Novel RAB3GAP1 compound heterozygous mutations in Japanese siblings with Warburg Micro syndrome

Novel RAB3GAP1 compound heterozygous mutations in Japanese siblings with Warburg Micro syndrome

Brain & Development xxx (2015) xxx–xxx www.elsevier.com/locate/braindev Case Report Novel RAB3GAP1 compound heterozygous mutations in Japanese sibli...

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Brain & Development xxx (2015) xxx–xxx www.elsevier.com/locate/braindev

Case Report

Novel RAB3GAP1 compound heterozygous mutations in Japanese siblings with Warburg Micro syndrome Miki Asahina a,⇑, Yusaku Endoh b, Tomoko Matsubayashi a, Tokiko Fukuda a, Tsutomu Ogata a b

a Department of Pediatrics, Hamamatsu University School of Medicine, Hamamatsu, Japan Department of Pediatrics, Hamamatsu City Welfare and Medical Center for Development, Hamamatsu, Japan

Received 31 July 2015; received in revised form 11 September 2015; accepted 12 September 2015

Abstract Background: Warburg Micro syndrome (WARBM) is a rare autosomal recessive disease characterized by postnatal growth retardation, microcephaly, severely delayed motor and intellectual development, microcornea, congenital cataracts, optic atrophy, and hypogonadism. While WARBM is a genetically heterogeneous condition, RAB3GAP1 mutations account for 40% of WARBM patients, and 69 different mutations of various types (nonsense, missense, frameshift, and splice site mutations) have been identified to date. Patients: Japanese siblings (a 7 years 3 months old male and a 2 years 1 month old female) were found to have WARBMcompatible phenotypes. Direct sequencing of RAB3GAP1 revealed novel compound heterozygous mutations in the siblings: a paternally inherited missense mutation (c.560G > C; p.Arg187Pro) in exon 7 and a maternally derived nonsense mutation (c.1009C > T; p.Arg337Ter) in exon 12. Conclusion: The siblings had WARBM caused by novel mutations in RAB3GAP1. Since molecular diagnosis permits adequate genetic counseling and appropriate management for predicted complications such as adequate sex steroid supplementation therapy for hypogonadism, in addition to standard supportive therapies for developmental delay and visual dysfunction, we recommend molecular studies for this rare condition. Ó 2015 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

Keywords: Warburg Micro syndrome; RAB3GAP1; Mutation

1. Introduction Warburg Micro syndrome (WARBM) is a rare autosomal recessive disease characterized by postnatal growth retardation, microcephaly, delayed motor development, severe intellectual disability, microcornea,

⇑ Corresponding author at: Department of Pediatrics, Hamamatsu University School of Medicine, 1-20-1-Handayama, Higashi-ku, Hamamatsu 431-3192, Japan. Tel./fax: +81 53 435 2312. E-mail address: [email protected] (M. Asahina).

congenital cataracts, optic atrophy, and hypogonadism [1]. WARBM is caused by mutations of RAB3GAP1 on chromosome 2q21.3, RAB3GAP2 on chromosome 1q41, RAB18 on chromosome 10p21.1, and TBC1D2 on chromosome 20p13 [2–4]. Of these, RAB3GAP1 mutations are most common and account for 40% of WARBM patients [5]. RAB3GAP1 consists of 24 exons and encodes the catalytic subunit of a Rab GTPase activating protein that has specificity for Rab3 subfamily (RAB3A, RAB3B, RAB3C and RAB3D) [2,6]. Rab3 proteins are required for normal eye and brain

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Please cite this article in press as: Asahina M et al. Novel RAB3GAP1 compound heterozygous mutations in Japanese siblings with Warburg Micro syndrome. Brain Dev (2015), http://dx.doi.org/10.1016/j.braindev.2015.09.006

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development [2]. Actually, they are involved in regulated exocytosis of neurotransmitters and hormones, and participate in neurodevelopmental processes such as proliferation, migration and differentiation before synapse formation, and non-synaptic vesicular release of neurotransmitters [7]. Here, we report novel RAB3GAP1 compound heterozygous mutations in siblings with WARBM, and review the RAB3GAP1 mutations reported to date. 2. Case reports The Japanese siblings, a boy aged 7 years 3 months old (case 1) and a girl aged 2 years 1 month old (case 2), were refereed to us because of severe developmental delay. The parents were non-consanguineous and clinically normal. The father was 42 years old and the mother was 27 years old when the elder brother was born. Clinical studies showed quite similar findings in the siblings, including normal birth size, compromised postnatal growth with microcephaly, markedly delayed gross motor development with no head control, severely impaired intellectual ability with no single words, axial hypotonia, limb spasticity, wide nasal bridge, ptosis, micrognathia, large anteverted ears, brain anomalies such as hypoplastic corpus callosum and cortical dysplasia, and ocular abnormalities with bilateral microphthalmia,

