Association Between Invisible Basal Ganglia and ZNF335 Mutations: A Case ReportRieko Sato, MD, a, b Jun-ichi Takanashi, MD, PhD, c Yu Tsuyusaki, MD, d Mitsuhiro Kato, MD, PhD, e, f Hirotomo Saitsu, MD, PhD, g Naomichi Matsumoto, MD, PhD, g Takao Takahashi, MD, PhDb

aDepartment of Pediatrics, National Hospital Organization

Tokyo Medical Center, Tokyo, Japan; bDepartment of

Pediatrics, Keio University School of Medicine, Tokyo,

Japan; cDepartment of Pediatrics, Yachiyo Medical Center,

Tokyo Women's Medical University, Yachiyo, Japan; dDepartment of Child Neurology, Kanagawa Children’s

Medical Center, Yokohama, Japan; eDepartment of

Pediatrics, Yamagata University Faculty of Medicine,

Yamagata, Japan; fDepartment of Pediatrics, Showa

University School of Medicine, Tokyo, Japan; and gDepartment of Human Genetics, Yokohama City University

Graduate School of Medicine, Yokohama, Japan

Dr Sato was an attending pediatrician, designed

the study, collected clinical information, and wrote

the paper; Dr Takanashi designed the study and

examined MRI; Dr Tsuyusaki was an attending

pediatrician and collected clinical information;

Dr Kato performed initial genetic testing, including

Sanger sequencing; Dr Saitsu performed exome

sequencing and in silico analysis; Dr Matsumoto

designed the study; Dr Takahashi designed the

study and reviewed the manuscript; and all authors

approved the fi nal manuscript for submission and

agree to be accountable for all aspects of the study.

DOI: 10.1542/peds.2016-0897

Accepted for publication May 24, 2016

Address correspondence to Takao Takahashi, MD,

PhD, Department of Pediatrics, Keio University

School of Medicine, 35 Shinanomachi, Shinjuku-ku,

Tokyo 160-8582, Japan. E-mail: ttakahashi@keio.jp

PEDIATRICS (ISSN Numbers: Print, 0031-4005; Online,

1098-4275).

Copyright © 2016 by the American Academy of

Pediatrics

FINANCIAL DISCLOSURE: The authors have

indicated they have no fi nancial relationships

relevant to this article to disclose.

ZNF335, a nuclear zinc finger protein,

is essential for methylation and

expression of brain-specific genes. 1

ZNF335 was reported in 2012 as a

causative gene for microcephaly. 1

Yang et al 1 identified homozygous

ZNF335 mutations in 7 microcephaly

patients of 1 consanguineous Arab–

Israeli pedigree, suggesting that it

demonstrates autosomal recessive

inheritance. Because only this single

pedigree has been reported to date,

the key clinical features associated

with ZNF335 mutations remain

unknown. In this study, we report a

second family with ZNF335 mutations

and describe the associated clinical

features.

CASE PRESENTATION

The proband of this study was a

33-month-old girl. She was the

first child of nonconsanguineous

Japanese parents. No family members

had microcephaly or presented

developmental delay. Prenatal history

was unremarkable. She was delivered

vaginally at full term without

asphyxia. Her birth weight was 3030 g

(+0.9 SD), length was 49.5 cm (+0.5 SD),

and head circumference was 32.0 cm

(−0.6 SD). She had a systolic murmur

and was diagnosed with a ventricular

septal defect (3 mm).

At 3 months of age, she was admitted

to our hospital because of an afebrile

abstractZNF335 was first reported in 2012 as a causative gene for microcephaly.

Because only 1 consanguineous pedigree has ever been reported, the key

clinical features associated with ZNF335 mutations remain unknown. In

this article, we describe another family harboring ZNF335 mutations. The

female proband was the first child of nonconsanguineous Japanese parents.

At birth, microcephaly was absent; her head circumference was 32.0 cm

(−0.6 SD). At 3 months, microcephaly was noted, (head circumference,

34.0 cm [−4.6 SD]). Brain MRI showed invisible basal ganglia, cerebral

atrophy, brainstem hypoplasia, and cerebellar atrophy. At 33 months, (head

circumference, 41.0 cm [−5.1 SD]), she had severe psychomotor retardation.

After obtaining informed consent from her parents, we performed exome

sequencing in the proband and identified 1 novel and 1 known mutation

in ZNF335, namely, c.1399T>C (p.C467R) and c.1505A>G (p.Y502C),

respectively. The mutations were individually transmitted by her parents,

indicating that the proband was compound heterozygous for the mutations.

Her brain imaging findings, including invisible basal ganglia, were similar to

those observed in the previous case with ZNF335 mutations. We speculate

that invisible basal ganglia may be the key feature of ZNF335 mutations.

