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Biallelic UNC80 mutations caused infantile hypotonia with psychomotor retardation and characteristic facies 2 in two Chinese patients with variable phenotypes
Gene 660 (2018) 13–17
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Research paper
Biallelic UNC80 mutations caused infantile hypotonia with psychomotor retardation and characteristic facies 2 in two Chinese patients with variable phenotypes ⁎
Yunjuan He, Xing Ji, Hui Yan, Xiantao Ye, Yu Liu, Wei Wei, Bing Xiao , Yu Sun
T
⁎
Department of Pediatric Endocrinology/Genetics, Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China Molecular Genetics Group, Shanghai Institute for Pediatric Research, Shanghai, China
A R T I C LE I N FO
A B S T R A C T
Keywords: UNC80 Developmental delay Whole exome sequencing
Biallelic UNC80 mutations cause infantile hypotonia with psychomotor retardation and characteristic facies 2 (IHPRF2), which is characterized by hypotonia, developmental delay (DD)/intellectual disability (ID), intrauterine growth retardation, postnatal growth retardation and characteristic facial features. We report two unrelated Chinese patients with compound heterozygous UNC80 mutations inherited from their parents, as identified by whole-exome sequencing (WES). Mutations c.3719G > A (p.W1240*)/c.4926_4937del (p.N1643_L1646del) and c.4963C > T (p.R1655C)/c.8385C > G (p.Y2795*) were identified in patient 1 and patient 2, respectively. Although both patients presented with DD/ID and hypotonia, different manifestations also occurred. Patient 1 presented with infantile hypotonia, epilepsy and hyperactivity without growth retardation, whereas patient 2 presented with persistent hypotonia, growth retardation and self-injury without epilepsy. Furthermore, we herein summarize the genotypes and phenotypes of patients with UNC80 mutations reported in the literature, revealing that IHPRF2 is a phenotypically heterogeneous disease. Common facial dysmorphisms include a thin upper lip, a tented upper lip, a triangular face, strabismus and microcephaly. To some extent, the manifestations of IHPRF2 mimic those of Angelman syndrome (AS)-like syndromes.
1. Introduction Infantile hypotonia with psychomotor retardation and characteristic facies 2 (IHPRF2; OMIM 616801) is a severe autosomal recessive neurodevelopmental disorder caused by biallelic mutations in the UNC80 gene (OMIM 612636), which was first described by Perez et al. (2016). This disorder is characterized by hypotonia, psychomotor retardation, facial dysmorphias, intrauterine growth retardation and postnatal growth retardation. There exists a different subtype of this disorder, infantile hypotonia with psychomotor retardation and characteristic facies 1 (IHPRF1), caused by NALCN (OMIM 611549) mutations. We report two Chinese patients with compound heterozygous UNC80 mutations whose characteristics are consistent with IHPRF2, with the exception of lack of postnatal growth retardation in one patient. Both patients exhibited hypotonia and developmental delay (DD)/intellectual disability (ID), but different manifestations occurred. Patient 1’s manifestations mimic those observed in Angelman syndrome (AS)
patients. In addition, we summarize common clinical characteristics of IHPRF2 and compare IHPRF2 with IHPRF1 and AS-like syndromes. 2. Materials and methods 2.1. Clinical reports Patient 1 (Fig. 1A–B) was a 9.5-year-old boy who was the only child of healthy and non-consanguineous parents with no family history of DD/ID. He was born via cesarean section following an uneventful and full-term pregnancy. His birth weight and body length were 3.35 kg (0SD) and 50 cm (0SD), respectively. He experienced physiologic jaundice one week after birth. Hypotonia and psychomotor retardation were observed. He could raise his head at the age of one year and sit by himself when he was two years of age; he ambulated when he was three years old. At approximately five years of age, he could use only simple speech. He also exhibited poor balance and displayed hyperactive
Abbreviations: ACMG, American College of Medical Genetics; AS, Angelman syndrome; DD/ID, developmental delay/intellectual disability; EEG, electroencephalograph; EVS, Exome Variant Server; ExAC, Exome Aggregation Consortium; IHPRF2, infantile hypotonia with psychomotor retardation and characteristic facies 2; IHPRF2, infantile hypotonia with psychomotor retardation and characteristic facies 2; LOVD, Leiden Open Variant Database; MRI, magnetic resonance imaging; WES, whole exome sequencing ⁎ Corresponding authors at: Room 803, Sci&Edu Bldg, 1665 Kongjiang Road, 200092 Shanghai, China. E-mail addresses: [email protected] (B. Xiao), [email protected] (Y. Sun). https://doi.org/10.1016/j.gene.2018.03.063 Received 20 November 2017; Received in revised form 8 February 2018; Accepted 19 March 2018 Available online 20 March 2018 0378-1119/ © 2018 Elsevier B.V. All rights reserved.
Gene 660 (2018) 13–17
Y. He et al.
Fig. 1. Clinical features of patient 1 and patient 2. [A] Frontal features of patient 1 include a long face, frontal bossing, strabismus, a broad nasal bridge, a short and smooth philtrum, and a tented upper lip. [B] Lateral features of patient 1 include a depressed nasal bridge. [C] Patient 1 has long and slender fingers. [D] Frontal features of patient 2 include a triangular face, frontal bossing, strabismus, a thin upper lip, and micrognathia. [E] Patient 2 presents with a depressed nasal bridge. [F] Pedigree of patient 2's family.
her head with her hands or bumping her head. Follow-up when the patient was 5 years and 9 months old showed that she could not walk or speak. At that time, her body length was 100 cm (−3.4 SD), and she weighed 11 kg (−4 SD). Chromosomal microarray tests revealed no pathogenic copy number variations. According to clinical records, the proband's younger brother (II-3 in Fig. 1F) presented similar phenotypes such as hypotonia and DD. He died at the age of one year due to severe pneumonia. He was diagnosed as IHPRF2 clinically. However, no genetic analysis was performed, as DNA was unavailable. Informed consent was provided by the parents of both patients for DNA study and the publication of clinical features. The study protocol was approved by the Ethical Review Board of Xin Hua Hospital (XHECD-2014-044).
