many reports involve CS patients without molecular or cellular confirmation of the diagnosis and little data is available on this topic in recent years. Based on existing data, renal complications can be grouped in chronic or acute manifestations. Chronic hypertension and moderate proteinuria are the most common symptoms and can lead to progressive renal failure. Nephronic reduction, arteriolosclerosis, glomerular hyalinosis are reported in pathological reports (Higginbottom et al., 1979; Hirooka et al., 1988; Sato et al., 1988). This type of renal lesions globally mimicks the lesions of ageing. Uric acid levels are often elevated in CS patients even without renal malfunction and the potential causative relationship between hyperuricemia and renal failure is unclear.

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Acute nephrotic syndrome and acute renal failure have also been reported in late stages of the disease and can lead to the death of the patient (Reiss et al., 1996; Funaki et al., 2006). It is not clear if this is an ultimate consequence of the chronic renal lesions or if it represents another pathological process. 2.9. Liver Liver enzymes (aspartate aminotransferase or ASAT and alanine aminotransferase or ALAT) are commonly elevated in CS patients (Nance and Berry, 1992), usually without any clinical significance or alteration in coagulation factors. ASAT and ALAT are typically around 2 to 10 times the upper limit of normal range and ASAT/ ALAT ratio is usually inferior to 1, which is consistent with a mild liver damage (unpublished results). Liver enlargement has been noticed occasionally. Asymptomatic cholestasis is detectable in a minority of CS patients (unpublished results). Acute liver failure has been reported as a cause of death in at least 2 CS patients (Natale, 2011). 2.10. Cardiovascular system Atherosclerosis has been mentioned in pathological reports of CS patients and may involve brain and coronary arteries and the aorta, but is not a constant feature (Rapin et al., 2006). Arteriosclerosis has also been described in brain and renal arteries. However, transient ischemic attacks and strokes have been very rarely reported (Shirasaki et al., 1986) and no clinical coronary disease or spontaneous myocardial infarction has been observed so far. Myocardial ischemia has been observed after general anaesthesia (Yuen et al., 2001). Hypertension may result from vascular changes and renal involvement. 2.11. Endocrine function Hyperinsulinemia and abnormal glucose tolerance curves have been mentioned in the review by Nance and Berry (1992) and diabetes mellitus has been reported in a few young adults presenting with CS type III (Rapin et al., 2006). Pubertal changes have been observed in most if not all CS patients who reach the appropriate age. A few successful pregnancies have been reported in mildly affected CS females but the small size of the mother can complicate the pregnancy and induce miscarriage or premature birth (Hashimoto et al., 2008; Kennedy et al., 1980; Lahiri and Davies, 2003). Undescended testis and underdeveloped genitalia are frequently reported in male patients. Growth hormone testings have yielded contradictory results in various studies but IGF-1 levels are usually within normal range. Growth hormone therapy did not result in significant growth in at least one patient. Thyroid hormone levels are usually within normal range (unpublished results). 2.12. Premature ageing symptoms? Loss of subcutaneous fat, progressive hearing loss, cognitive decline, nephronic reduction, atherosclerosis, arteriolosclerosis, chronic hypertension, diabetes represent CS features that are reminiscent of specific signs of normal ageing and occur at a much younger age in CS patients than in the global population. However, all other neurological signs observed in CS, as well as retinal pigmentary degeneration or specific teeth anomalies do not belong to the usual clinical spectrum of ageing. Whether those symptoms are the consequence of the same pathophysiological mechanisms in CS and in normal ageing will require specific studies both at clinical and molecular levels.

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3. Severity groups in CS The large variety of symptoms and the wide range of severity are among the most striking features of CS (Fig. 3 and Suppl. Fig. 3). Most symptoms are present in all CS subtypes but the time of onset and the rate of progression vary between the different subtypes. Growth failure, neurological impairment and decline, visual and hearing impairment, joint contracture eventually occur in all patients presenting with CS type I, II or III and always progress with time. Only the most severe COFS and the mildest UVSS patients may not show the same panel of symptoms and therefore require specific diagnostic criteria. These various subtypes have initially been described as discrete groups of patients but accumulating clinical data clearly indicate that there is no threshold between the groups and that every intermediate level of severity can be observed in CS patients. The clinical pictures of all CS patients identified so far definitely build a continuous spectrum of severity. Variations in quantitative parameters such as head circumference, height, weight, life expectancy, mental disability (IQ), retinal dysfunction (ERG responses), hearing loss, show a continuous distribution (unpublished results). Provided this underlying continuous spectrum is kept in mind, it still seems relevant to keep a classification in distinct severity groups for diagnostic purposes and for prognostic information. Such classification is also necessary to build homogenous groups in clinical trials. In general, the degrees of severity of the clinical symptoms seem to parallel with one another in a given CS patient: the degrees of microcephaly, growth failure, neurological and sensorial impairment are usually grossly correlated. Renal and respiratory complications are also more frequent in the more severe subtypes.