For infants presenting with both microcephaly and invisible basal ganglia,

ZNF335 mutations should be considered as a differential diagnosis.

CASE REPORTPEDIATRICS Volume 138 , number 3 , September 2016 :e 20160897

To cite: Sato R, Takanashi J, Tsuyusaki Y, et al.

Association Between Invisible Basal Ganglia and

ZNF335 Mutations: A Case Report. Pediatrics. 2016;

138(3):e20160897

by guest on August 27, 2018www.aappublications.org/newsDownloaded from

SATO et al

seizure. Microcephaly, with a head

circumference of 34.0 cm (−4.6

SD), was noted; her weight was

4685 g (−2.0 SD) and her length

was 56.4 cm (−1.6 SD) ( Fig 1). Her

facial characteristics included a low

sloping forehead and micrognathia.

Neurologic examination showed

increased muscle tone, dystonic

posture, few voluntary movements

with an involuntary sucking-like

movement, and no eye tracking

movement. Phenobarbital was

effective for controlling the seizure.

An EEG showed no abnormal

findings. Brain MRI revealed invisible

basal ganglia, hypomyelination, and

brainstem hypoplasia ( Fig 2). We

performed several examinations for

differential diagnosis of microcephaly

and complications. Congenital

infections, such as cytomegalovirus,

toxoplasma, rubella, and measles,

were excluded by measuring

antibody titers. Mitochondrial

diseases and metabolic diseases were

not suspected based on the levels of

serum lactate and urine organic acid

analysis. Her auditory brainstem

response threshold was 80 dB in

both ears, which was considered

as moderate sensorineural hearing

impairment. Ophthalmologic

examination showed a subtle

corneal scar because of entropium

ciliarum. Cardiac ultrasound

sonography revealed closure of the

ventricular septal defect. Abdominal

ultrasonography showed no

abnormalities in the kidney, liver,

and spleen.

At 5 months of age, she could

not hold up her head. Brain MRI

showed invisible basal ganglia,

hypomyelination, brainstem

hypoplasia, and cerebellar atrophy

( Fig 2). At 9 months of age, her

weight was 6785 g (−2.0 SD), length

was 69.0 cm (−1.3 SD), and head

circumference was 37.5 cm (−4.4 SD)

( Fig 1). We introduced tube feeding

because she could not suck well and

poor weight gain was apparent. At

16 months of age, brain MRI revealed

progressive cerebral and cerebellar

atrophy and brainstem hypoplasia

( Fig 2). At 25 months of age, she

underwent Nissen fundoplication and

gastrostomy due to gastroesophageal

reflux. Later, she required

cardiopulmonary resuscitation 3

times, because of bradycardia or

asystole, while receiving oral and

nasal suctioning. At 33 months of

age, her length was 82.0 cm (−2.5

SD), weight was 11.5 kg (−0.8 SD),

and head circumference was 41.0

cm (−5.1 SD) ( Fig 1). She presented

with no voluntary movement,

showed spastic paralysis, and could

not speak any words. Levodopa

treatment improved the rigidity in

her extremities. Partial epilepsy with

ocular deviation was well controlled

with lamotrigine.

Mutation Analysis

The family pedigree is shown in Fig

3A. After obtaining written informed

consent from the proband’s parents,

we extracted genomic DNA from

peripheral blood samples of both

the proband and the parents. No

mutations in ARX and NKX2-1 were

found by Sanger sequencing of the

proband’s DNA. When we performed

exome sequencing of ZNF335 in the

proband, we identified 2 missense

mutations in exon 9 of the gene,

namely, c.1399T>C and c.1505A>G.

These 2 nucleotide substitutions,

confirmed by Sanger sequencing,

are predicted to lead to amino acid

substitutions of cysteine to arginine

at codon 467 and tyrosine to cysteine

at codon 502, respectively ( Fig 3B).

These mutations were transmitted

individually from her father and

mother, respectively, indicating

that the proband was compound

heterozygous for these mutations

( Fig 3A).

These mutations cause amino acid

substitutions in the zinc finger

domain of the protein ( Fig 3B). Both

Cys467 and Tyr502 residues are

evolutionarily conserved across

species ( Fig 3C). c.1399T>C is

not present in the 1000 Genomes

database, the Human Genetic

Variation database, or the National

Center for Biotechnology Information

dbSNP database, whereas c.1505A>G

is observed in 1 in 121 412 alleles,

according to the dbSNP144. In silico

analyses with Polyphen-2 (Harvard

University, Cambridge, MA), SIFT (J.