behavior and a happy and excitable disposition with frequent smiling and laughing. In addition, he suffered from constipation. Seizures occurred when he was two years old and were effectively treated with valproate and levetiracetam. When he was referred to our clinic at 6 years and 5 months of age, his height and weight were 120 cm (0 SD) and 20 kg (0 SD), respectively. When followed up at the age of 9.5 years, his height, weight and head circumference were 135 cm (−0.5 SD), 25 kg (−1.3 SD) and 51 cm (−0.5 SD), respectively. His facial features included frontal bossing, strabismus, a tented upper lip, a broad nasal bridge, and a short, smooth philtrum. He had long, slender fingers (Fig. 1C). An electroencephalograph (EEG) revealed excessively slow background. AS was initially suspected, but methylation-specific multiplex ligation-dependent probe amplification revealed no abnormalities in the 15q11-q13 region. Chromosomal microarray tests also showed no pathogenic copy number variations. Patient 2 (Fig. 1D-F) was born in a healthy, non-consanguineous Chinese family after a full-term pregnancy. Her mother had no history of exposure to teratogenic pathogens or drugs during gestation. Her mother's first pregnancy (II-1 in Fig. 1F) terminated during the first trimester due to an unknown cause. The patient was referred to our department for DD and failure to thrive without feeding difficulty at approximately three years of age. She could speak only simple words such as “baba” and “mama” and could not walk. A physical examination revealed hypotonia. Her head circumference was 44 cm (−3.7 SD). Her facial dysmorphisms included a triangular face; light, sparse hair; frontal bossing; strabismus; a thin upper lip; and micrognathia. Echocardiographic examination, brain magnetic resonance imaging (MRI) and an EEG revealed no abnormalities. Metabolic screening and assessments of the patient's thyroid function at age three produced normal results. At the age of five, she could stand with support but remained unable to ambulate or speak. She suffered from sleep disturbance and a tendency toward self-injury behaviors, such as hitting
2.2. Next-generation sequencing Genomic DNA was extracted from peripheral whole blood using a QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA, USA), following standard procedures. Whole exome sequencing (WES) was performed as previously described (Sun et al., 2017). We excluded variants from the candidate variant list that were observed with a frequency of > 1% in the 1000 Genomes Project, Exome Aggregation Consortium (ExAC), or Exome Variant Server (EVS) database or a frequency of > 5% in a local database containing 150 exomes. Variants of patient 1 were subsequently screened using autosomal recessive and autosomal dominant/ de novo inheritance patterns. Variants of patient 2 were filtered using an autosomal recessive inheritance pattern based on the pedigree. 2.3. Sanger sequencing Candidate variants were confirmed using Sanger sequencing. PCR primer sequences and protocols are available upon request. Amplified 14
15 − + + + + + +
− − −
+ + +
+
Other clinical features Scoliosis
Neuroaxonal dystrophy Unmyelinated sural nerve Spheroid formation Sural nerve edema Behavioral findings
−
NA NA Self-injury
NA NA Arm flapping, hyperactivity
−
NA NA
−
At birth
NA NA
+
− −
+ −
Seizures
8/15 8/15
+ + − − + − − − − + + − +
− + − − + − − − + − − + −
2–3 months
16/19 11/19 10/19 10/19 15/19 8/19 8/19 8/19 17/19 12/19 18/19 17/19 16/19
−
−
Hypotonia first noted
5y 9m Female − + 3100 g (−0.4 SD) 100 cm (−3.4 SD)
9.5 y Male − − 3350 g (0 SD) 135 cm (−0.5 SD)
+ + +
− − 5/6
+
+ +
− −
7/14
+
+
+ + +
− − +
− −
+ + + − + − − + + + + − +
−
NALCN
IHPRF1b
At birth/in early infancy 11/19
19/19
19/19 19/19 19/19
7/19 15/19 12/15
8/19
UNC80
Age at last review Gender Parental consanguinity Family history Birth weight(SD) Body length (SD) Facial features Downslanting palpebral fissures Triangular face Frontal bossing Low-set ears Posteriorly rotated ears Strabismus Anteverted nasal tip Depressed nasal bridge Broad nasal bridge Short/smooth philtrum Micrognathia Thin upper lip Tented upper lip Microcephaly Limb features Long, thin fingers Tapering of the distal phalanx Growth findings IUGRc FTTd PGRe Developmental findings Intellectual disability Speech delay Motor delay Neurological findings Hypotonia
UNC80
UNC80
Gene
IHPRF2a in the literature
Patient 1
Subject
Patient 2
EIEE2: +; KDVS: ± ; RTT: +; ATRX: +;AS: +; FOXG1 haploinsufficiency syndrome: +
NA NA KDVS: +; PTHS: +; MRXSCH: +; RTT: +; PHMDS: +;AS: +
PHMDS: +; KDVS: +; PTHS: +; MRXSCH: +; MOWS: +; RTT: +; MRXS13: +; Kleefstra syndrome: + MRT38: +; EIEE2: +; MRD20: +; ATRX: +; FOXG1 haploinsufficiency syndrome: +; AS: + at birth/ in infancy MRD1: +; KDVS: +; PTHS: +; MRXSCH: +; MOWS: +; RTT: +; Kleefstra syndrome: +; ADSLD: + FOXG1 haploinsufficiency syndrome: +; MRXS13: +; MRD20: +;AS: + NA NA
All All All
KDVS: + KDVS: +; MRXS13: + MRD1: +; ADSLD: +; ATRX: +; FOXG1 haploinsufficiency syndrome: +
KDVS: ± ; MRXSCH: + PTHS: +; EIEE2: +; ATRX: +
NA MRD1: + MRD1: +; ADSLD: +; MRXS13: +; MRD20: +; ATRX: + ATRX: + KDVS: ± ; PTHS: +; MOWS: +; MRT38: +; ADSLD: +; AS: + KDVS: +; Kleefstra syndrome: +; ADSLD: +; EIEE2: +; MRD20: +; ATRX: + PTHS: + PTHS: + PTHS: +; MRD20: + MRD1: +; PHMDS: +; MRD20: + MRD1: +; ADSLD: + MRXS13: + MRXSCH: +; MOWS: +; ADSLD: +; RTT: +; ATRX: +; AS: +; FOXG1 haploinsufficiency syndrome: +
MOWS: +
(continued on next page)
22q13.3 deletion;2q23.1 deletion MBD5;17q21.31 deletion KANSL1; HERC2; ADSL; MECP2; CDKL5; FOXG1; MEF2C;17q21.31 deletion KANSL4; TCF4; SLC9A6; ZEB2;9q34.3 deletion EHMT1; ATRX; maternal deletion or paternal uniparental disomy of 15q11.2-q13; imprinting defects; UBE3A
AS/AS-like syndromesf
Table 1 Clinical features of our two Chinese IHPRF2 patients, IHPRF2 patients in the literature, IHPRF1, and AS/AS-like syndromes.
