- Email: [email protected]
EXOME REPORT: Novel mutation in ATP6V1B2 segregating with autosomal dominant epilepsy, intellectual disability and mild gingival and nail abnormalities
Journal Pre-proof EXOME REPORT: Novel mutation in ATP6V1B2 segregating with autosomal dominant epilepsy, intellectual disability and mild gingival and nail abnormalities Marie Shaw, Anna Winczewska-Wiktor, Magdalena Badura-Stronka, Sunita Koirala, Alison Gardner, Łukasz Kuszel, Piotr Kowal, Barbara Steinborn, Monika Starczewska, Sarah Garry, Ingrid Scheffer, Samuel F. Berkovic, Jozef Gecz PII:
S1769-7212(19)30275-7
DOI:
https://doi.org/10.1016/j.ejmg.2019.103799
Reference:
EJMG 103799
To appear in:
European Journal of Medical Genetics
Received Date: 17 April 2019 Revised Date:
2 October 2019
Accepted Date: 20 October 2019
Please cite this article as: M. Shaw, A. Winczewska-Wiktor, M. Badura-Stronka, S. Koirala, A. Gardner, Ł. Kuszel, P. Kowal, B. Steinborn, M. Starczewska, S. Garry, I. Scheffer, S.F. Berkovic, J. Gecz, EXOME REPORT: Novel mutation in ATP6V1B2 segregating with autosomal dominant epilepsy, intellectual disability and mild gingival and nail abnormalities, European Journal of Medical Genetics (2019), doi: https://doi.org/10.1016/j.ejmg.2019.103799. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Masson SAS.
EXOME REPORT: Novel mutation in ATP6V1B2 segregating with autosomal dominant epilepsy, intellectual disability and mild gingival and nail abnormalities. #1
#2
3
1,4
1
Marie Shaw , Anna Winczewska-Wiktor , Magdalena Badura-Stronka , Sunita Koirala , Alison Gardner , 3
5
2
2
7
7
Łukasz Kuszel , Piotr Kowal , Barbara Steinborn Monika Starczewska , Sarah Garry , Ingrid Scheffer , Samuel. 6
1,8,9
F Berkovic , Jozef Gecz
1.
*
Adelaide Medical School, Faculty of Health and Medical Sciences, The University of Adelaide, Adelaide, Australia SA 5000.
2.
Department of Developmental Neurology, Poznan University of Medical Sciences, Poznan, Poland.
3.
Department of Medical Genetics, Poznan University of Medical Sciences, Poznan, Poland.
4.
Central Department of Biotechnology, Tribhuvan University, Kathmandu, Nepal.
5.
Department of Neurology, Poznan University of Medical Sciences, Poznan, Poland.
6.
Departments of Medicine and Paediatrics, The University of Melbourne, Austin Health and Royal
7.
Epilepsy Research Centre, Department of Medicine, University of Melbourne, Melbourne, VIC, Australia.
8.
Robinson Research Institute, The University of Adelaide, Adelaide, SA, Australia SA 5000.
9.
Healthy Mothers, Babies and Children, South Australian Health and Medical Research Institute,
Children's Hospital and Florey Institute, Victoria, Australia.
Adelaide SA 5000, Australia.
# Equal First Author. * Corresponding Author
Key words: ATP6V1B2; epilepsy; autosomal dominant inheritance; Zimmerman-Laband Syndrome; DeafnessOnychodystrophy Syndrome.
1
1.
Abstract.
Mutations in ATP6V1B2, which encodes the B2 subunit of the vacuolar H+ ATPase have previously been associated with Zimmermann-Laband syndrome 2 (ZLS2) and deafness-onychodystrophy (DDOD) syndrome. Recently epilepsy has also been described as a potentially associated phenotype. Here we further uncover the role of ATP61VB2 in epilepsy and report autosomal dominant inheritance of a novel missense variant in ATP6V1B2 in a large Polish family with relatively mild gingival and nail problems, no phalangeal hypoplasia and with generalized epilepsy. In light of our findings and review of the literature, we propose that the ATP6V1B2 gene should be considered in families with autosomal dominant epilepsy both with or without intellectual disability, and that presence of subtle gingival and nail problems may be another characteristic calling card of affected individuals with ATP6V1B2 mutations.
2.
Introduction.