Craig Venter Institute, La Jolla, CA),

e2

FIGURE 1Growth chart of the proband. The proband had postnatal microcephaly and appropriate growth.

by guest on August 27, 2018www.aappublications.org/newsDownloaded from

PEDIATRICS Volume 138 , number 3 , September 2016

and Mutation Taster (Charité, Berlin,

Germany) predicted that p.C467R

and p.Y502C are pathogenic (data not

shown).

DISCUSSION

We have identified compound

heterozygous ZNF335 mutations,

consisting of a novel mutation

p.C467R and a known mutation

p.Y502C, in an infant with

microcephaly, spastic paralysis,

afebrile seizure, and severe

psychomotor retardation.

Perioperative management of this

patient during Nissen fundoplication

and gastrostomy has been reported

by Nishida et al. 2 To our knowledge,

this is only the second reported

pedigree with microcephaly and

ZNF335 mutations.

MRI findings in our patient, including

invisible basal ganglia, were similar

to those observed in the previous

report of an individual with ZNF335

mutations. 1 It is noteworthy that the

diagnosis of invisible basal ganglia in

both our case and the previous case

was made in infancy. To the best of

our knowledge, hypomyelination

with atrophy of the basal ganglia and

cerebellum (known as H-ABC), is the

only disease showing a decreased

basal ganglia size in infancy. 3 Thus,

it is important to consider ZNF335

mutations as a differential diagnosis

when encountering infants with both

microcephaly and invisible basal

ganglia.

Our case presented with a low

sloping forehead, micrognathia, and

extremity contracture. These physical

features are similar to those seen in

previous cases, but the severity of

microcephaly and prognosis seemed

to be milder in our case. 1 Our patient

had postnatal microcephaly and

appropriate growth. At 33 months

of age, she had microcephaly (head

circumference, –5.1 SD) and was still

alive. On the other hand, the previous

cases had microcephaly from

birth and growth restriction in the

prenatal period. One patient showed

severe microcephaly (−9.0 SD) at 3

months of age. 1 Most previous cases

(6 of 7 patients) died within 1 year. 1

We speculate that these clinical

differences depend on the functional

effect of the ZNF335 mutations. The

previous cases were homozygous for

splice-site mutations, whereas our

patient was compound heterozygous

for missense mutations. 1

We could not determine whether

the basal ganglia in our patient and

in the previous cases were aplastic/

hypoplastic, atrophic, or both. In

the ZNF335-conditional knockout

mouse, neural cell migration defects

occur in the cerebral and cerebellar

cortex due to a deficiency in the

neuron-restrictive silencer factor

(NRSF), which is known to be a

critical epigenetic regulator of

neurogenesis. 1, 4 – 6 We speculate that

if neural cell migration from the

ganglionic eminence is also affected,

the basal ganglia would become

aplastic/hypoplastic. In addition,

our patient’s cerebral and cerebellar

cortex regressed postnatally,

raising the possibility that ZNF335

deficiency may also lead to atrophy

of the basal ganglia. A previous

study showed that in Huntington

e3

FIGURE 2Brain MRI of the proband. The yellow arrows indicate the invisible basal ganglia. At 3 months of age, invisible basal ganglia, brainstem hypoplasia, and hypomyelination were noted. At 5 and 16 months of age, progression of cerebral and cerebellar atrophy could be observed. T1WI, T1-weighted imaging; T2WI, T2-weighted imaging.

by guest on August 27, 2018www.aappublications.org/newsDownloaded from

disease characterized by striatal

and cerebral atrophy, aberrant

accumulation of NRSF in the nucleus

would lead to loss of expression of

neuronal genes regulated through the

neuron-restrictive silencer element.7

This implies that fine regulation of

NRSF is essential for maintenance

of the striatum and cerebral cortex.

Therefore, we speculate that invisible

basal ganglia in patients with ZNF335

mutations may result from aplasia/

hypoplasia and atrophy of the basal

ganglia via a deficiency in NRSF.

In summary, we report an infant with

microcephaly and ZNF335 mutations.

Invisible basal ganglia may be the key

clinical feature of ZNF335 mutations.

ACKNOWLEDGMENTS

We thank the family for

participating in this study. We

also thank Drs A. James Barkovich,

Masayuki Sasaki, Osamu Komiyama,

Tomohide Goto, Kazuki Yamazawa,

and Takeshi Sato for fruitful

discussions.

SATO et al e4

ABBREVIATION

NRSF:  neuron-restrictive silencer

factor

FUNDING: This work was supported in part by a grant for Research on Measures for Intractable Diseases from the Japan Agency for Medical Research and

Development.

POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential confl icts of interest to disclose.