Y. He et al.
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Gene 660 (2018) 13–17
IHPRF2, infantile hypotonia with psychomotor retardation and characteristic facies 2. IHPRF1, infantile hypotonia with psychomotor retardation and characteristic facies 1. c IUGR, intrauterine growth retardation. d FTT, failure to thrive. e PGR, postnatal growth retardation. f AS-like syndromes include ADSLD: adenylosuccinase deficiency; ATRX: alpha-thalassemia/intellectual disability syndrome; EIEE2: CDKL5 syndrome; KDVS: 17q21.31 deletion KANSL1 haploinsufficiency syndrome; MOWS: Mowat–Wilson syndrome; MRD1:2q23.1 deletion MBD5 haploinsufficiency syndrome; MRD20:MEF2C haploinsufficiency syndrome; MRT38: HERC2 deficiency syndrome; MRXS13: MECP2 duplication; MRXSCH: Christianson syndrome; PHMDS: 22q13.3 deletion syndrome; PTHS: Pitt-Hopkins syndrome; and RTT: typical Rett syndrome. NA: not available.
fragments were sequenced using a 96-capillary 3730xl system (Applied Biosystems, Foster, CA, USA). 3. Results and discussion Patient 1 and patient 2 carried the c.4963C > T (p.R1655C)/ c.8385C > G (p.Y2795*) and c.3719G > A (p.W1240*)/c.4926_4937del (p.N1643_L1646del) mutations, respectively. The relevant transcript accession number is NM_032504.1. These mutations were not present in the 1000 Genomes Project, gnomAD, EVS or in-house databases and were not previously reported in the literature. The mutations are highly conserved across vertebrates and are predicted to have deleterious effects by PolyPhen, SIFT, and MutationTaster. According to American College of Medical Genetics (ACMG) guidelines for variant interpretation (Richards et al., 2015), three of the four mutations, c.8385C > G (PM2+PM4+PP1+PP4), c.3719G > A (PM2+PP1+PP3+PP4) and c.4926_4937del (PM2+PM4+PP1+PP4), can be classified as likely pathogenic; c.4963C > T is a variant of uncertain significance (PM2+PP1+PP3+PP4). The clinical features and identified mutations observed in these two cases were submitted to the UNC80 variant database (https://databases.lovd.nl/shared/individuals/UNC80) in the Leiden Open Variant Database (LOVD) (Fokkema et al., 2011). To date, 14 UNC80 mutations in 21 IHPRF2 patients have been reported, including the mutations found in the present study (Perez et al., 2016; Shamseldin et al., 2016; Stray-Pedersen et al., 2016; Valkanas et al., 2016). These mutations include nonsense, missense, and frameshift mutations as well as in-frame deletions and are spread across the entire gene. Thus far, no mutation hot spots have been detected. The observed mutations include not only truncating mutations but also missense mutations that affect amino acids that have been highly conserved throughout evolution (Supplementary Fig. SII), suggesting that IHPRF2-related mutations might disrupt the normal physiological function of UNC80. The genotypes and phenotypes of the 21 reported IHPRF2 patients, including the two patients in this study, are summarized in detail in Supplementary Table SI. All of these patients presented with DD/ID, severe speech impairment and hypotonia. Other common phenotypes caused by UNC80 mutations include the following facial dysmorphisms: a thin upper lip (19/21); a tented upper lip (18/21); a triangular face (17/21); a short, smooth philtrum (18/21); strabismus (17/21); microcephaly (17/21); micrognathia (13/21); and frontal bossing (13/ 21). In addition, postnatal growth retardation, EEG abnormalities, seizures, intrauterine growth retardation, and brain MRI abnormalities have been observed in 80% (12/15), 78% (14/18), 57% (12/21), 33% (7/21), and 12% (2/17) of IHPRF2 patients, respectively. We observed variability in some common phenotypes; for example, the extent of hypotonia between the two patients in this report varied. Patient 1 exhibited infantile hypotonia, whereas patient 2 displayed persistent hypotonia. In addition, 11 patients in four studies with examined growth records all exhibited postnatal growth retardation. However, patient 1 in this study showed normal postnatal growth. Other frequently reported phenotypes, such as microcephaly, facial features, and seizures, are variable among individuals and between family members. Moreover, our two patients presented with diverse behavioral abnormalities, such as arm flapping and a happy disposition for patient 1 and self-injury behaviors for patient 2. One common and major manifestation of UNC80 mutations is severe speech impairment, which is also a major feature of AS. Such impairment was particularly evident in patient 1, who exhibited many characteristics that mimic those of AS and was first suspected of having AS. There are also AS-like genetic syndromes, including microdeletion/ duplication syndromes and single-gene syndromes in which severe DD/ ID and speech impairment are commonly present, and characteristics such as hypotonia, growth retardation, microcephaly, seizures, sleep disturbance, and behavior disturbance, among others, have frequently been observed in these syndromes, which may confound diagnosis (Table 1). Tan et al. (2014) listed key features of such syndromes to
b
a
PHMDS: +; KDVS: +; AS: + MRD1: +; AS: + Kleefstra syndrome: +; RTT: +; EIEE2: +; FOXG1 haploinsufficiency syndrome: +; AS: + PTHS: +; MRXSCH: +; MOWS: +; MRT38: +; ADSLD: +; RTT: +; MRD20: +; ATRX: +; FOXG1 haploinsufficiency syndrome: +; AS: + RTT: +; EIEE2: +; MRXS13: +; MRD20: +; FOXG1 haploinsufficiency syndrome: +; AS: + + + − − − 5/6 5/6 2/6 2/16 13/16 − − + − − − + − NA + Feeding difficulties Constipation Sleep disturbance Brain MRI abnormalities EEG abnormalities
Patient 2 Patient 1 Subject
Table 1 (continued)
