A case of Cockayne syndrome with unusually mild clinical manifestations

We present a mild case of Cockayne syndrome that was referred to us with an extreme sunburn at the age of 3. In early teens, although her cutaneous symptoms alleviated without any medications, she developed tremor and dysarthria. Neurological examination and brain imaging suggested demyelination disorders. The patient's cells indicated a reduced recovery of RNA synthesis, which was partially restored by the introduction of CSB (Cockayne Syndrome B)‐cDNA. In addition, her cells indicated a substantially reduced level of CSB protein. Despite the insidious progression of neurological symptoms, she gave birth to a child. Such mild cases of Cockayne syndrome may be misdiagnosed.

CS-related genes constitute or interact with Transcription factor II Human which is required to initiate transcription by RNA polymerase II and the nucleotide excision repair system that is involved in removing dipyrimidine photoproducts, major DNA lesions caused by UV exposure. In Japan, the majority of patients with CS have type I disease characterized by marked developmental retardation and emaciation, sensorineural deafness, typical dysmorphic features, including long nose, aged face, large ears, and sunken eyes; most of them die in their teens. Here, we report an extremely mild case of CS, manifested by photosensitivity and limited neurological symptoms.

| Case history
A female patient was first referred to Kobe University Hospital at the age of 3 with a history of extreme sunburns after minimum sun exposure without apparent neurological symptoms (Figure 1a). She was tentatively diagnosed with XP, and strict sun protection was encouraged. Thereafter, her cutaneous symptoms became milder, and she did not experience exaggerated sunburn reactions except one incident, when she visited Okinawa, the southernmost island of Japan, and was exposed to intense sunlight there. Although she did not show apparent mental or developmental/growth retardation, her scholastic ability was not high. At 11 years, she suddenly experienced intention tremor, dysgraphia, and dysarthria which did not progress and remitted for a while. They did not consult until 15 years. Neurological examination revealed saccadic eye movement, dysdiadochokinesis, intention tremor of hands, asynergy, and brisk deep tendon reflex, without joints dislocations. symptoms appeared and disappeared without apparent progress.
She lived without any medication, although her tremor, occasional stumbling, and dysarthria were often observed until 23 years.
When she again visited us at the age of 28, after 5 years of absence, dysarthria and tremor appeared more frequently and became apparent. She did not present other typical manifestations of CS, such as dwarfism, seizures, cataracts, caries, and retinal pigmentation. With age, she developed insidious mild cerebral atrophy, which was indicated by brain magnetic resonance imaging ( Figure 1b). She gave birth to a child at the age of 32, without any trouble during pregnancy (Table S1).

| Measurement of minimal erythema doses
Minimal erythema dose (MED) was examined using standard phototesting procedures with Sunlamps (Toshiba FL 32 S E-30), and erythema was assessed after 24, 48, and 72 h. All cells were maintained as previously described. 5 The study was approved by the Institutional Review Boards of the Medical Ethical Committees of Kobe University and Nagoya University, and was conducted according to the Declaration of Helsinki.

| Flow cytometry-based global genomenucleotide excision repair assay
The global genome-nucleotide excision repair (GG-NER) was determined by quantifying the removal of the pyrimidine-pyrimidone  photoproducts ((6-4)PP) as previously described. 6,7 Cells were irra- at 0 h was set as 100%, whereas that at 6 h was considered as the remaining (6-4)PP. Finally, the ratio of (6-4)PP removal was calculated.

| Recovery of RNA synthesis and genetic complementation test
Recovery of RNA synthesis (RRS) was assessed as previously described. 8 Fibroblasts were grown in a 96-well plate, irradiated with 13 J/m 2 of UV-C, incubated for 18 h, and labeled with ethynyl uridine (EU; Thermo Fisher Scientific) for 2 h. The incorporated EU was detected by reaction with Alexa Fluor 488 azide (Thermo Fisher Scientific) and 4′,6-diamidino-2-phenylindole (Dojindo).
Nuclear fluorescence intensity was measured using CellInsight (Thermo Fisher Scientific). RRS was presented as the percentage of the average fluorescence intensity in irradiated cells to that in nonirradiated cells.
For genetic complementation testing, patient-derived cells were infected with lentiviruses expressing wild-type CSA, CSB, or UVSSA genes prior to the RRS test, as previously described. 9

| Western blotting
Total proteins were extracted from the fibroblasts derived from the patient and reference cells. They were analyzed by Western blotting using anti-CSB antibodies (10R-1587; Fitzgerald) and anti-beta actin (sc-47 778; Santa Cruz Biotechnology).

| Minimal erythema doses
The MED for the patient was 55.8 mJ/cm 2 , which was slightly below the normal range for healthy Japanese individuals (60-140 mJ/cm 2 ) established in our institution. 10 MED at 48 and 72 h were same (27.9 mJ/cm 2 ) indicating a delay in the peak of UVinduced erythema.

| RRS and genetic complementation
The level of RRS in the patient's cells irradiated with 13 J/m 2 of UV-C was 30% of that in control cells (Figure 2a), but was restored to 90% after the introduction of CSB whereas that in cells introduced with UVSSA, CSA genes remained at 30% of that in control (Figure 2b).

| CSB expression
Western blotting analysis indicated that the CSB level in the patientderived fibroblasts was much lower than in 48BR (Figure 2c).

| DISCUSS ION
We present a mild case of CS in a Japanese female patient. She had a normal level of GG-NER but a reduced level of RRS, which suggested CS and this was confirmed by the genetic complementation test. The clinical signs were marked sunburns in early childhood and mild neurological symptoms starting in the early teens. Her MED was slightly reduced, and the UV erythema peak was delayed to 48-72 h after UV-B irradiation, which is typical for patients with a deficiency in transcription-coupled-NER (TC-NER). Therefore, she was tentatively diagnosed with TC-NER-deficient XP, but her visits to the hospital were discontinued because photosensitivity became less prominent. However, she consulted us at 15, claiming she expe-  (Table S2).
The genetic complementation test revealed that the patient had a deficiency in CSB, which was further confirmed by a substantially decreased CSB protein level. The reason for the mild symptoms of the patient remains to be investigated in future. As we occasionally encounter mild cases of CS, 11 it should be kept in mind that the disease may have a wide spectrum of clinical manifestations ranging from mild to severe. This report presents a mild case of CS with a decreased level of CSB. Further accumulation and analysis of CS cases are expected to elucidate the mechanisms behind the variations in CS severity.

CO N FLI C T O F I NTE R E S T
The authors declare that there are no conflicts of interest.