ZNF335 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.
- NRSF —
- neuron-restrictive silencer factor
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 al1 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.
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 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.
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), and Mutation Taster (Charité, Berlin, Germany) predicted that p.C467R and p.Y502C are pathogenic (data not shown).
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 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.
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.
- Accepted 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:
FINANCIAL DISCLOSURE: The authors have indicated they have no financial relationships relevant to this article to disclose.
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 conflicts of interest to disclose.
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- Copyright © 2016 by the American Academy of Pediatrics