IHPRF2a in the literature
IHPRF1b
AS/AS-like syndromesf
Y. He et al.
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Gene 660 (2018) 13–17
Y. He et al.
Municipal Commission of Health and Family Planning Foundation (201540054 to BX; 20154Y0153 and 2017YQ020 to YS), and Shanghai Jiao Tong University School of Medicine (2014XJ10044 to YS).
help clinicians determine which of these syndromes should be further considered in differential diagnosis. To an extent, the clinical features of UNC80-related IHPRF2 mimic those of these AS-like syndromes. Therefore, when patients manifest hypotonia, speech impairment and inappropriately happy demeanors that include frequent smiling and excitability, UNC80-related IHPRF2 should be considered. The other subtype of IHPRF is IHPRF1, which is caused by biallelic NALCN mutations. With the exception of peripheral nervous system symptoms such as neuroaxonal dystrophy, sural nerve edema and decreased motor nerve conduction velocity, the clinical manifestations of IHPRF1 caused by NALCN mutations overlap with those of IHPRF2 and include hypotonia, DD/ID, facial characteristics and postnatal growth retardation (Table 1). This extensive similarity can be explained based on the intimate relationship between UNC80 and NALCN. UNC79 and NALCN are bridged by UNC80 to form a functional complex that is involved in the formation and maintenance of neuromembrane resting potential (Cochet-Bissuel et al., 2014; Lu et al., 2010). Defects in UNC80/NALCN disrupt the integrity of this complex and therefore result in related, similar phenotypes. In summary, we provide the first report of two unrelated Chinese patients with compound heterozygous UNC80 mutations. Most of our patients' phenotypic characteristics were consistent with IHPRF2, with the exception of a lack of postnatal growth retardation in one patient. Although both patients carried UNC80 mutations, they exhibited some variable phenotypes. Furthermore, we summarized the diverse phenotypes of IHPRF2 patients documented in the literature, and we found the phenotypes of IHPRF2 to be heterogeneous. To a certain degree, the manifestations of UNC80-related IHPRF2 resemble those of AS-like syndromes. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.gene.2018.03.063.
References Cochet-Bissuel, M., Lory, P., Monteil, A., 2014. The sodium leak channel, NALCN, in health and disease. Front. Cell. Neurosci. 8, 132. Fokkema, I.F., Taschner, P.E., Schaafsma, G.C., Celli, J., Laros, J.F., den Dunnen, J.T., 2011. LOVD v.2.0: the next generation in gene variant databases. Hum. Mutat. 32 (5), 557–563. Lu, B., Zhang, Q., Wang, H., Wang, Y., Nakayama, M., Ren, D., 2010. Extracellular calcium controls background current and neuronal excitability via an UNC79-UNC80NALCN cation channel complex. Neuron 68 (3), 488–499. Perez, Y., Kadir, R., Volodarsky, M., Noyman, I., Flusser, H., Shorer, Z., Gradstein, L., Birnbaum, R.Y., Birk, O.S., 2016. UNC80 mutation causes a syndrome of hypotonia, severe intellectual disability, dyskinesia and dysmorphism, similar to that caused by mutations in its interacting cation channel NALCN. J. Med. Genet. 53 (6), 397–402. Richards, S., Aziz, N., Bale, S., Bick, D., Das, S., Gastier-Foster, J., Grody, W.W., Hegde, M., Lyon, E., Spector, E., Voelkerding, K., Rehm, H.L., Committee Acmg Laboratory Quality Assurance, 2015. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet. Med. 17 (5), 405–424. Shamseldin, H.E., Faqeih, E., Alasmari, A., Zaki, M.S., Gleeson, J.G., Alkuraya, F.S., 2016. Mutations in UNC80, encoding part of the UNC79-UNC80-NALCN channel complex, cause autosomal-recessive severe infantile encephalopathy. Am. J. Hum. Genet. 98 (1), 210–215. Stray-Pedersen, A., Cobben, J.M., Prescott, T.E., Lee, S., Cang, C., Aranda, K., Ahmed, S., Alders, M., Gerstner, T., Aslaksen, K., Tetreault, M., Qin, W., Hartley, T., Jhangiani, S.N., Muzny, D.M., Tarailo-Graovac, M., van Karnebeek, C.D., Lupski, J.R., Ren, D., Yoon, G., 2016. Biallelic mutations in UNC80 cause persistent Hypotonia, encephalopathy, growth retardation, and severe intellectual disability. Am. J. Hum. Genet. 98 (1), 202–209. Sun, Y., Hu, G., Liu, H., Zhang, X., Huang, Z., Yan, H., Wang, L., Fan, Y., Gu, X., Yu, Y., 2017. Further delineation of the phenotype of truncating KMT2A mutations: the extended Wiedemann-Steiner syndrome. Am. J. Med. Genet. A 173 (2), 510–514. Tan, W.H., Bird, L.M., Thibert, R.L., Williams, C.A., 2014. If not Angelman, what is it? A review of Angelman-like syndromes. Am. J. Med. Genet. A 164A (4), 975–992. Valkanas, Elise, Schaffer, Katherine, Dunham, Christopher, Maduro, Valerie, du Souich, Christèle, Rupps, Rosemarie, Adams David, R., Baradaran-Heravi, Alireza, Flynn, Elise, Malicdan May, C., Gahl William, A., Toro, Camilo, Boerkoel Cornelius, F., 2016. Phenotypic evolution of UNC80 loss of function. Am. J. Med. Genet. A170 (12), 3106–3114.