Fig. 3. Continuous spectrum of severity in CS.

However, weight and dental anomalies are partly accessible to external intervention and therefore may not totally correlate with the rest of the symptoms. Photosensitivity is another clear exception and may be present or absent, mild or severe, independently of other clinical symptoms, and this absence of correlation between photosensitivity and overall clinical severity is not solely explained by differences in solar exposure or protection. The overall severity of the clinical picture of CS and the life expectancy of the patients are also grossly correlated with the age of onset of the first symptoms. In general, patients with congenital symptoms have the more severe clinical course and the shorter life expectancy. However, some rare exceptions to this general rule have been described and some patients with early-onset symptoms show an unexpectedly slow rate of progression of the disease. Long-lived patients were recently shown to have had congenital cataracts at birth. Still, the equation between severe type II CS and early-onset CS on the one hand and mild type III CS and late-onset CS on the other hand remains valid in most cases. Type II CS patients typically show symptoms at birth (congenital microcephaly, hypotonia or cataracts), severe feeding difficulties, very limited neurological development, severe contractures, severe growth failure and an average age of death of 5 or 6 years (mainly by respiratory failure or less frequently renal failure). Type I or moderate CS patients usually experience the first signs of the disease at the end of the first year of life (delayed developmental milestones or failure to thrive), often learn to sit, stand, walk a few steps, and speak a few words or simple sentences, show severe growth failure and progressive sensory and neurological impairment. Mean life expectancy is around 16 years and death mostly occurs from respiratory or renal failure. Type III CS patients may only manifest the first symptoms after several years of life (growth failure, learning difficulties, ataxia) and show mild developmental delay and limited growth failure. Cognitive decline, progressive cerebellar symptoms and hearing loss may be prominent symptoms in this subtype. Death occurs at a mean age of 30 years, mostly by respiratory failure (Nance and Berry, 1992; Natale, 2011; unpublished results). The most severe and the mildest ends of the clinical spectrum do not represent distinct entities either and should be considered in continuity with the other subtypes of CS. However, their clinical presentation is far enough from the canonical clinical picture of CS to require specific attention. Genetic and clinical evidence show that COFS syndrome should be regarded as the most severe and prenatal allelic variant of CS. The presentation of COFS syndrome is overlapping with CS type II (as defined supra) but the presence of arthrogryposis in COFS is in our mind an important and distinctive feature that should still justify keeping a specific label for these patients. Type II CS patients often show some degree of peripheral hypertonia at birth and progressive postnatal joint contractures, but the presence of arthrogryposis in COFS patients strongly suggests preexisting fetal akinesia and thus an even earlier onset of the neurological signs of the disease than in CS type II. Moreover COFS patients often never fulfill the diagnostic criteria for CS at any point during their life and specific diagnostic criteria have been proposed. Keeping COFS as a syndromic arthrogryposis and an allelic variant of CS may favor the recognition of the disease by clinicians and will subsequently facilitate molecular confirmation. At the opposite end of the spectrum, the identification of very mild or adult-onset cases of CS and UVSS patients also raises specific questions. Rare cases of very mild or adult-onset CS have been identified and linked to mutations in CSB (Hashimoto et al., 2008; Laugel, personal communication). Although they are again in continuity with the rest of the clinical spectrum, their clinical presentation is remarkable (and distinct from CS type III) by the complete absence of symptoms until adulthood (apart from short