REFERENCES

1. Yang YJ, Baltus AE, Mathew RS, et al.

Microcephaly gene links trithorax and

REST/NRSF to control neural stem cell

proliferation and differentiation. Cell.

2012;151(5):1097–1112

2. Nishida T, Mihara T, Horiki T, Ka K.

Anaesthesia and orphan disease:

primary autosomal recessive

microcephaly-10 caused by a mutation

in the ZNF335 gene: A case report. Eur

J Anaesthesiol. 2015; 33(7):543–545

3. Hamilton EM, Polder E, Vanderver

A, et al; H-ABC Research Group.

Hypomyelination with atrophy of the

basal ganglia and cerebellum: further

delineation of the phenotype and

genotype-phenotype correlation. Brain.

2014;137(Pt 7):1921–1930

4. Chong JA, Tapia-Ramírez J, Kim S,

et al. REST: a mammalian silencer

protein that restricts sodium channel

FIGURE 3A, Pedigree of the family. c.1399T>C and c.1505A>G in ZNF335 were transmitted individually from the parents, indicating that the proband was compound heterozygous for these mutations. B, Protein structure of ZNF335. c.1399T>C and c.1505A>G are predicted to lead to amino acid substitutions of cysteine to arginine at codon 467, and of tyrosine to cysteine at codon 502, respectively. C467R and Y502C are located in the zinc fi nger domain of the protein. C, Comparison of conservation of Cys467 and Try502 residues across species. These 2 residues are evolutionarily conserved across species.

by guest on August 27, 2018www.aappublications.org/newsDownloaded from

PEDIATRICS Volume volume , number issue , pub-date e5

gene expression to neurons. Cell.

1995;80(6):949–957

5. Schoenherr CJ, Anderson DJ. Silencing

is golden: negative regulation in the

control of neuronal gene transcription.

Curr Opin Neurobiol. 1995;5(5):566–571

6. Sun YM, Greenway DJ, Johnson R,

et al. Distinct profi les of REST

interactions with its target genes

at different stages of neuronal

development. Mol Biol Cell.

2005;16(12):5630–5638

7. Zuccato C, Tartari M, Crotti A,

et al. Huntingtin interacts with

REST/NRSF to modulate the

transcription of NRSE-controlled

neuronal genes. Nat Genet.

2003;35(1):76–83

by guest on August 27, 2018www.aappublications.org/newsDownloaded from

DOI: 10.1542/peds.2016-0897 originally published online August 18, 2016; 2016;138;Pediatrics 

Naomichi Matsumoto and Takao TakahashiRieko Sato, Jun-ichi Takanashi, Yu Tsuyusaki, Mitsuhiro Kato, Hirotomo Saitsu,

Report Mutations: A CaseZNF335Association Between Invisible Basal Ganglia and

ServicesUpdated Information &

http://pediatrics.aappublications.org/content/138/3/e20160897including high resolution figures, can be found at:

Referenceshttp://pediatrics.aappublications.org/content/138/3/e20160897#BIBLThis article cites 7 articles, 1 of which you can access for free at:

Subspecialty Collections

subhttp://www.aappublications.org/cgi/collection/neurologic_disorders_Neurologic Disordershttp://www.aappublications.org/cgi/collection/neurology_subNeurologyhttp://www.aappublications.org/cgi/collection/genetics_subGeneticsfollowing collection(s): This article, along with others on similar topics, appears in the

Permissions & Licensing

http://www.aappublications.org/site/misc/Permissions.xhtmlin its entirety can be found online at: Information about reproducing this article in parts (figures, tables) or

Reprintshttp://www.aappublications.org/site/misc/reprints.xhtmlInformation about ordering reprints can be found online:

by guest on August 27, 2018www.aappublications.org/newsDownloaded from

DOI: 10.1542/peds.2016-0897 originally published online August 18, 2016; 2016;138;Pediatrics 

Naomichi Matsumoto and Takao TakahashiRieko Sato, Jun-ichi Takanashi, Yu Tsuyusaki, Mitsuhiro Kato, Hirotomo Saitsu,

Report Mutations: A CaseZNF335Association Between Invisible Basal Ganglia and

http://pediatrics.aappublications.org/content/138/3/e20160897located on the World Wide Web at:

The online version of this article, along with updated information and services, is

1073-0397. ISSN:60007. Copyright © 2016 by the American Academy of Pediatrics. All rights reserved. Print

the American Academy of Pediatrics, 141 Northwest Point Boulevard, Elk Grove Village, Illinois,has been published continuously since 1948. Pediatrics is owned, published, and trademarked by Pediatrics is the official journal of the American Academy of Pediatrics. A monthly publication, it

by guest on August 27, 2018www.aappublications.org/newsDownloaded from