Keywords (3-5)
Cockayne Syndrome; Whole Genome Sequencing; ERCC6 ; DNA Repair
Molecular diagnosis of children with rare neurodegenerative and complex
multi-system disease is challenging especially when the phenotypic
presentation deviates from what is reported in the literature. Next
generation sequencing (NGS) techniques have improved diagnostic rates by
providing an unbiased diagnostic approach guided, but not limited by,
phenotype. Nevertheless, for rare disorders for which few variants may
be reported and the phenotypic spectrum may not yet be fully elucidated,
variants of uncertain significance (VUS) remain a vexing problem for NGS
interpretation. Functional studies are often unavailable to investigate
VUSs and even when available, are often beyond the diagnostic scope of a
clinical testing lab.
Cockayne syndrome (CS) is a spectrum diagnosis that is characterized by
growth deficiency, premature aging, pigmentary retinal degeneration as
well as multiple other neurologic and systemic findings. Standard CS
classification is based on the age at onset and severity of symptoms and
progression. The classifications are: CS-I, canonical; CS-II (including
C-Oculo-Facio-Skeletal (COFS) syndrome and Pena-Shokeir type 2 syndrome
(PS-2)), very severe; CS-III, mild; CS-IV, late onset; and xeroderma
pigmentosum–Cockayne syndrome (XP-CS) (Laugel, 2000).
CS-I is the classic form with symptom onset early in life. CS-II is more
severe with symptoms evident pre-natally or at birth. The majority of CS
cases are caused by mutations in ERCC6 or ERCC8 .ERCC6 mutations are most common in Caucasian CS patients, but in
other ethnic groups, ERCC8 mutations are more prevalent (Wilson,
2016). Mutations in ERCC6 are also seen in cerebro-oculular
facial-skeletal syndrome (COFS) (Laugel, 2008), and Ultraviolet
(UV)-Sensitive Syndrome (Horibata, 2004). Recessive mutations in five
additional DNA repair genes may cause CS (ERCC3/XPB, ERCC2/XPD,
ERCC5/XPG, ERCC4/XPF and ERCC1 ) in association with XP
(Kashiyama, 2013; Laugel, 2010) (Figure 1A).
All seven genes that if mutated can cause CS are critical for nucleotide
excision repair (NER), the pathway responsible for the repair of
helix-distorting DNA adducts including UV-induced photolesions (Scharer,
2013). NER consists of two sub-pathways: global genome NER (GG-NER) and
transcription-coupled NER (TC-NER) (Figure 1A) (Gillet, 2006;
Hanawalt,
2008). CSA (ERCC8 ) and CSB (ERCC6 ) are key TC-NER factors
that participate in the repair of transcription-blocking DNA lesions
that stall RNA polymerase II (Scharer, 2013). Once the lesion is
recognized in the genome or transcribed areas of the genome, the two
sub-pathways converge, utilizing additional proteins to unwind and
stabilize the DNA around the lesion, enabling two endonucleases to
excise the lesion as part of a single-stranded oligonucleotide. The gap
left after the removal of the damaged oligonucleotide is filled by
templated DNA synthesis by the replication machinery. Generally
speaking, mutations in genes needed for GG-NER cause xeroderma
pigmentosum, a cancer predisposition syndrome, while mutations in genes
needed for TC-NER cause CS. However, the impact of a particular mutation
or sequence variant on protein expression and function complicates this
generalization (Laugel, 2010).
The only CS symptom clearly caused by defective NER is photosensitivity.
Despite decades of research, there is no clear explanation for the
mechanistic basis of CS. A variety of roles outside of TC-NER have been
proposed for CSA and CSB (Wilson, 2016), though at present none may be
conclusively mechanistically linked to the spectrum of symptoms observed
in CS patients. Roughly 65% of patients with CS have mutations inERCC6 (Laugel, 2000). The true prevalence of CS is unknown. A
study in 2008 estimated the incidence at 2.7 per million births in
western Europe (Wilson, 2016), though this is likely an underestimate in
part due to reliance on the presence of photosensitivity as a diagnostic
feature (Kleijer, 2008). Genomic sequencing has allowed expansion of the
phenotype as in this case where the presentation is atypical.
The patient (INE4CC) is a 7-year-old Korean female with multisystem
disease including: failure to thrive, congenital microcephaly, global
developmental delay with motor and language regression, tremor, ataxia,
cardiomyopathy, renal dysfunction, chronic lung disease with oxygen
requirement, diabetes, hypothyroidism and hypertension. Pregnancy was
unremarkable until 36 weeks of gestation when intrauterine growth
retardation was identified, prompting an uncomplicated delivery by
Cesarean section at 37 weeks of gestation. Birth weight was 2126 grams
(z=-2.37) and birth length was 43.2 cm (z=-2.87). Weight gain was
appropriate for the first 6 months of life while breast fed. Poor weight
gain was noted after transition to formula due to poor oral intake and a
G-tube was placed at 23 months. Sitting was attained at 6 months and
pulling to stand at 12 months. Delays in development were noted at 1
year of age at which time she had difficulty with fine motor movements
due to tremor. Independent ambulation was achieved at 29 months. Though
she spoke a single word - “dada” at nine months, by 33 months she had
only a 40-word vocabulary and was not combining words. Regression in
motor skills was noted at age 3 years in association with a two-week
hospitalization for RSV infection, with increased tremor and progressive
difficulty with gait and balance. Following a complicated
hospitalization at age 4.5 years, including multiple infections and a
prolonged mechanical ventilation, she lost the ability to ambulate.
