Atypical features in two adult patients with Cockayne syndrome and analysis of genotype–phenotype correlation : Chinese Medical Journal

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

Cheng, Haoling1; Chen, Dianfu2; Wu, Zhiying2; Wang, Ning1,3

Editor(s): Ni, Jing

Author Information
Chinese Medical Journal ():10.1097/CM9.0000000000002245, April 28, 2023. | DOI: 10.1097/CM9.0000000000002245

To the Editor: Cockayne syndrome (CS; Mendelian Inheritance in Man # 133540, 216400) is a rare autosomal recessive neurodegenerative disorder described by Edward Cockayne in 1936.[1] The prevalence of CS is 2.7 per million live births,[2] and the disease is probably underdiagnosed. The major clinical features are progressive growth failure and microcephaly as well as other characteristics such as a “cachectic dwarfism” appearance with sunken eyes, cutaneous photosensitivity, mental retardation, demyelinating peripheral neuropathy, pigmentary retinopathy, cataracts, deafness, dental anomalies, and premature death.[1,3] There is considerable variation in the severity of the disorder, leading to classification into three main types.

Given the high heterogeneity of symptoms, precise diagnosis of CS relies mainly on molecular genetic testing. CS has been found to be mainly caused by biallelic pathogenic variants in ERCC6 (65% of the cases) and ERCC8 (35% of the cases).[4] The ERCC6 gene, also known as Cockayne Syndrome Group B (CSB), is comprised of 21 exons that encode a 168 kDa protein named CSB that contains an acidic domain, a glycine-rich region, two putative nuclear localized signal sequences, and seven characteristic helicase adenosine triphosphatase domains.[4]

To date, over 150 variants in the ERCC6 gene have been identified worldwide (Human Gene Mutation Database [HGMD®] Professional 2021.1, Here, we report two adult CS siblings with mild phenotypes from a consanguineous Chinese family.

Two siblings and their biological parents were recruited from the Department of Neurology, Second Affiliated Hospital of Zhejiang University, School of Medicine in 2017. The clinical evaluations and neurological examinations were performed by at least two senior neurologists. Written informed consent was obtained from all subjects and ethics committee approval was provided by local hospital ethics committees (protocol code 2015–048, approved on 11 August 2015).

Genomic DNA was extracted from this family for whole-exome sequencing, and subsequent annotation and analysis were performed according to our previously reported protocol.[5] By co-segregation and phenotype analysis, we identified a presumed splice site variant (c.543+4A>T) in ERCC6 (NM_000124). The variant resided on the splicing donor site in intron 3 and has never been previously reported in public databases or the literature. The genotype for ERCC6 c.543+4A>T was co-segregated with the phenotype in this family [Figure 1A]. The genotype was also confirmed by Sanger sequencing using an ABI 3500xl Dx Genetic Analyzer (Applied Biosystems, Forster City, USA), and was homozygous in both affected siblings and heterozygous in the parents [Figure 1B].

Figure 1:
Biallelic ERCC6 splicing mutations c.543+4A>T were identified in a Chinese CS family. (A) The pedigree shows segregation of variants confirmed by Sanger sequencing, and the arrow indicates the proband. (B) Chromatograms of the c.543+4A>T variant in the family and normal control, the arrow indicates the site of the variant. (C, D) Splicing alteration was identified by a minigene assay: (C), cDNA products were separated by agarose gel electrophoresis, Lane 1 was the marker; Lane 2 was the WT (263 bp + 121 bp [exon 3]) and Lane 3 was the c.543+4A>T variant (263 bp); (D), Schematic diagram of minigene construction and sequencing results for the bands. SD and SA were two exons of the pSPL3 vector. (E) Clinical features and brain MRI of the proband: E1, the sunken eyes; E2, the normal teeth; E1 and E3, many abnormal dermal pigmentations on face, neck, and arms; E4, mild cerebral atrophy; E5, enlargement of the ventricles; E6, hippocampus atrophy; E7, abnormal signals of bilateral thalamic. (F) Clinical features and brain MRI of the proband's sister: F1, the sunken eyes; F2, decayed incisor teeth and anomalies of tooth size and shape; F1 and F3, more severe hyperpigmentation on the face, neck, and arms; F4, cerebral atrophy and demyelinating leukoencephalopathy; F5, enlargement of the ventricles; F6, hippocampus atrophy; F7, abnormal signal on the right parietal surface. cDNA: Complementary DNA; CS: Cockayne syndrome; MRI: Magnetic resonance imaging; MU: Mutant; SA: Splice acceptor; SD: Splice donor; pSPL3: Phosphorylated squamosa promoter binding protein-like 3; WT: Wild-type.