Mutations in ATP6V1B2, which encodes the B2 subunit of the vacuolar H+ ATPase have thus far been reported in two affected individuals with a severe form of Zimmermann-Laband syndrome [Castori, et al., 2013, Kortum, et al., 2015] and four affected individuals with deafness-onychodystrophy (DDOD) syndrome [Yuan, et al., 2014 , Menendez, et al., 2017]. All of these cases were reported to have aplastic or hypoplastic nails, and most of the affected individuals also presented with hearing loss and aplastic or hypoplastic phalanges. Other features identified in previously reported individuals include gingival enlargement, coarse facies, hypertrichosis, triphalangeal thumb, scoliosis and intellectual disability. The clinical picture amongst these reported cases is highly variable, and all previously identified mutations appear de novo. Recently, an additional de novo variant in ATP6V1B2 was reported [Popp, et al., 2017] in an individual with severe ID, hypotonia, microcephaly and seizures. However, the authors inferred an uncertainty or “suspicion” of causality of the finding as the affected individual lacked some of the typical features of Zimmermann-Laband syndrome (hypertrichosis, gingival hyperplasia, and dystrophic nails). In this report, we describe a large, multigenerational Polish family with relatively mild (inconsistent) gingival and nail problems, no phalangeal hypoplasia, all with epilepsy, and exhibiting autosomal dominant inheritance of an ATP6V1B2 missense variant. This variant, identified by Whole Exome Sequencing (WES), further establishes evidence of the association of ATP6V1B2 with epilepsy without the typical features of ZLS, and exposes a novel inheritance mechanism for ATP6V1B2 mutation and disease.
3.
Clinical description
2
We have clinically examined 6 of 7 of the affected individuals from this family. We were unable to examine affected individual I:1 at this time. A three-generational family pedigree is presented in Figure 1, and a summary of the main clinical characteristics is presented in comparison to those cases reported thus far (Table 1). Affected individuals II:1, II:3, II:4 and III:3 present with nail morphology of ‘half-and-half nails’, typically associated with severe renal or liver insufficiency (Supplementary Figure 1(a+b)). Renal and liver function is normal however in all studied affected individuals. In two affected individuals (II:4 and III:3) we observed onychodystrophy of the thumb nails and fifth toe nails (Supplementary Figure 1(a+b)). X-ray of hands and feet showed no abnormalities in all affected individuals. Three out of the six affected individuals examined have mild gingival enlargement, whilst the remaining demonstrate only subtle signs of mild gingivitis (frequent bleeding, red rim surrounding dental crowns, aphtha) (Supplementary Figure 1(c)). In all cases examined, pregnancy, delivery, and early psychomotor development were described as normal. A detailed description of the neurological aspects in this family is presented in Table 2. Female affected individual I:1. Seizures developed in early childhood and remitted at 14 years of age. Treatment with valproic acid was successful. There was no EEG data and an epilepsy syndrome could not be determined. She no longer takes any antiepileptic drugs and remains seizure-free. Female affected individual II:1. Generalized, tonic-clonic seizures occurred at 14 years of age and were wellcontrolled with valproic acid, with a frequency of seizures of 1 every 3-4 years. The EEG background activity was normal, but typical generalized spike-and-wave discharges in wakefulness and sleep were observed; hyperventilation and intermittent photic stimulation did not activate the discharges. The electroclinical phenotype was of Epilepsy with generalized with tonic-clonic seizures alone [Berg, et al., 2010]. There were no dysmorphic features. Male affected individual II:3. Generalized tonic–clonic seizures began at 9 years of age and was wellcontrolled on valproic acid; the last attack was at age 44 years. The EEG showed normal background activity with generalized spike-and-wave at 2.5-3 Hz, not activated by photic stimulation or hyperventilation. The electroclinical phenotype was also of Epilepsy with generalized with tonic-clonic seizures alone. Dysmorphological examination revealed moderate hirsutism, thick eyebrows, and a thin upper lip. Male affected individual II:4. Convulsive seizures were observed in infancy. EEG testing was not obtainable, and a syndrome diagnosis could not be made. Currently, he is not prescribed any medication and is seizure-free. Dysmorphological examination showed thick eyebrows and a thin upper lip. Female affected individual III:1. She had her first generalized tonic–clonic seizure at age 16 years and is well controlled on valproic acid. Her EEG background was normal but generalized epileptiform spike-and-wave or polyspike-and-wave (3-3.5 Hz) lasting up to 3 seconds were observed without clinical signs. These were noted from age 15 when she was investigated for difficulties at school, when the epilepsy began and when last examined in middle life.
Spike-wave discharges were activated by photic stimulation. but not by hyperventilation. The
3
electroclinical phenotype was Epilepsy with generalized with tonic-clonic seizures alone.
She has mild
hypertelorism, posteriorly rotated ears, a pit on ear helix, wide nasal bridge, and mildly hypoplastic nostrils. Female affected individual III:2. She developed typical absence seizures at the age of 3. At 5 years old, myoclonic and atonic seizures appeared. Interictal EEG background was normal with generalized spike-and-wave and polyspike-and-wave discharges. Hyperventilation did not provoke EEG abnormality. A clinical absence with upper limb myoclonus was recorded and showed generalized 2.5-3Hz spike-and-wave and polyspike-and-wave, lasting 20 seconds. She was treated using valproic acid without satisfactory control of seizures. After 8 years without seizures, valproic acid was ceased. The electroclinical phenotype was consistent with Epilepsy with myoclonic atonic seizures. She has a wide nasal bridge, thick eyebrows, posteriorly rotated ears, right facial palsy, short philtrum. Female affected individual III:3. She underwent neurological assessment due to behavioural issues at 6 years of age. An EEG showed normal background with brief bursts of fast (3-6 Hz) generalized polyspike-andwave and spike-and-wave discharges . Absence seizures with eyelid myoclonia were recorded (Supplementary Figure 1d). She was refractory to treatment with valproic acid, topiramate and lamotrigine. At 9 years of age, ethosuximide led to some improvement with shortening of absence attacks. Behavioural disturbances increased and in teenage, generalized tonic-clonic seizures occurred and absence seizures intensified. At the age of 16, non-convulsive status epilepticus of absence seizures occurred. Her EEG showed active generalized epileptiform discharges, with some focal discharges, without activation by hyperventilation or intermittent photic stimulation. The electroclinical phenotype was Absences with eyelid myoclonias. She has no dysmorphic features.