Acknowledgments The authors thank both patients' families for participating in the present study. This work was funded by grants from the Shanghai
17
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Research paper
Biallelic UNC80 mutations caused infantile hypotonia with psychomotor retardation and characteristic facies 2 in two Chinese patients with variable phenotypes ⁎
Yunjuan He, Xing Ji, Hui Yan, Xiantao Ye, Yu Liu, Wei Wei, Bing Xiao , Yu Sun
T
⁎
Department of Pediatric Endocrinology/Genetics, Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China Molecular Genetics Group, Shanghai Institute for Pediatric Research, Shanghai, China
A R T I C LE I N FO
A B S T R A C T
Keywords: UNC80 Developmental delay Whole exome sequencing
Biallelic UNC80 mutations cause infantile hypotonia with psychomotor retardation and characteristic facies 2 (IHPRF2), which is characterized by hypotonia, developmental delay (DD)/intellectual disability (ID), intrauterine growth retardation, postnatal growth retardation and characteristic facial features. We report two unrelated Chinese patients with compound heterozygous UNC80 mutations inherited from their parents, as identified by whole-exome sequencing (WES). Mutations c.3719G > A (p.W1240*)/c.4926_4937del (p.N1643_L1646del) and c.4963C > T (p.R1655C)/c.8385C > G (p.Y2795*) were identified in patient 1 and patient 2, respectively. Although both patients presented with DD/ID and hypotonia, different manifestations also occurred. Patient 1 presented with infantile hypotonia, epilepsy and hyperactivity without growth retardation, whereas patient 2 presented with persistent hypotonia, growth retardation and self-injury without epilepsy. Furthermore, we herein summarize the genotypes and phenotypes of patients with UNC80 mutations reported in the literature, revealing that IHPRF2 is a phenotypically heterogeneous disease. Common facial dysmorphisms include a thin upper lip, a tented upper lip, a triangular face, strabismus and microcephaly. To some extent, the manifestations of IHPRF2 mimic those of Angelman syndrome (AS)-like syndromes.
1. Introduction Infantile hypotonia with psychomotor retardation and characteristic facies 2 (IHPRF2; OMIM 616801) is a severe autosomal recessive neurodevelopmental disorder caused by biallelic mutations in the UNC80 gene (OMIM 612636), which was first described by Perez et al. (2016). This disorder is characterized by hypotonia, psychomotor retardation, facial dysmorphias, intrauterine growth retardation and postnatal growth retardation. There exists a different subtype of this disorder, infantile hypotonia with psychomotor retardation and characteristic facies 1 (IHPRF1), caused by NALCN (OMIM 611549) mutations. We report two Chinese patients with compound heterozygous UNC80 mutations whose characteristics are consistent with IHPRF2, with the exception of lack of postnatal growth retardation in one patient. Both patients exhibited hypotonia and developmental delay (DD)/intellectual disability (ID), but different manifestations occurred. Patient 1’s manifestations mimic those observed in Angelman syndrome (AS)
patients. In addition, we summarize common clinical characteristics of IHPRF2 and compare IHPRF2 with IHPRF1 and AS-like syndromes. 2. Materials and methods 2.1. Clinical reports Patient 1 (Fig. 1A–B) was a 9.5-year-old boy who was the only child of healthy and non-consanguineous parents with no family history of DD/ID. He was born via cesarean section following an uneventful and full-term pregnancy. His birth weight and body length were 3.35 kg (0SD) and 50 cm (0SD), respectively. He experienced physiologic jaundice one week after birth. Hypotonia and psychomotor retardation were observed. He could raise his head at the age of one year and sit by himself when he was two years of age; he ambulated when he was three years old. At approximately five years of age, he could use only simple speech. He also exhibited poor balance and displayed hyperactive
Abbreviations: ACMG, American College of Medical Genetics; AS, Angelman syndrome; DD/ID, developmental delay/intellectual disability; EEG, electroencephalograph; EVS, Exome Variant Server; ExAC, Exome Aggregation Consortium; IHPRF2, infantile hypotonia with psychomotor retardation and characteristic facies 2; IHPRF2, infantile hypotonia with psychomotor retardation and characteristic facies 2; LOVD, Leiden Open Variant Database; MRI, magnetic resonance imaging; WES, whole exome sequencing ⁎ Corresponding authors at: Room 803, Sci&Edu Bldg, 1665 Kongjiang Road, 200092 Shanghai, China. E-mail addresses: [email protected] (B. Xiao), [email protected] (Y. Sun). https://doi.org/10.1016/j.gene.2018.03.063 Received 20 November 2017; Received in revised form 8 February 2018; Accepted 19 March 2018 Available online 20 March 2018 0378-1119/ © 2018 Elsevier B.V. All rights reserved.
Gene 660 (2018) 13–17
Y. He et al.
Fig. 1. Clinical features of patient 1 and patient 2. [A] Frontal features of patient 1 include a long face, frontal bossing, strabismus, a broad nasal bridge, a short and smooth philtrum, and a tented upper lip. [B] Lateral features of patient 1 include a depressed nasal bridge. [C] Patient 1 has long and slender fingers. [D] Frontal features of patient 2 include a triangular face, frontal bossing, strabismus, a thin upper lip, and micrognathia. [E] Patient 2 presents with a depressed nasal bridge. [F] Pedigree of patient 2's family.
her head with her hands or bumping her head. Follow-up when the patient was 5 years and 9 months old showed that she could not walk or speak. At that time, her body length was 100 cm (−3.4 SD), and she weighed 11 kg (−4 SD). Chromosomal microarray tests revealed no pathogenic copy number variations. According to clinical records, the proband's younger brother (II-3 in Fig. 1F) presented similar phenotypes such as hypotonia and DD. He died at the age of one year due to severe pneumonia. He was diagnosed as IHPRF2 clinically. However, no genetic analysis was performed, as DNA was unavailable. Informed consent was provided by the parents of both patients for DNA study and the publication of clinical features. The study protocol was approved by the Ethical Review Board of Xin Hua Hospital (XHECD-2014-044).