To examine the potential pathogenic effect of the novel variant c.543+4A>T in ERCC6, we carried out a minigene splice assay with the exon trapping cloning vector phosphorylated squamosa promoter binding protein-like 3 (pSPL3). Exon 3 (121 bp) and flanking 5′ (266 bp) and 3′ (519 bp) intronic sequences in the ERCC6 gene were amplified from genomic DNA of the proband [II-2, Figure 1A] and control, and then subcloned into the pSPL3 expression vector. Following forty-eight hours post-transfection of HEK293T cells with mutant and control minigene plasmids, total RNA was extracted and reversely transcribed into complementary DNA. Agarose gel electrophoresis of polymerase chain reaction products showed that the band of the mutant was smaller than that of the wild-type strain [Figure 1C]. Sequencing analysis revealed a loss of exon 3 (121 bp) of ERCC6 in the mutant minigene [Figure 1D]. These findings confirmed the impact of c.543+4A>T on pre-mRNA.

The siblings were born at full term by spontaneous vaginal delivery to healthy parents, and birth weight and height scores were normal. Both had a typical neonatal period and early childhood development. The major clinical features of CS patients are summarized in Supplementary Table 1 []. The proband [II-2; Figure 1A] is a 30-year-old man. At the age of 6, action related tremor of his hands was observed bilaterally, especially when he was nervous, and it was relieved at rest. Since then, he has been stable without any other symptoms or aggravation of tremors. On neurological examination at the age of 26, his height and weight were 168 cm and 49 kg, respectively, and both were below the mean (169.7 cm, 69.6 kg) according to the “Report on the Nutrition and Chronic Disease Status of Chinese Residents (2020),” but his head circumference was 55 cm, which was in the normal range. He had an unusual face with sunken eyes [Figure 1E1], but his teeth were normal [Figure 1E2]. Many abnormal dermal pigmentations in the form of macules were observed on the exposed parts of the body [Figure 1E1, E3]. He also had horizontal nystagmus, increased deep tendon reflexes in the lower extremities, and difficulties walking in a straight line. Examination of vision and hearing revealed normal results. In addition, brain magnetic resonance imaging (MRI) showed mild cerebral and hippocampal atrophy, enlargement of the ventricles, and abnormal bilateral thalamic signals [Figure 1E4–7]. Electroencephalography was normal. A nerve conduction study showed demyelinating peripheral neuropathies [Supplementary Table 2,].

The affected sister [II-1; Figure 1A] is a 33-year-old female with more severe clinical features. Unlike her brother, she presented with tremor of the hands regardless of action or rest at the age of 8, and it was aggravated when she was nervous or excited. She was diagnosed with paranoid schizophrenia when she was 26 years old because she experienced self-talk, auditory hallucinations, and persecutory delusions. The psychiatric symptoms were controlled well by treatment with olanzapine, aripiprazole, and escitalopram. At the age of 29, she experienced an aggravation of tremors in both her jaw and hands, which was not alleviated even after receiving treatment with trihexyphenidyl. At the age of 30, her height and weight were 147 cm and 42.5 kg, respectively. Both height and weight were below the mean (158.0 cm, 59.0 kg), and her head circumference was 52 cm, which falled in the normal range. She had sunken eyes, dental caries, and anomalies of tooth size and shape [Figure 1F1, F2]. In addition, hyperpigmentation was more severe on her exposed skin [Figure 1F1, F3] than on her brother. Neurological examinations revealed dysarthria, lack of tendon reflexes, mental impairment (the Mini-mental State Examination score was 17, the Montreal Cognitive Assessment score was 10), and cerebellar ataxia. Brain MRI revealed marked cerebral and hippocampal atrophy, sulcus widening, dilated ventricles, demyelinating leukoencephalopathy, and abnormal signals on the right parietal surface [Figure 1F4–7].