4.
Methods.
Genetic studies were approved by all local human research ethics committees and written informed consent was obtained from all participating individuals. DNA from II:3 and III:1 (Figure. 1(a)) was extracted from whole-blood (QIAamp DNA blood maxi kit; Qiagen, Limburg, Netherlands) and screened by WES. WES (Agilent SureSelect v6) was performed on a HiSeq2500 (Illumina) by the Australian Genome Research Facility. Reads were mapped to the human genome (Hg19) using BWA-MEM [Li and Durbin, 2009] and mapping refined using Genome Analysis Toolkit (GATK) version 3.5 [DePristo et al., 2011]. Mapping achieved a minimum median target coverage depth of 39 reads/sample and covered 94% of intended targets with at least 10 reads. Single nucleotide variants and small insertions and deletions were called by the GATK haplotype caller version 3.5 [DePristo et al., 2011]. Larger copy number and structural variants were analysed by CoNIFER [Krumm et al., 2012]; however, no segregating, rare copy number variants were detected. All variants were annotated for allele frequency, clinical significance, locus identity, and likely pathogenicity using ANNOVAR [Wang et al., 2010]. Novel genotypes shared between affected individuals,
4
but absent from a control set of 5 exomes, were separated using the vcf-contrast module from VCFtools [Danecek et al., 2011]. Amino acid sequences were aligned using ClustalW (MegAlign; DNAStar, Wisconsin, USA). Sanger sequencing using standard methods was used to confirm and segregate the ATP6V1B2 variant in genomic DNA through the family, using primers flanking the ATP6V1B2 gene variant: F - 5’- GAC AGT AGG ATT CAT CTA GGA G -3’ and R - 5’- ATA GCA TTA GAC ATG GGT TTC -3’.
5.
Results
Variants remaining after filtering (Supplementary Table 1) were manually assessed based on frequency / presence in known SNP databases, any previous association with disease, predicted functional impact, any nucleotide / amino acid conservation, and the potential detrimental biochemistry of any amino acid substitution observed (CADD>25). Only one variant remained after the filtering process, ATP6V1B2. Alignment
and
coverage
statistics
are
in
Supplementary
Table
2.
The
ATP6V1B2:
NG_047013.1(NM_001693):c.1192C>G variant was confirmed by Sanger sequencing (Figure 1 (b)) and segregated with the phenotype in this family. The leucine (Leu) change to valine (Val) at position 398 in the protein occurs at a base which is completely conserved in available vertebrate species, and is predicted to be “damaging” (score 0: SIFT, http://blocks.fhcrc.org/sift/SIFT.html) and “probably damaging” (score 0.999: PolyPhen2: http://genetics.bwh.harvard.edu/pph2/) with a CADD [Kircher et al., 2014] score prediction of “deleterious”, at 25.2 This variant affects a conserved residue in the V-type (H+) ATP-ase domain. There are no missense
variants
at
this
position
currently
(http://exac.broadinstitute.org/gene/ENSG00000147416)
reported or
in
ExAC gnomAD
(http://gnomad.broadinstitute.org/gene/ENSG00000147416). Information regarding this variant has been submitted to the LOVD (Leiden Open Variation Database), genomic variant #0000473757. Amino acid sequences from both ATP6V1B2 orthologs and paralogs were aligned using ClustalW method. Alignment of orthologs showed that the mutated ATP6V1B2 amino acid residue at position 398 is in fact invariable across all species, however only selected representative species were shown (Figure 1(c)). Upon aligning the paralogous ATP6V1B2 gene family, some variation was observed at position 398. Upon closer examination of phylogeny though (Figure 1(d)), it was clear that the ATP6V1B2 and ATP6V1B1 proteins cluster together evolutionarily and share the same residue at this position, whilst it is only those less-closely related proteins which vary at residue 398 as well as the surrounding amino acids. A summary of the main clinical characteristics (Table 1), in comparison to previously reported cases indicates that there are three major phenotypic subgroupings of patient with variants in ATP6V1B2. Those individuals with autosomal dominant congenital deafness with onychodystrophy (DODD) and Zimmermann-Laband syndrome 2
5
(ZLS2) have variants clustering towards the carboxy terminal of the ATP6V1B2 protein / V-ATPase_V1_B Domain (Figure 2). Conversely, individuals reported to have intellectual disability, epilepsy and milder ZLS2-type characteristics, cluster together further towards the middle of the ATP6V1B2 protein / V-ATPase_V1_B Domain.