microcornea, congenital cataract, persistently constricted pupils, and optic nerve atrophy (Table 1 and Fig. 1A). In addition, case 1 had an episode of febrile convulsion, and exhibited hypogenitalism with micropenis and bilateral cryptorchidism indicative of hypogonadotropic hypogonadism that has been reported in WARBM [1]. His testis volume was 2 ml bilaterally (normal for his age). Thus, after consultation with the parents, we planned to perform endocrine studies for both the siblings in their pubertal ages, to allow for an appropriate hormone replacement therapy. Case 2 showed abnormal electroencephalogram but had no convulsion. On the basis of these findings, the siblings were diagnosed as having WARBMcompatible phenotype. Since the genital findings of the elder brother were consistent with hypogonadotropic hypogonadism indicated in WARBM. 3. Mutation analysis of RAB3GAP1 This study was approved by the Institutional Review Board Committee at Hamamatsu University School of Medicine. After obtaining written informed consent, direct sequencing was performed for all the 24 coding exons and their splice sites of RAB3GAP1, using leukocyte genomic DNA samples of the siblings and the parents. The primer sequences are available on request. The siblings were found to be compound heterozygotes for a paternally inherited missense variant

Table 1 WARBM-compatible clinical features of the siblings.

Age at assessment (yr:mos) Sex Karyotype Gestational age (week) Birth length (cm) (SDS) Birth weight (g) (SDS) Birth OFC (cm) (SDS) Present height (cm) (SDS) Present weight (kg) (SDS) Present OFC (cm) (SDS) Delayed motor development Intellectual disability Axial hypotonia Limb spasticity Seizures EEG Brain MRI

Microphthalmia Microcornea Congenital cataract Persistently constricted pupils Optic nerve atrophy Hypogenitalism

Case 1

Case 2

7:03 Male 46, XY 40 49.8 (+0.2) 3246 (+0.4) 31.8 ( 1.2) 105.0 ( 3.3) 12.2 ( 2.7) 45.3 ( 4.4) + (No head control) + (no single words) + + + (Febrile seizure) Not examined Hypoplastic corpus callosum Cortical dysplasia Ventricular deformity + (Bilateral) + (Bilateral) + (Bilateral) + (Bilateral) + (Bilateral) Micropenis Cryptorchidism (bilateral)

2:01 Female 46, XX 39 49.5 (+0.1) 2970 ( 0.3) 31.5 ( 1.4) 73.9 ( 3.7) 7.3 ( 4.8) 42.0 ( 3.6) + (No head control) + (no single words) + + Abnormal Hypoplastic corpus callosum Frontal cortical dysplasia + (Bilateral) + (Bilateral) + (Bilateral) + (Bilateral) + (Bilateral) Normal (female)

SDS: standard deviation score; OFC: occipitofrontal circumference; EEG: electroencephalogram; and MRI: magnetic resonance imaging.

Please cite this article in press as: Asahina M et al. Novel RAB3GAP1 compound heterozygous mutations in Japanese siblings with Warburg Micro syndrome. Brain Dev (2015), http://dx.doi.org/10.1016/j.braindev.2015.09.006

M. Asahina et al. / Brain & Development xxx (2015) xxx–xxx

A

Case 1

T2WI

B Case 1

Case 2

Father

gov/blast/Blast.cgi) (Fig. 1C). The p.Arg187Pro was predicted as ‘‘damaging” with a score of 0.02 by Sorting Intolerant From Tolerant (SIFT, http://sift.bii.a-star. edu.sg/) [8] and as ‘‘probably damaging” with a score of 1.00 (sensitivity 0.00 and specificity 1.00) by Polymorphism Phenotyping-2 (PolyPhen-2, http://genetics.bwh. harvard.edu/pph2/) [9]. The p.Arg337Ter was predicted to satisfy the condition for the occurrence of nonsensemediated mRNA decay [2,5]. In addition, a known SNP (c.2265T > C; p.Phe755Leu, rs17261772) were identified in both of the siblings.