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stature and skin photosensitivity in some cases) and thus by a completely normal intellectual development (Czeizel and Marchalko, 1995). The disease may only be revealed by progressive ataxia, early dementia and hearing loss in their 30s or 40s. At the very end of the spectrum, UVSS patients have been defined by a defect in the NER pathway and skin photosensitivity only, without any other clinical symptom. Yet, it may be questioned if these patients or some of these patients might experience cognitive decline, hearing loss or other very late-onset neurological symptoms in their 50s or 60s, or if they are truly constitutively free of extra-dermatological symptom. 4. Diagnostic criteria The criteria defined by Nance and Berry in 1992, before the molecular era, are still the standard reference for the clinical diagnosis of CS. In a personal series of 120 consecutive cases sent to our laboratory with a clinical suspicion of CS, among which 29 cases received a cellular and molecular confirmation of CS, these criteria had a specificity of 88% and a sensitivity of 90%. In our sample, the classical clinical criteria had a negative predictive value of 96% and a positive predictive value of 70% (unpublished results). We propose the following modifications to decrease the number of clinically false positive cases, and therefore improve specificity and positive predictive value. We propose that progressive microcephaly should be considered a major diagnostic criteria, that progressive growth failure would be more specific than undefined growth failure, that dental decay is not part of the disease per se and should be replaced by enamel hypoplasia as a minor diagnostic criteria and that the most distinctive facial feature of CS is the bilateral and progressive enophthalmia (Table 1). Should these modified criteria have been applied, specificity and sensitivity would have been 98% and 90% respectively. Positive predictive value and negative predictive value would have been 97% in our series. Brain imaging criteria might also be additionally helpful for clinicians: white matter hypomyelination and atrophy, cerebellar atrophy or hypoplasia, bilateral calcifications of the putamina had a great diagnostic value in our series. Malformations of the brain and especially abnormal cortical gyration have not been reported in confirmed CS and should be considered as a negative sign for the diagnosis of CS. Similarly, the presence of heart or kidney malformations should lead to question the diagnosis of CS. These improved criteria would still only apply for type I, II and III CS patients and would overlook the most severe and the mildest patients of the spectrum. COFS syndrome should be considered upon the following criteria: arthrogryposis, congenital microcephaly, congenital cataracts and/or microphthalmia, severe developmental delay, severe postnatal growth failure (Laugel et al., 2008). To better identify the underdiagnosed adult-onset cases at the

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other end of the spectrum, we propose that the combination of short stature or photosensitivity with progressive cognitive decline, progressive ataxia or progressive hearing loss should suffice to prompt DNA repair testing. It should however be underlined that if these criteria are very relevant for the full-blown clinical pictures of CS, they may not be met in the very early stages of the disease. The progressive course of CS will remain a diagnostic challenge in any case, as CS patients would only fulfill the required criteria several months or even years after the onset of the very first symptoms. In the early stages of the disease, requiring two minor criteria rather than three would increase the sensitivity of the clinical screening (but would decrease its specificity). Reduced recovery of RNA synthesis in fibroblasts after UV irradiation remains the gold standard to confirm the clinical suspicion of CS. If this assay confirms the defect in transcriptioncoupled repair or if fibroblasts are not available, a molecular analysis of the CS genes should be routinely proposed to all patients. Conventional genomic or cDNA sequencing is available in a few research or diagnostic laboratories worldwide and are progressively being replaced by next-generation sequencing strategies. Quantitative techniques are useful to detect large deletions (Ghai et al., 2011; Zhang et al., 2011). Prenatal testing is available by through functional or molecular testing (Conte et al., 2009; Kleijer et al., 2006). Preimplantation genetic diagnosis is theoretically feasible and available but has never been performed in CS families to the best of our knowledge. 5. Molecular genetics At least 78 different mutations are known in the CSB gene and 30 in the CSA gene to date, in more than 120 genetically confirmed CS patients reported in the literature or in our series (Figs. 4 and 5). Approximately 65% of these patients have mutations in the CSB gene (Laugel et al., 2010). The clinical spectra of CSA- and CSBlinked phenotypes are largely overlapping and there is no specific symptom or severity group linked to one gene in particular (Stefanini et al., 1996; Laugel et al., 2010). All CS subtypes can be associated with either one of the genes. However, these phenotypic spectra are not strictly identical and a significant number of CSB patients are affected by the most severe forms of the disease (COFS or CS type II) whereas CSA mutations seem to be linked preferentially to CS type I: in 101 CS patients for which the clinical subtype could be determined from the literature or from personal unpublished data (28 CSA patients and 73 CSB patients), 56% of the CSB patients met the criteria of CS type II, whereas 75% of the CSA patients could be classified as type I (Fig. 6). These results should however be repeated on a larger scale to confirm this trend. Most families have private mutations in the CS genes but some founder effects have been identified in specific populations (Laugel et al., 2010). In the CSA gene, c.618-1G>A (p.Ala207_Ser209del)

Table 1 Clinical diagnostic criteria of CS. Classical diagnostic criteria (Nance and Berry, 1992)

Modified criteria

Major criteria (mandatory)

Developmental delay Growth failure

Developmental delay Progressive growth failure Progressive microcephaly

Minor criteria (3 out of 5)

Cutaneous photosensitivity Pigmentary retinopathy and/or cataracts Sensorineural hearing loss

Cutaneous photosensitivity Pigmentary retinopathy and/or cataracts Progressive sensorineural hearing loss Enamel hypoplasia Enophthalmia

Dental caries Cachectic dwarfism Exclusion criteria

Absence of microcephaly

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Fig. 4. Updated linear map of the mutations in the CSB/ERCC6 gene. Missense mutations are represented above the protein, other point mutations and deletions are represented beneath the protein. Helicase motifs (I–VI), acidic domain (A) and nuclear localization signal (N) are shown in grey.