6.
Discussion.
Using WES, we have identified a novel missense variant in ATP16V1B2 in a family with epilepsy, intellectual disability and mild gingival and nail abnormalities. In silico analyses, segregation testing and the online resources Polyphen2, Sift and CADD provide additional evidence of this variant’s pathogenicity. Mutations in ATP6V1B2 are a well-established cause of autosomal dominant Zimmermann-Laband syndrome 2, as well as autosomal dominant congenital deafness with onychodystrophy [Castori, et al., 2013, Yuan, et al., 2014, Kortum, et al., 2015, Menendez, et al., 2017]. The typical symptoms of ZLS2 include significant gingival hyperplasia, hypoplasia of terminal phalanges and nails, whilst DDOD is associated with deafness, anonychia, hypoplasia of nails, dystrophic nails, phalangeal hypoplasia and/or other hand and feet malformations [Castori, et al., 2013, Yuan, et al., 2014, Kortum, et al., 2015, Menendez, et al., 2017]. We have reported here a family where 3 out of 7 affected individuals exhibit mild gingival enlargement. Other gingival and teeth abnormalities such as severe caries, early loss of teeth, gingivitis and aphtha were detected in 5 cases. None of our family members had either hypoplastic/aplastic nails nor hypoplastic/aplastic phalanges, the latter was confirmed by radiograms of hands and feet (data not shown). We have observed mild nail involvement in 4 out of 7 affected individuals from the studied family. We frequently saw whitening of almost the entire nail bed in these individuals, or half-and-half nails (Lindsay`s nails) which is traditionally observed in chronic kidney disease or Terry`s nails, which is observed in affected individuals with chronic congestive heart failure, cirrhosis or diabetes [Holzberg, 1984, Lindsay, 2015]. Two of our affected individuals also presented with onychodystrophy of single toes. Dysmorphic features have been previously described in affected individuals with ZLS including soft, thick ear helices and lobules, thick laterally flared eyebrows, long eyelashes, bulbous, fleshy nose with soft nose cartilage, broad nasal bridge, prominent nasal septum or even bifid nasal tip or cutaneous-cartilaginous ridging on nasal tip [Castori, et al., 2013]. In this family however, dysmorphism was present in some but not all affected persons and only comprised thick eyebrows, thin upper lip, posteriorly rotated ears, ear pits, prominent nose with wide nasal bridge. In our opinion these dysmorphic features are too subtle to inspire clinical geneticists to suspect diagnosis of ZLS in the absence of severe gingival component. In this family, all 7 affected subjects had epilepsy as the main symptom. All affected individuals described have experienced or still experience epileptic seizures, with the first seizure appearing between early childhood and adolescence (16 years). In 5/7 the epilepsy type was proven generalized epilepsy based on the seizure semiology and EEG; in the other two generalized epilepsy was suspected but not proven. In 3 cases the specific
6
syndrome was Epilepsy with generalized tonic-clonic seizures, with single cases of Epilepsy with myoclonic-atonic seizures. and Absences with eyelid myoclonia. EEG examination, in 5 studied individuals showed normal background activity with interictal generalised discharges (spike-and- wave and polyspike-and-wave) lasting 1-2 seconds. In one affected individual focal discharges were also detected. In families with generalized epilepsy heterogeneous syndromes are often seen [Epi4K Consortium, 2017] as shown here. In most such families the genetic architecture is believed to be oligogenic or polygenic, but rare families segregate an autosomal dominant gene. These include SLC2A1, GABRG2 and GABRA1 [Mullen, et al., 2018]. ATP6V1B2 can now be added to this list and should be suspected in families where intellectual disability, and dysmorphic features especially of the gums and nails are observed in some affected subjects. In addition to intellectual disability, observed in 4 affected individuals, neurological examination of this family revealed a postural tremor, seen in 4 out of 7 affected individuals. It may also be a side effect of treatment with antiepileptic drugs, especially valproic acid. MRI examination was not characteristic, two pineal cysts and enlargement of subarachnoid space in frontal spaces were visualised. All affected members had epilepsy. The only other individual reported thus far, with a likely pathogenic variant in ATP6V1B2 and seizure comorbidity, was recently published by Popp et al. [2017]. This affected individual was described as having severe intellectual disability, hypotonia, microcephaly and three seizures. No further seizure detail was provided. It is also worth noting that, Baulac et al. [2008] identified a 13-Mb interval by linkage analysis on chromosome 8p23-p21 in a large family with generalised epilepsy with febrile seizures plus (GEFS+). The members of this family presented with febrile seizures but also afebrile generalized tonic-clonic seizures or absence epilepsy, however, sequencing of the coding sequence of the ATP6V1B2 gene revealed no pathogenic variant [Baulac, et al., 2008]. Review of the current literature, together with this data, suggests that DNA variation in the ATP6V1B2 gene is associated with variable, inter- and intra- familial clinical outcomes. Pathogenic variants in other genes have previously been shown to cause similar variability in clinical expressivity involving nail / orodental abnormalities and epilepsy / intellectual disability. A prime example is the TBC1D24 gene, known to cause DOORS syndrome, with onychodystrophy, deafness, intellectual disability and seizures, but also familial infantile myoclonic epilepsy (FIME), progressive myoclonus epilepsy (PME), early-infantile epileptic encephalopathy 16 (EIEE16), autosomal recessive nonsyndromic hearing loss, DFNB86, and autosomal dominant nonsyndromic hearing loss [Corbett, et al., 2010, Campeau, et al., 2014(1), Campeau, et al., 2014(2)]. The vacuolar ATPase (V-ATPase) family are membrane-bound protein complexes which use the free energy released when ATP is hydrolysed, to drive the transmembrane movement of protons [Harrison, et al., 2018] and are critical to normal cell functioning. The V-ATPases have been implicated in a number of human genetic diseases, including distal renal tubule acidosis and deafness [Karet, et al., 1999], osteoporosis [Frattini, et al., 2000] and of course DDOD and ZLS2 [Castori, et al., 2013, Yuan, et al., 2014, Kortum, et al., 2015, Menendez,
7
et al., 2017], and more recently de novo ID with hypotonia and epilepsy [Popp et al., 2017]. All variants in ATP6V1B2 reported thus far, associated with disease, have arisen de novo. Our multigenerational family is the first example of an autosomal dominant inheritance of epilepsy and ID due to a mutation in ATP6V1B2. This mutation occurs in an amino residue which is invariable across all orthologs, as well as the only closely related member of the human gene family. Interestingly, we also examined that within the ATP6V1B2 protein, variants giving rise to DDOS / ZLS2 cluster towards the carboxy terminal of the ATP6V1B2 protein / V-ATPase_V1_B Domain, whilst variants associated with ID and epilepsy (and in our case milder ZLS2-llike characteristics) reside further central to the protein. However, no major conclusions about genotype/phenotype correlation can yet be made due to the scarcity of ATP6V1B2 variants reported so far. Diagnosis, and in particular prognosis prediction, in affected individuals with mutation in the ATP6V1B2 gene remains a challenge. In light of our findings, we propose that ATP6V1B2 gene variation should certainly be considered in singletons or families with autosomal dominant epilepsy with or without intellectual disability. The presence of subtle gingival and nail problems in the affected individuals might be another useful clinical sign of the ATP6V1B2 gene associated disorder.
7.
Facultative sections.
7.1. Acknowledgements. We thank the members of the families studied for their participation. This work was supported by the Australian National Health and Medical Research Council grants APP101593 and APP1041920, and a statutory grant of Chair and Department of Medical Genetics of PUMS, Poznan, Poland.
7.2. Conflict of Interest statement. Authors do not have any conflict of interest. 7.3. Appendix A. Supplementary data Supplementary data related to this article can be found below.
8.
Figure Headings
Figure 1. (a) Pedigree of the Polish family with variant in ATP6V1B2. Whole exome sequenced individuals are indicated by red asterisks. ATP6V1B2 genotype is indicated where DNA analysis was carried out. (b) ATP6V1B2 Sanger sequencing from family members and wild-type control (WT). Arrow indicates nucleotide variant site (c.1192C>G). (c) Amino acid sequence alignments flanking the p.(Leu398Val) mutated residue (red box) against selected orthologs (top panel) and all paralogs (bottom panel). Sequence alignment of amino acids was performed using CLUSTALW. (d) Phylogenetic tree displaying amino acid substitution per 100 residues.
8
Figure 2. All currently known mutations annotated on a schematic of the ATP6V1B2 protein, associated phenotypes are indicated. A total of 4 different mutations have been reported, including the one presented in this report (in bold). Asterisks denote mutations identified more than once. Supplementary Figure 1. Clinical photographs of affected individuals with mutation in the ATP6V1B2. (a) Halfand-half nails in affected individuals III:3, II:1, II:3 and II:4 with mutation in ATP6V1B2 gene. Onychodystrophy of great toes and 5th toes in affected individuals III:3 and II:4. (b) Half-and-half nails in hands of affected individual III:3 and II:4. (c) Gingivas and teeth of affected individuals with ATP6V1B2 mutation. Mild gingival enlargement and severe caries in affected individuals II:1, II:3, II:4. Signs of mild gingivitis without gingival enlargement in affected individuals III:1, III:2 and III:3. (d) Affected individual III:3 (12 years old at the moment of EEG examination) awake EEG examination. At normal background activity, generalized high amplitude spike and polyspike wave discharges recorded during two subsequent absence episodes with myoclonus of eyelids.
9
9.