Case 2

T1WI

T1WI

T2WI

Exon 7

4. Discussion

Exon 12

*

*

TGGTGTACGAA CTGA TGGTGTACC

TTGCCGTCGAAAGGA TTGCCGTT

TGGTGTACGAA CTGA TGGTGTACC

TTGCCGTCGAAAGGA TTGCCGTT

TGGTGTACGAA CTGA TGGTGTACC

TTGCCGTCGAAAGGA

TGGTGTACGAA CTGA

TTGCCGTCGAAAGGA TTGCCGTT

*

*

*

Mother

*

C

3

Homo sapiens Pan troglodytes Mus musculus Gallus gallus Takifugu rubripes Drosophila melanogaster

Q Q Q Q L L

G G G G G A

P P P P P P

G G G G G A

V V V V L L

187 R R R R R R

T T T T T T

D D D D D N

F F F F F F

E E E E E R

M M M M M L

Homo sapiens Pan troglodytes Mus musculus Gallus gallus Takifugu rubripes Drosophila melanogaster

F F L F F -

K K K K K -

I I I L L -

C C C C C V

R R R R C A

337 R R R R I E

K K K K K V

E E E E E T

S S S S T R

T T T T A S

D D D D E L

Fig. 1. Clinical and molecular findings of the siblings. (A) Facial photographs and magnetic resonance imaging scans. (B) Electrochromatograms showing a paternally derived missense mutation in exon 7 (c.560G > C; p.Arg187Pro) and a maternally inherited nonsense mutation in exon 12 (c.1009C > T; p.Arg337Ter) (shown with asterisks). (C) Conservation of the arginine residues at exon 7 and exon 12.

(c.560G > C; p.Arg187Pro) in exon 7 and a maternally derived nonsense variant (c.1009C > T; p.Arg337Ter) in exon 12 (Fig. 1B). The p.Arg187Pro and the p. Arg337Ter were absent from public databases including Japanese SNP Database (http://snp.ims.u-tokyo.ac.jp/), and the 187th and the 337th arginine residues were evolutionary conserved (http://www.ncbi.nlm.nih.

The siblings had WARBM-compatible phenotype and compound heterozygous variants in RAB3GAP1. For phenotypic assessment, while most features such as growth and developmental delay and hypogenitalism are rather non-specific, ocular manifestations appear to be pathognomonic for WARBM. Indeed, the constellation of ocular features permitted us to make a clinical diagnosis of WARBM in the siblings. In addition, while the MRI findings were somewhat different between the siblings, both of them showed hypoplastic corpus callosum and cortical dysplasia such as the frontal and parietal polymicrogyria that appear to be typical for WARBM [5], although ventricular deformity in case 1 would be rather non-specific. To our knowledge, however, such MRI findings are commonly observed in WARBM irrespective of the type of mutant genes, and there is no MRI findings characteristic of RAB3GAP1 mutations. For molecular studies, although functional studies have not been performed for the variants, in silico analysis suggests that both variants are pathologic mutations. These findings imply that the siblings have molecularly confirmed WARBM. Molecular diagnosis would have a clinical significance. Indeed, an adequate genetic counseling becomes possible for a next child. Furthermore, appropriate management would become available for predicted complications, in addition to standard supportive therapies for developmental delay and visual dysfunction. In this regard, WARBM is associated with hypogonadism presumably of hypothalamic origin [1]. Actually, hypogenitalism such as micropenis and cryptorchidism in case 1 is consistent with hypogonadotropic hypogonadism of hypothalamic origin [10]. Thus, adequate sex steroid supplementation therapy is suggested from the pubertal period of the siblings, to keep good body conditions such as normal bone mineral density. To date, a total of 69 different mutations of various types (nonsense, missense, frameshift, and splice site mutations) have been identified in RAB3GAP1 of 65 families (Supplementary Table 1) [2,5]. Notably, there is no mutation hot spot. This implies that each patient has his/her own private mutations, so that all the 24

Please cite this article in press as: Asahina M et al. Novel RAB3GAP1 compound heterozygous mutations in Japanese siblings with Warburg Micro syndrome. Brain Dev (2015), http://dx.doi.org/10.1016/j.braindev.2015.09.006

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exons should be examined in each patient. Indeed, both mutations identified in the siblings have not been reported previously. It is also notable that apparently homozygous mutations are more prevalent than compound heterozygous mutations. In summary, we identified novel compound heterozygous mutations of RAB3GAP1 in siblings WARBM. Further studies will permit better understanding of the genetic background for WARBM. Disclosure The authors have nothing to declare. Funding This work was supported by Student Grant application of Hamamatsu University School of Medicine 2013–2014.

Acknowledgments We thank the patients and the parents for participating to this study. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10. 1016/j.braindev.2015.09.006.

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Please cite this article in press as: Asahina M et al. Novel RAB3GAP1 compound heterozygous mutations in Japanese siblings with Warburg Micro syndrome. Brain Dev (2015), http://dx.doi.org/10.1016/j.braindev.2015.09.006