Fig. 5. Updated linear map of the mutations in the CSA/ERCC8 gene. Missense mutations are represented above the protein, other point mutations and deletions are represented beneath the protein. WD domains are shown in grey.

has been found in Portuguese/Brazilian patients, c.551-1G>A (p.Gly184AspfsX28) in Somali families, p.Asp93LeufsX26 in Japanese families, and c.313_314delGT (p.Val105ThrfsX6) in Brazilian families, c.598_600delinsAA (p.Tyr200LysfsX12) in North African families. In the CSB gene, a deletion in the 50 untranslated region is specifically found in the population of Reunion Island, c.544-1G>A (p.Glu182AsnfsX4) has been identified in unrelated French families, and c.2008C>T (p.Arg670Trp) and (p.Phe665_Gln723) in British families (Caucasian). All types of mutations have been detected in CS patients (missense, nonsense, frameshift, splicing mutations, as well as large deletions). In the CSA genes, all missense mutations are

located in the WD motifs, underlining the likely prominent role of these motifs which are needed to build circularized beta propeller structures and serve as a scaffold for protein–protein interactions. The fourth WD motif contains half of all CSA missense mutations. In the CSB gene, four missense mutations are clustered in a conserved amino acid stretch between position 670 and 687 in helicase motif III. All missense mutations and in-frame deletions are located within the two RecA-like domains of CSB. Novel large deletions involving the ERCC6 locus have been identified recently (Ghai et al., 2011; Zhang et al., 2011). Genotype–phenotype correlations remain elusive in CS patients. CS patients from the same family usually show similar

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Fig. 6. Distribution of the clinical subtypes of CS in CSA and CSB mutated patients.

phenotypes. There are only very few examples of unrelated CS patients who carry the same mutations on both alleles, but even in this case, the clinical phenotypes seem similar. In CSB patients, the role of a piggyBac3 (PGBD3) transposable element nested in intron 5 of the CSB gene has recently come under scrutiny (Bailey et al., 2012; Newman et al., 2008). It has been proposed that the fusion protein consisting of the first five exons of CSB and of the PGBD3 transposon could play a deleterious role in the absence of the full length CSB protein and could trigger the CS phenotype. According to this paradigm, mutations downstream of intron 5 would cause CS whereas mutations upstream of intron 5 would only cause the mildest forms of CS or UVSS. Very-late onset CS and UVSS patients have been reported with early-truncating mutations in CSB, in compliance with this paradigm but other CSB patients do not follow the same rule (Hashimoto et al., 2008; Laugel et al., 2010). Alternatively, the severity of the CS phenotype may also be dependent on other genetic or environmental factors. More clinical and molecular data will be needed to confirm this hypothesis. 6. Conclusion CS encompasses an expanding spectrum of clinical presentations from the most severe prenatal subtype to the adult-onset subtype. Accumulating clinical data show that all CS patients build a continuous spectrum of clinical severities. A specifically defective transcription-coupled repair serves as a unifying hallmark for all clinical subtypes. This huge clinical variability which has been unraveled since 1936 is a great challenge to describe the natural history of the disease but also to build homogenous patient groups for future clinical trials. A clear overview of all the clinical characteristics of the disease and the underlying mutations is a necessary prerequisite for any further progress towards the complete understanding of the disease. Acknowledgements The author wishes to express his profound gratitude to all CS patients and families for their participation, to Ms Cathy Obringer for her excellent technical support, to Dr Nadege Calmels for fruitful discussions and ongoing collaboration, to Dr Meriam Koob for the imaging studies and to all clinicians involved in the care of CS patients in Strasbourg University Hospital and worldwide. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.mad.2013.02.006. References Arenas-Sordo, M., Herna´ndez-Zamora, E., Montoya-Pe´rez, L.A., Aldape-Barrios, B.C., 2006. Cockayne’s syndrome: a case report. Medicina Oral, Patologia, Oral y Cirugia Bucal 11, E236–E238.

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