Tables
Table 1. Clinical characteristics of all reported affected individuals with ATP6V1B2 variants. AD- autosomal dominant, ID- intellectual disability, ND- no data. Affected individual S7
Affected individual S8
3 affected individuals
Affected individual
Affected individual S_065
c.1454G>C
c.1454G>C
c.1516C>T
c.1516C>T
c.1120G>C
p.(Arg485Pro)
p.(Arg485Pro)
p.(Arg506*)
p.(Arg506*)
p.(Glu374Gln)
[Kortum F. et al., 2015]
[Kortum F. et al., 2015]
[Yuan Y. et al., 2014]
[Menendez I. et al., 2017]
[Popp B. et al., 2017]
II:1
II:3
II:4
III:1
III:2
III:3
This study
This study
This study
This study
This study
This study
Features already described in affected individuals with ATP6V1B2 gene variants ID
+
+
-
-
severe
-
moderate
borderline
-
moderate
mild
Coarse face
+
+
ND
-
ND
-
-
-
-
-
-
Gingival enlargement
+
+
ND
-
-
mild
mild
mild
-
-
-
Hearing loss
+
-
+
+
-
-
-
-
-
-
-
Aplastic or hypoplastic nails
+
+
+
+
-
-
-
-
-
-
-
Aplastic or hypoplastic phalanges
+
+
+
(triphalangeal thumb bilaterally)
-
-
-
-
-
-
-
Scoliosis
+
ND
ND
ND
ND
-
-
-
-
-
-
Hypertrichosis
+
+
ND
-
ND
-
+
+
-
-
-
Features not described so far in affected individuals with ATP6V1B2 gene variants Seizures
-
-
-
EEG normal
+
+
+
+
+
+
+
Half-and-half nails
-
-
-
-
-
+ (feet)
+ (feet)
+ (feet and hands)
-
-
+ (feet)
10
Onychodystrophy
-
-
-
-
-
-
-
+ (1st and 5th toenail)
-
-
+ (1st and 5th toenail)
severe caries, early loss of teeth
gingivitis, aphtha
gingivitis
gingivitis
Other gingival and teeth abnormalities
-
-
-
-
-
-
severe caries, early loss of teeth
Height (cm)
-
-
-
-
-
ND
168
ND
170
172
160
BMI (Z-score)
-
-
-
-
-
ND
1.47
ND
0.50
-0.84
2.68
Birth Weight (Z-score)
-
-
-
-
-
ND
ND
ND
-1.0 > 0.0
-1.0 > 0.0
0.0 > 1.0
Head Circumference (centile)
-
-
-
-
-
ND
>97
ND
75
25-50
25-50
Other features
-
-
-
-
microcephaly, hypotonia
ND
lipoma on right shoulder, lip hematoma
ND
higharched palate
-
brachydactyly
Inheritance
De novo
De novo
De novo
De novo
De novo
AD
AD
AD
AD
AD
AD
Table 2. Detailed presentation of neurological findings in analysed affected individuals. GTCS – generalized tonic-clonic seizures, M – myoclonus , AB – generalized absence, typical, AEM –absence with eyelid myoclonia, BA – background activity, GD – generalised discharges, SWD – spike-and-wave discharges, PSWD – polyspike-and-wave discharges, FD – focal discharges, EEG – electroencephalography, AED – antiepileptic drugs, VPA – valproic acid, TPM – topiramate, LMT – lamotrigine, ETS – ethosuximide, CLB – clobazam, NE – neurological examination, N – normal, T- positional tremor, A- absence, ID- intellectual disability, ND- no data. Affected individual, this study I:1 II:1 II:3 II:4 III:1 III:2
Age of first seizures (in years) ? 14 9 ? 16 3
Type of first seizures ND GTCS GTCS GTCS GTCS AB
III:3
6
AEM
Other seizures M AT GTCS
BA
+
+
+ + + + + +
ND + + ND + +
+
-
+
+
EEG GD SWD PSWD ND ND + + + + ND ND + + + +
+
+
FD
VPA
TPM
AED LMT
ETS
CLB
ID
NE
Brain MRI
Without AED
ND ND -
+ + + + + +
-
+
+
-
+ + +
ND normal ND ND small pineal cysts normal
+ + -
+
+
N N T N T T, facial nerve paresis (since birth) T
+
+
+
+
+
enlargement of subarachnoid spaces in frontal regions, small pineal cyst
-
11
Supplementary Table 1. Summary of variants identified (after filtering) by whole exome sequencing. P – Present. A -Absent. E – Whole Exome Sequencing. S - Sanger Sequencing. Chromosome
Chr8
Chr14
ChrX
Chr12
Chr6
Chr3
Chr2
Chr19
Position
20074761
92537353
135333480
2622093
42932606
4718380
24916179
42880522
Gene name
ATP6V1B2
ATXN3
MAP7D3
CACNA1C
PEX6
ITPR1
NCOA1
MEGF8
refseq
NM_001693
NM_001164782
NM_001173517
NM_000719
NM_000287
NM_001168272
NM_003743
NM_001410
Reference
C
-
C
G
T
G
G
G
50
31
0
35
41
37
36
24
G
G
-
A
C
T
A
A
31
7
13
31
35
33
36
29
Other relative
Sister: P-S
-
-
-
-
-
-
-
tested (NGS or
Brother: P-S
-
-
-
-
-
-
-
Sanger) with the
Sister: A-S
-
-
-
-
-
-
-
result
Daughter: P-E-S
-
-
-
-
-
-
-
Daughter: P-S
-
-
-
-
-
-
-
sequence PROBAND: number of reads with reference PROBAND : alternative PROBAND: num ber of reads with alternative
Mutation type
Niece: P-S
-
-
-
-