To investigate the genotype–phenotype correlation, we classified patients into two groups: patients harboring splicing mutations and patients harboring nonsplicing mutations. In reviewing the previous investigations from January 1, 1992 to April 10, 2021, we discovered that 47 patients carried one (25 cases) or two (22 cases) ERCC6 splicing mutations with detailed information on genotype and phenotype [Supplementary Table 1,]. In addition, we ascertained that 48 and 46 patients carried nonsplicing mutations, respectively, from the studies of Calmels et al[3] and Laugel et al.[4] Including the two patients in our study, the numbers of patients with splicing mutations (49 patients in total) assigned to CS types I, II, and III were 7, 21, and 19, respectively, and two patients were difficult to classify [Supplementary Table 3,]. Combining the results from the above two studies, the numbers of patients with nonsplicing mutations (94 patients in total) assigned to CS types I, II, and III were 31, 49, and 7, respectively, and no CS typing information was available for seven patients [Supplementary Table 3,]. After comparison analysis, splicing mutations in ERCC6 were significantly associated with a milder CS type (CS III) than nonsplicing mutations (P <0.001), but nonsplicing mutations were more related to the classical phenotype (CS type I) than splicing mutations (P = 0.011) [Supplementary Table 3,]. To further explore the diversity of phenotypes, a comparison of the detailed clinical features between the two groups was conducted. The percentage of mental retardation and arthrogryposis in the nonsplicing group tended to be greater, but no statistically significant differences were observed, similar to other clinical features [Supplementary Table 3,].

The siblings presented in this study display many features consistent with CS, including special faces, growth failure, cutaneous photosensitivity, and neurological dysfunction (demyelinating leukoencephalopathy, brain atrophy, and demyelinating peripheral neuropathy). The relatively late onset and mild disease course allow them to live well into adulthood, which is most consonant with CS type III. Intriguingly, the sister had later onset but developed a more severe course of disease, and she had more symptoms than the proband, such as psychosis, mental retardation, and resting tremor. These symptoms appeared only in the sister but did not affect the proband. It was also reported previously that patients displayed a variation in clinical phenotype even though they carried the same ERCC6 mutation and were from the same family.[2,3] Therefore, in addition to CSB dysfunction, other unknown genetic and/or environmental factors may be responsible for the diverse clinical symptoms and disease severity of CS. Moreover, psychiatric symptoms in the sister had never been reported in CS cases with previously identified causative mutations in ERCC6. In 1971, Crome and Kanjilal[6] reported a clinically diagnosed CS patient who gradually developed behavior disturbances related to delusions of persecution and auditory and visual hallucinations after 16 years of age but lacked a genetic diagnosis. However, it is still unclear whether psychiatric symptoms are a new phenotype of CS, which requires more research to determine.

Among the 157 variants reported in ERCC6 from HGMD, splice site mutations account for 14% of the variants. Based on the summary and comparison of the clinical features of CS patients with or without splicing mutations, we demonstrate that splicing mutations appear to be more frequently associated with a mild phenotype (CS type III) than nonsplicing mutations. One hypothesis is that splicing mutations would represent a partial loss of function, which accounts for the milder phenotype.[7] However, a total loss of CSB protein has been associated not only with a mild phenotype consisting of photosensitivity without short stature or any neurologic findings, but also with a severe phenotype,[4] confounding the clear pathogenicity mechanism of the genotype–phenotype correlation of CS.

In conclusion, we first reported a novel splicing mutation (c.543+4A>T) of ERCC6 in two Chinese adults with a mild form of CS and further investigated the correlation between genotypes of ERCC6 and phenotypes of CS. This study not only expands but also provides valuable insights into the genotype–phenotype correlation of ERCC6 in CS patients.


The authors sincerely thank all the participants.


This research was supported by grants from the National Natural Science Foundation of China (No. U2005201 to Wang N. and No. 82071260 to Wu Z.Y.).

Conflicts of interest



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