behavior and a happy and excitable disposition with frequent smiling and laughing. In addition, he suffered from constipation. Seizures occurred when he was two years old and were effectively treated with valproate and levetiracetam. When he was referred to our clinic at 6 years and 5 months of age, his height and weight were 120 cm (0 SD) and 20 kg (0 SD), respectively. When followed up at the age of 9.5 years, his height, weight and head circumference were 135 cm (−0.5 SD), 25 kg (−1.3 SD) and 51 cm (−0.5 SD), respectively. His facial features included frontal bossing, strabismus, a tented upper lip, a broad nasal bridge, and a short, smooth philtrum. He had long, slender fingers (Fig. 1C). An electroencephalograph (EEG) revealed excessively slow background. AS was initially suspected, but methylation-specific multiplex ligation-dependent probe amplification revealed no abnormalities in the 15q11-q13 region. Chromosomal microarray tests also showed no pathogenic copy number variations. Patient 2 (Fig. 1D-F) was born in a healthy, non-consanguineous Chinese family after a full-term pregnancy. Her mother had no history of exposure to teratogenic pathogens or drugs during gestation. Her mother's first pregnancy (II-1 in Fig. 1F) terminated during the first trimester due to an unknown cause. The patient was referred to our department for DD and failure to thrive without feeding difficulty at approximately three years of age. She could speak only simple words such as “baba” and “mama” and could not walk. A physical examination revealed hypotonia. Her head circumference was 44 cm (−3.7 SD). Her facial dysmorphisms included a triangular face; light, sparse hair; frontal bossing; strabismus; a thin upper lip; and micrognathia. Echocardiographic examination, brain magnetic resonance imaging (MRI) and an EEG revealed no abnormalities. Metabolic screening and assessments of the patient's thyroid function at age three produced normal results. At the age of five, she could stand with support but remained unable to ambulate or speak. She suffered from sleep disturbance and a tendency toward self-injury behaviors, such as hitting
2.2. Next-generation sequencing Genomic DNA was extracted from peripheral whole blood using a QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA, USA), following standard procedures. Whole exome sequencing (WES) was performed as previously described (Sun et al., 2017). We excluded variants from the candidate variant list that were observed with a frequency of > 1% in the 1000 Genomes Project, Exome Aggregation Consortium (ExAC), or Exome Variant Server (EVS) database or a frequency of > 5% in a local database containing 150 exomes. Variants of patient 1 were subsequently screened using autosomal recessive and autosomal dominant/ de novo inheritance patterns. Variants of patient 2 were filtered using an autosomal recessive inheritance pattern based on the pedigree. 2.3. Sanger sequencing Candidate variants were confirmed using Sanger sequencing. PCR primer sequences and protocols are available upon request. Amplified 14
15 − + + + + + +
− − −
+ + +
+
Other clinical features Scoliosis
Neuroaxonal dystrophy Unmyelinated sural nerve Spheroid formation Sural nerve edema Behavioral findings
−
NA NA Self-injury
NA NA Arm flapping, hyperactivity
−
NA NA
−
At birth
NA NA
+
− −
+ −
Seizures
8/15 8/15
+ + − − + − − − − + + − +
− + − − + − − − + − − + −
2–3 months
16/19 11/19 10/19 10/19 15/19 8/19 8/19 8/19 17/19 12/19 18/19 17/19 16/19
−
−
Hypotonia first noted
5y 9m Female − + 3100 g (−0.4 SD) 100 cm (−3.4 SD)
9.5 y Male − − 3350 g (0 SD) 135 cm (−0.5 SD)
+ + +
− − 5/6
+
+ +
− −
7/14
+
+
+ + +
− − +
− −
+ + + − + − − + + + + − +
−
NALCN
IHPRF1b
At birth/in early infancy 11/19
19/19
19/19 19/19 19/19
7/19 15/19 12/15
8/19
UNC80
Age at last review Gender Parental consanguinity Family history Birth weight(SD) Body length (SD) Facial features Downslanting palpebral fissures Triangular face Frontal bossing Low-set ears Posteriorly rotated ears Strabismus Anteverted nasal tip Depressed nasal bridge Broad nasal bridge Short/smooth philtrum Micrognathia Thin upper lip Tented upper lip Microcephaly Limb features Long, thin fingers Tapering of the distal phalanx Growth findings IUGRc FTTd PGRe Developmental findings Intellectual disability Speech delay Motor delay Neurological findings Hypotonia
UNC80
UNC80
Gene
IHPRF2a in the literature
Patient 1
Subject
Patient 2
EIEE2: +; KDVS: ± ; RTT: +; ATRX: +;AS: +; FOXG1 haploinsufficiency syndrome: +
NA NA KDVS: +; PTHS: +; MRXSCH: +; RTT: +; PHMDS: +;AS: +
PHMDS: +; KDVS: +; PTHS: +; MRXSCH: +; MOWS: +; RTT: +; MRXS13: +; Kleefstra syndrome: + MRT38: +; EIEE2: +; MRD20: +; ATRX: +; FOXG1 haploinsufficiency syndrome: +; AS: + at birth/ in infancy MRD1: +; KDVS: +; PTHS: +; MRXSCH: +; MOWS: +; RTT: +; Kleefstra syndrome: +; ADSLD: + FOXG1 haploinsufficiency syndrome: +; MRXS13: +; MRD20: +;AS: + NA NA
All All All
KDVS: + KDVS: +; MRXS13: + MRD1: +; ADSLD: +; ATRX: +; FOXG1 haploinsufficiency syndrome: +
KDVS: ± ; MRXSCH: + PTHS: +; EIEE2: +; ATRX: +
NA MRD1: + MRD1: +; ADSLD: +; MRXS13: +; MRD20: +; ATRX: + ATRX: + KDVS: ± ; PTHS: +; MOWS: +; MRT38: +; ADSLD: +; AS: + KDVS: +; Kleefstra syndrome: +; ADSLD: +; EIEE2: +; MRD20: +; ATRX: + PTHS: + PTHS: + PTHS: +; MRD20: + MRD1: +; PHMDS: +; MRD20: + MRD1: +; ADSLD: + MRXS13: + MRXSCH: +; MOWS: +; ADSLD: +; RTT: +; ATRX: +; AS: +; FOXG1 haploinsufficiency syndrome: +
MOWS: +
(continued on next page)
22q13.3 deletion;2q23.1 deletion MBD5;17q21.31 deletion KANSL1; HERC2; ADSL; MECP2; CDKL5; FOXG1; MEF2C;17q21.31 deletion KANSL4; TCF4; SLC9A6; ZEB2;9q34.3 deletion EHMT1; ATRX; maternal deletion or paternal uniparental disomy of 15q11.2-q13; imprinting defects; UBE3A
AS/AS-like syndromesf
Table 1 Clinical features of our two Chinese IHPRF2 patients, IHPRF2 patients in the literature, IHPRF1, and AS/AS-like syndromes.