-
-
-
Nonsynonymous
Frameshift
Frameshift
Nonsynonymous
Nonsynonymous
Nonsynonymous
Synonymous
Synonymous
SNV
insertion
deletion
SNV
SNV
SNV
SNV
SNV
Mutation : DNA
c.1192C>G
c.68_69insC
c.38delG
c.1333G>A
c.2728A>G
c.2817G>T
c.771G>A
c.7932G>A
Mutation : protein
p.(Leu398Val)
p.(Pro25Thrfs*24)
p.(Ser13Thrfs*4)
p.(Glu445Lys)
p.(Thr910Ala)
p.(Met939Ile)
p.(Thr257Thr)
p.(Pro2644Pro)
Prediction < SIFT
Damaging
-
-
Deleterious
Deleterious
Tolerated
-
-
Prediction <
Probably
-
-
Deleterious
Deleterious
Benign
-
-
PolyPhen-2
Damaging
Prediction <
Deleterious
-
-
Deleterious
Deleterious
Not Deleterious
-
-
-
-
-
-
-
-
-
rs370578585
CADD dbSNP
12
Supplementary Table 2. Alignment Duplicate and Coverage Statistics. Description Reads Total Read pairs mapped Unmapped reads Read length (bp) Duplicate reads Mapped reads singletons Cover over 10x Cover over 50x Number of reads with < q30 bases
II:3
III:1
74728047 74459940 268107 100 24780674 123023 98.84 86.48 5014204
81664087 81454198 209889 100 27560000 112623 98.87 89.2 5314868
13
8. References. Baulac, S., et al. A novel locus for generalized epilepsy with febrile seizures plus in French families. Arch Neurol, 2008. 65(7): p. 943-51. Berg, AT., et al. Revised terminology and concepts for organization of seizures and epilepsies: report of the ILAE Commission on Classification and Terminology, 2005-2009. Epilepsia, 2010. 51(4): p. 676-85. Campeau (1), P.M., et al. DOORS syndrome: phenotype, genotype and comparison with Coffin-Siris syndrome. Am J Med Genet C Semin Med Genet, 2014. 166C(3): p. 327-32. Campeau (2), P.M., et al. The genetic basis of DOORS syndrome: an exome-sequencing study. Lancet Neurol, 2014. 13(1): p. 44-58. Castori, M., et al. Clinical and genetic study of two patients with Zimmermann-Laband syndrome and literature review. Eur J Med Genet, 2013. 56(10): p. 570-6. Corbett MA., et al. A focal epilepsy and intellectual disability syndrome is due to a mutation in TBC1D24. Am J Hum Genet. 2010. 87: p. 371–5. Danecek, et al. The variant call format and VCF tools. Bioinformatics. 2011. 27(15): p. 2156-8. DePristo, et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet. 2011. 43(5): p. 491-8. The Epi4K Consortium. Phenotypic Analysis of 303 Multiplex Families with Common Epilepsies. Brain 2017. 140: p. 2144-2156 Frattini A, et al. Defects in TCIRG1 subunit of the vacuolar proton pump are responsible for a subset of human autosomal recessive osteopetrosis. Nat Genet. 2000. 25(3): p. 343-6. Harrison M.A. Muench S.P. The Vacuolar ATPase – A Nano-scale Motor That Drives Cell Biology. In: Harris J., Boekema E. (eds) Membrane Protein Complexes: Structure and Function. Subcellular Biochemistry. 2018. p. 87. Holzberg M, Walker HK. Terry’s nails: revised definition and new correlations. Lancet. 1984. 1(8382): p. 896–899. Karet F.E., et al. Mutations in the gene encoding B1 subunit of H+-ATPase cause renal tubular acidosis with sensorineural deafness. Nat Genet. 1999. (1): p. 84-90. Kircher, et al. A general framework for estimating the relative pathogenicity of human genetic variants. Nat Genet. 2014. 46(3): p. 310-5. Kortṻm F., et al. Mutations in KCNH1 and ATP6V1B2 cause Zimmermann‐Laband syndrome. Nat. Genet. 2015. 47: p. 661–667. Krumm, et al. Copy number variation detection and genotyping from exome sequence data. Genome Res. 2012. 22(8): p. 1525-32. Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics, 2009. 25(14): p. 1754-60. Lindsay PG. The half-and-half nail. Arch Intern Med. Dermatological Manifestations of Kidney Disease. Place of Publication Not Identified: Springer, 2015. 1967;119(6): p. 583–587. Menendez, I. et al., Dominant deafness-onychodystrophy syndrome caused by an ATP6V1B2 mutation. Clin Case Rep, 2017. 5(4): p. 376-379. Mullen SA, Berkovic SF. ILAE Genetics Commission. Genetic generalized epilepsies. Epilepsia, 2018. 59: p. 1148-1153. Popp, B., et al. Exome Pool-Seq in neurodevelopmental disorders. Eur J Hum Genet, 2017. 25(12): p. 1364-1376. Wang, et al. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 2010. 38(16). e164.