Since that time, she made forward developmental progress and at age 7
was able to walk short distances with a walker and was able to speak in
short sentences. She has had poor somatic and cerebral growth with a
steady decline in her z-scores for linear growth with most recent
measurement at -8. At 7 years her weight z score was
~3.3, and her head circumference z score was
~3.5. Her course has been further complicated by
hypothyroidism (onset age 3 years), insulin-dependent diabetes
(diagnosed at age 4 years), cardiomyopathy, fibrotic kidney disease,
chronic lung disease with oxygen requirement, hypertension, dental
caries, and movement disorder characterized by action-induced tremor and
paroxysmal tremulous episodes. Audiogram at age 6 showed bilateral
absent oto-acoustic emissions and reduced compliance on tympanometry;
visual reinforced audiometry and conditioned play audiometry were felt
to be unreliable. Dilated ophthalmologic evaluation at ages 3 and 4
years showed bilateral hyperopia with secondary refractive amblyopia, no
early cataracts and normal appearing retinas. Dilated ophthalmologic
evaluation, with a different pediatric ophthalmologist (SLR), at age 5
in the office setting and later under anesthesia, revealed decreased
lacrimation, small corneas with scarring consistent with exposure
keratopathy, prominent Schwalbe’s line with normal intraocular pressure,
severe hyperopia (farsightedness) requiring eyeglass correction, optic
nerve atrophy and foveal hypoplasia. Additionally, bilateral reticulated
retinal pattern with central vascular sheathing of both retinal
arterioles and venules was observed. Neurologic examination at age 7
revealed a small child able to speak in phrases and follows simple
commands. She displayed gaze-evoked nystagmus in all fields of gaze.
There was increased appendicular tone, hypoactive reflexes, titubation
and tremor while reaching for objects. She was able to stand with
support.
At age two, brain MRI showed inferior vermian hypoplasia with cerebellar
atrophy, diffuse white matter signal abnormality and mineralization of
the basal ganglia. Repeat brain MRI at age four showed progression of
supratentorial volume loss. Computed Tomography Scan at age 5 showed
dense calcification of the globi pallidi and lentiform nuclei and
calcification of the parietal, occipital and frontal cortices, with
stable vermian hypoplasia and supratentorial volume loss (Figure 1B).
Radiographic review identified diffuse platyspondyly and acetabular
dysplasia. Extensive laboratory evaluations including whole exome
sequencing, electromyogram nerve conductions and muscle biopsy were
unremarkable.
Whole genome sequencing (WGS) initially revealed two variants of
uncertain significance (VUS) in the
ERCC6 gene (NM_00124.3): a
maternally inherited c.1583G>A, p.Gly528Glu missense
variant and a paternally inherited c.-15+3G>T upstream
intronic variant (Supplemental Figure 1). Both variants were absent from
the gnomAD population database. At the time of initial analysis, both
variants were unreported in the literature. However, in silicoprediction algorithms were suggestive of pathogenicity with the
p.Gly528Glu variant predicted to be damaging by SIFT and Polyphen, and
the c.-15+3G>T variant was predicted to alter splicing by
multiple splicing algorithms utilized by Alamut (Interactive
Biosoftware, Rouen, France). Based on the predicted consequence of both
variants and the phenotypic overlap between the patient and CS, the
family was approached to discuss the possibility of further functional
testing. Patient dermal fibroblast line was evaluated for the expression
of ERCC6 and CSB protein levels (Figure 2A and B). ERCC6expression was significantly reduced in INE4CC patient cells compared to
control C5RO fibroblasts (Figure 2A) and immunoblotting revealed nearly
undetectable CSB protein in the patient cells (Figure 2B). These data
suggest both ERCC6 variant alleles contribute to significantly
reduced ERCC6 /CSB levels.
ERCC6 is required for TC-NER. Thus, diagnosis of CS requires
measurement of NER and more specifically TC-NER. To determine if the
patient’s sequence variants had a functional impact on NER, unscheduled
DNA synthesis (UDS) was measured following UV-irradiation of the patient
cells. UV-induced UDS is a direct measure of GG-NER capacity. Defective
GG-NER is pathognomonic for XP and defective TC-NER is pathognomonic for
CS and trichothiodystrophy, whereas mutations in common components of
both GG-NER and TC-NER can result clinically in CS, XP, COFS, or even
Fanconi anemia (Kashiyama, 2013). UDS was not impaired in INE4CC patient
fibroblasts compared to a normal control (C5RO used to set normal levels
of NER at 100%) (Figure 2C). Cells from a patient with mutations in
XPF, known to have an NER capacity of ~5% (XP51RO),
were utilized as an NER-deficient control (Niedernhofer, 2006). Lack of
an NER-defect in the patient fibroblasts is consistent with a diagnosis
of CS.