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IHPRF2, infantile hypotonia with psychomotor retardation and characteristic facies 2. IHPRF1, infantile hypotonia with psychomotor retardation and characteristic facies 1. c IUGR, intrauterine growth retardation. d FTT, failure to thrive. e PGR, postnatal growth retardation. f AS-like syndromes include ADSLD: adenylosuccinase deficiency; ATRX: alpha-thalassemia/intellectual disability syndrome; EIEE2: CDKL5 syndrome; KDVS: 17q21.31 deletion KANSL1 haploinsufficiency syndrome; MOWS: Mowat–Wilson syndrome; MRD1:2q23.1 deletion MBD5 haploinsufficiency syndrome; MRD20:MEF2C haploinsufficiency syndrome; MRT38: HERC2 deficiency syndrome; MRXS13: MECP2 duplication; MRXSCH: Christianson syndrome; PHMDS: 22q13.3 deletion syndrome; PTHS: Pitt-Hopkins syndrome; and RTT: typical Rett syndrome. NA: not available.
fragments were sequenced using a 96-capillary 3730xl system (Applied Biosystems, Foster, CA, USA). 3. Results and discussion Patient 1 and patient 2 carried the c.4963C > T (p.R1655C)/ c.8385C > G (p.Y2795*) and c.3719G > A (p.W1240*)/c.4926_4937del (p.N1643_L1646del) mutations, respectively. The relevant transcript accession number is NM_032504.1. These mutations were not present in the 1000 Genomes Project, gnomAD, EVS or in-house databases and were not previously reported in the literature. The mutations are highly conserved across vertebrates and are predicted to have deleterious effects by PolyPhen, SIFT, and MutationTaster. According to American College of Medical Genetics (ACMG) guidelines for variant interpretation (Richards et al., 2015), three of the four mutations, c.8385C > G (PM2+PM4+PP1+PP4), c.3719G > A (PM2+PP1+PP3+PP4) and c.4926_4937del (PM2+PM4+PP1+PP4), can be classified as likely pathogenic; c.4963C > T is a variant of uncertain significance (PM2+PP1+PP3+PP4). The clinical features and identified mutations observed in these two cases were submitted to the UNC80 variant database (https://databases.lovd.nl/shared/individuals/UNC80) in the Leiden Open Variant Database (LOVD) (Fokkema et al., 2011). To date, 14 UNC80 mutations in 21 IHPRF2 patients have been reported, including the mutations found in the present study (Perez et al., 2016; Shamseldin et al., 2016; Stray-Pedersen et al., 2016; Valkanas et al., 2016). These mutations include nonsense, missense, and frameshift mutations as well as in-frame deletions and are spread across the entire gene. Thus far, no mutation hot spots have been detected. The observed mutations include not only truncating mutations but also missense mutations that affect amino acids that have been highly conserved throughout evolution (Supplementary Fig. SII), suggesting that IHPRF2-related mutations might disrupt the normal physiological function of UNC80. The genotypes and phenotypes of the 21 reported IHPRF2 patients, including the two patients in this study, are summarized in detail in Supplementary Table SI. All of these patients presented with DD/ID, severe speech impairment and hypotonia. Other common phenotypes caused by UNC80 mutations include the following facial dysmorphisms: a thin upper lip (19/21); a tented upper lip (18/21); a triangular face (17/21); a short, smooth philtrum (18/21); strabismus (17/21); microcephaly (17/21); micrognathia (13/21); and frontal bossing (13/ 21). In addition, postnatal growth retardation, EEG abnormalities, seizures, intrauterine growth retardation, and brain MRI abnormalities have been observed in 80% (12/15), 78% (14/18), 57% (12/21), 33% (7/21), and 12% (2/17) of IHPRF2 patients, respectively. We observed variability in some common phenotypes; for example, the extent of hypotonia between the two patients in this report varied. Patient 1 exhibited infantile hypotonia, whereas patient 2 displayed persistent hypotonia. In addition, 11 patients in four studies with examined growth records all exhibited postnatal growth retardation. However, patient 1 in this study showed normal postnatal growth. Other frequently reported phenotypes, such as microcephaly, facial features, and seizures, are variable among individuals and between family members. Moreover, our two patients presented with diverse behavioral abnormalities, such as arm flapping and a happy disposition for patient 1 and self-injury behaviors for patient 2. One common and major manifestation of UNC80 mutations is severe speech impairment, which is also a major feature of AS. Such impairment was particularly evident in patient 1, who exhibited many characteristics that mimic those of AS and was first suspected of having AS. There are also AS-like genetic syndromes, including microdeletion/ duplication syndromes and single-gene syndromes in which severe DD/ ID and speech impairment are commonly present, and characteristics such as hypotonia, growth retardation, microcephaly, seizures, sleep disturbance, and behavior disturbance, among others, have frequently been observed in these syndromes, which may confound diagnosis (Table 1). Tan et al. (2014) listed key features of such syndromes to
b
a
PHMDS: +; KDVS: +; AS: + MRD1: +; AS: + Kleefstra syndrome: +; RTT: +; EIEE2: +; FOXG1 haploinsufficiency syndrome: +; AS: + PTHS: +; MRXSCH: +; MOWS: +; MRT38: +; ADSLD: +; RTT: +; MRD20: +; ATRX: +; FOXG1 haploinsufficiency syndrome: +; AS: + RTT: +; EIEE2: +; MRXS13: +; MRD20: +; FOXG1 haploinsufficiency syndrome: +; AS: + + + − − − 5/6 5/6 2/6 2/16 13/16 − − + − − − + − NA + Feeding difficulties Constipation Sleep disturbance Brain MRI abnormalities EEG abnormalities
Patient 2 Patient 1 Subject
Table 1 (continued)
IHPRF2a in the literature
IHPRF1b
AS/AS-like syndromesf
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Y. He et al.