Impaired recovery of RNA synthesis (RRS) post-UV irradiation of cells is
pathognomonic for a TC-NER defect, which is present in CS patients.
Expression of the housekeeping genes DHFR and GAPDH were
measured 6 and 24 hrs post-UV irradiation in the patient cells (INE4CC)
and compared to C5RO and XP51RO fibroblasts as well as those from a CS
patient (CS20LO) (Kashiyama, 2013). In all cell lines, DHFR andGAPDH mRNA was significantly reduced 6 hr post-UV irradiation
compared to sham-irradiated cells (Figure 2D). However, in
TC-NER-proficient C5RO cells, by 24 hrs, mRNA levels had recovered to
levels equivalent to unirradiated cells. Similar results were obtained
with XP51RO fibroblasts derived from a NER-defective patient with no
clinical signs of CS. As expected, RNA synthesis recovery was
significantly reduced in the CS patient fibroblasts (CS20LO). mRNA
expression in the patient (INE4CC) fibroblasts also failed to return to
normal levels by 24 hrs post-UV irradiations, indicating impaired RRS,
consistent with a TC-NER defect and CS (Figure 2D).
Re-curation of the variants after functional testing revealed the
p.Gly528Glu was recently reported in a patient with Cockayne syndrome in
the compound heterozygous state (Calmels, 2018), and the variant was
upgraded to pathogenic based on American College of Medical Genetics
(ACMG) guidelines (PM2, PP2, PP3, PS3, PP4). The c.-15+3G>T
remained unreported in the literature. However, with the results from
functional studies and re-curation of the variant, the
c.-15+3G>T variant was re-classified to likely pathogenic
(PM2, PP3, PS3, PM3, PP4). The new diagnosis was communicated to the
multiple specialists involved in the patient’s care and to the family.
The differential diagnosis for patients with neurodegenerative symptoms
is broad. Determining etiology is further hampered when classic
phenotypic features are absent or have not yet emerged. Next generation
sequencing techniques have addressed this problem by providing an
unbiased diagnostic approach guided, but not limited, by phenotype. Our
patient displays many features characteristic of CS-II, but several
common features that may have led to diagnosis were absent (Supplemental
Table 1). Family did not initially report photosensitivity, though in
retrospect, after the diagnosis was made, they noted that the child
sunburns easily. In addition, the child did not develop cataracts or
characteristic cachexic birdlike facies. These features are present in
62%, 55%, and 70% of CS-II patients respectively (Kou, 2018). She
does display early signs of pigmentary retinopathy seen in 47% of
patients, though full descriptive identification of that retinal finding
required examination under anesthesia due to decreased cooperation in
keeping with cognitive and behavioral aspects of the disease. To our
knowledge, the additional ophthalmologic findings of prominent
Schwalbe’s line and central retinal vascular sheathing have not been
previously reported in Cockayne Syndrome and microcornea has been
reported in only a single patient (Nance, 1992). In addition, she is
less severely developmentally delayed than other children with CS-II as
she achieved the ability to ambulate and speak in sentences. A
previously non-diagnostic trio whole exome sequence further hampered
diagnosis in this case.
Whole Genome Sequencing done as part of a research protocol identified
two variants of uncertain significance (VUS) in ERCC6 including
an upstream intronic variant that would not have been captured by
traditional exome sequencing tests, or potentially not reported due to
lack of variant evidence or patient phenotypic overlap. The
pathogenicity of these variants was initially uncertain for multiple
reasons including: absence of a classical phenotype, novelty of variants
and absence of evidence of a functional impact of the variants on the
gene product. Molecular analysis demonstrated impaired ERCC6expression and scant abundance of CSB protein in patient fibroblasts
(INE4CC) relative to the normal control cell line. Pathogenicity was
established based upon functional studies demonstrating normal UDS but
reduced recovery of RNA synthesis after UV-irradiation in patient cells,
pathognomonic for a diagnosis of Cockayne Syndrome. This case report
highlights how WGS in conjunction with functional testing can lead to a
clinically unexpected diagnosis. Evaluation and confirmation of
pathogenicity of VUS remains a vexing challenge to clinicians and
diagnostic sequencing laboratories.
A recent article summarized the molecular and clinical findings of 85
patients with mutations in ERCC6 (Calmels, 2018). The majority of
the mutations identified to date are truncation mutations and although
no strong genotype-phenotype correlation is observed, a higher
proportion of severely affected patients had mutations in ERCC6compared to ERCC8 . Our study demonstrates that intronic variation
may account for a yet to be determined percentage of pathogenic variants
in ERCC6 . This study supports the joint approach of molecular
analysis in conjunction with robust functional testing and highlights
the future promise of the additional value of whole genome sequencing
compared to whole exome sequencing.