Municipal Commission of Health and Family Planning Foundation (201540054 to BX; 20154Y0153 and 2017YQ020 to YS), and Shanghai Jiao Tong University School of Medicine (2014XJ10044 to YS).
help clinicians determine which of these syndromes should be further considered in differential diagnosis. To an extent, the clinical features of UNC80-related IHPRF2 mimic those of these AS-like syndromes. Therefore, when patients manifest hypotonia, speech impairment and inappropriately happy demeanors that include frequent smiling and excitability, UNC80-related IHPRF2 should be considered. The other subtype of IHPRF is IHPRF1, which is caused by biallelic NALCN mutations. With the exception of peripheral nervous system symptoms such as neuroaxonal dystrophy, sural nerve edema and decreased motor nerve conduction velocity, the clinical manifestations of IHPRF1 caused by NALCN mutations overlap with those of IHPRF2 and include hypotonia, DD/ID, facial characteristics and postnatal growth retardation (Table 1). This extensive similarity can be explained based on the intimate relationship between UNC80 and NALCN. UNC79 and NALCN are bridged by UNC80 to form a functional complex that is involved in the formation and maintenance of neuromembrane resting potential (Cochet-Bissuel et al., 2014; Lu et al., 2010). Defects in UNC80/NALCN disrupt the integrity of this complex and therefore result in related, similar phenotypes. In summary, we provide the first report of two unrelated Chinese patients with compound heterozygous UNC80 mutations. Most of our patients' phenotypic characteristics were consistent with IHPRF2, with the exception of a lack of postnatal growth retardation in one patient. Although both patients carried UNC80 mutations, they exhibited some variable phenotypes. Furthermore, we summarized the diverse phenotypes of IHPRF2 patients documented in the literature, and we found the phenotypes of IHPRF2 to be heterogeneous. To a certain degree, the manifestations of UNC80-related IHPRF2 resemble those of AS-like syndromes. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.gene.2018.03.063.
References Cochet-Bissuel, M., Lory, P., Monteil, A., 2014. The sodium leak channel, NALCN, in health and disease. Front. Cell. Neurosci. 8, 132. Fokkema, I.F., Taschner, P.E., Schaafsma, G.C., Celli, J., Laros, J.F., den Dunnen, J.T., 2011. LOVD v.2.0: the next generation in gene variant databases. Hum. Mutat. 32 (5), 557–563. Lu, B., Zhang, Q., Wang, H., Wang, Y., Nakayama, M., Ren, D., 2010. Extracellular calcium controls background current and neuronal excitability via an UNC79-UNC80NALCN cation channel complex. Neuron 68 (3), 488–499. Perez, Y., Kadir, R., Volodarsky, M., Noyman, I., Flusser, H., Shorer, Z., Gradstein, L., Birnbaum, R.Y., Birk, O.S., 2016. UNC80 mutation causes a syndrome of hypotonia, severe intellectual disability, dyskinesia and dysmorphism, similar to that caused by mutations in its interacting cation channel NALCN. J. Med. Genet. 53 (6), 397–402. Richards, S., Aziz, N., Bale, S., Bick, D., Das, S., Gastier-Foster, J., Grody, W.W., Hegde, M., Lyon, E., Spector, E., Voelkerding, K., Rehm, H.L., Committee Acmg Laboratory Quality Assurance, 2015. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet. Med. 17 (5), 405–424. Shamseldin, H.E., Faqeih, E., Alasmari, A., Zaki, M.S., Gleeson, J.G., Alkuraya, F.S., 2016. Mutations in UNC80, encoding part of the UNC79-UNC80-NALCN channel complex, cause autosomal-recessive severe infantile encephalopathy. Am. J. Hum. Genet. 98 (1), 210–215. Stray-Pedersen, A., Cobben, J.M., Prescott, T.E., Lee, S., Cang, C., Aranda, K., Ahmed, S., Alders, M., Gerstner, T., Aslaksen, K., Tetreault, M., Qin, W., Hartley, T., Jhangiani, S.N., Muzny, D.M., Tarailo-Graovac, M., van Karnebeek, C.D., Lupski, J.R., Ren, D., Yoon, G., 2016. Biallelic mutations in UNC80 cause persistent Hypotonia, encephalopathy, growth retardation, and severe intellectual disability. Am. J. Hum. Genet. 98 (1), 202–209. Sun, Y., Hu, G., Liu, H., Zhang, X., Huang, Z., Yan, H., Wang, L., Fan, Y., Gu, X., Yu, Y., 2017. Further delineation of the phenotype of truncating KMT2A mutations: the extended Wiedemann-Steiner syndrome. Am. J. Med. Genet. A 173 (2), 510–514. Tan, W.H., Bird, L.M., Thibert, R.L., Williams, C.A., 2014. If not Angelman, what is it? A review of Angelman-like syndromes. Am. J. Med. Genet. A 164A (4), 975–992. Valkanas, Elise, Schaffer, Katherine, Dunham, Christopher, Maduro, Valerie, du Souich, Christèle, Rupps, Rosemarie, Adams David, R., Baradaran-Heravi, Alireza, Flynn, Elise, Malicdan May, C., Gahl William, A., Toro, Camilo, Boerkoel Cornelius, F., 2016. Phenotypic evolution of UNC80 loss of function. Am. J. Med. Genet. A170 (12), 3106–3114.
Acknowledgments The authors thank both patients' families for participating in the present study. This work was funded by grants from the Shanghai
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