14
Yuan Y., et al. De novo mutation in ATP6V1B2 impairs lysosome acidification and causes dominant deafness‐ onychodystrophy syndrome. Cell Res. 2014. 24: p. 1370–1373.
15
(a)
∂
∂
*
C/G
C/G
C/G
C/C
∂
*
C/G
C/G
C/G
(b)
(c)
(d)
236.8 200
150
100
50
0
1
Exons Amino Acids
1
2 45
3
4
5
6
7
8
9
10
11
12
13
V-ATPase_V1_B Domain
14 509
DEAFNESSONYCHODYSTROPHY SYNDROME
R506X**** (de novo)
R485P** (de novo)
ZIMERMANN-LABAND SYNDROME 2
ID, EPILEPSY, (MILD ZLS FEATURES)
511
E374Q (de novo)
L398V (AD inherited)
S1769-7212(19)30275-7
DOI:
https://doi.org/10.1016/j.ejmg.2019.103799
Reference:
EJMG 103799
To appear in:
European Journal of Medical Genetics
Received Date: 17 April 2019 Revised Date:
2 October 2019
Accepted Date: 20 October 2019
Please cite this article as: M. Shaw, A. Winczewska-Wiktor, M. Badura-Stronka, S. Koirala, A. Gardner, Ł. Kuszel, P. Kowal, B. Steinborn, M. Starczewska, S. Garry, I. Scheffer, S.F. Berkovic, J. Gecz, EXOME REPORT: Novel mutation in ATP6V1B2 segregating with autosomal dominant epilepsy, intellectual disability and mild gingival and nail abnormalities, European Journal of Medical Genetics (2019), doi: https://doi.org/10.1016/j.ejmg.2019.103799. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Masson SAS.
EXOME REPORT: Novel mutation in ATP6V1B2 segregating with autosomal dominant epilepsy, intellectual disability and mild gingival and nail abnormalities. #1
#2
3
1,4
1
Marie Shaw , Anna Winczewska-Wiktor , Magdalena Badura-Stronka , Sunita Koirala , Alison Gardner , 3
5
2
2
7
7
Łukasz Kuszel , Piotr Kowal , Barbara Steinborn Monika Starczewska , Sarah Garry , Ingrid Scheffer , Samuel. 6
1,8,9
F Berkovic , Jozef Gecz
1.
*
Adelaide Medical School, Faculty of Health and Medical Sciences, The University of Adelaide, Adelaide, Australia SA 5000.
2.
Department of Developmental Neurology, Poznan University of Medical Sciences, Poznan, Poland.
3.
Department of Medical Genetics, Poznan University of Medical Sciences, Poznan, Poland.
4.
Central Department of Biotechnology, Tribhuvan University, Kathmandu, Nepal.
5.
Department of Neurology, Poznan University of Medical Sciences, Poznan, Poland.
6.
Departments of Medicine and Paediatrics, The University of Melbourne, Austin Health and Royal
7.
Epilepsy Research Centre, Department of Medicine, University of Melbourne, Melbourne, VIC, Australia.
8.
Robinson Research Institute, The University of Adelaide, Adelaide, SA, Australia SA 5000.
9.
Healthy Mothers, Babies and Children, South Australian Health and Medical Research Institute,
Children's Hospital and Florey Institute, Victoria, Australia.
Adelaide SA 5000, Australia.
# Equal First Author. * Corresponding Author
Key words: ATP6V1B2; epilepsy; autosomal dominant inheritance; Zimmerman-Laband Syndrome; DeafnessOnychodystrophy Syndrome.
1
1.
Abstract.
Mutations in ATP6V1B2, which encodes the B2 subunit of the vacuolar H+ ATPase have previously been associated with Zimmermann-Laband syndrome 2 (ZLS2) and deafness-onychodystrophy (DDOD) syndrome. Recently epilepsy has also been described as a potentially associated phenotype. Here we further uncover the role of ATP61VB2 in epilepsy and report autosomal dominant inheritance of a novel missense variant in ATP6V1B2 in a large Polish family with relatively mild gingival and nail problems, no phalangeal hypoplasia and with generalized epilepsy. In light of our findings and review of the literature, we propose that the ATP6V1B2 gene should be considered in families with autosomal dominant epilepsy both with or without intellectual disability, and that presence of subtle gingival and nail problems may be another characteristic calling card of affected individuals with ATP6V1B2 mutations.
2.
Introduction.
Mutations in ATP6V1B2, which encodes the B2 subunit of the vacuolar H+ ATPase have thus far been reported in two affected individuals with a severe form of Zimmermann-Laband syndrome [Castori, et al., 2013, Kortum, et al., 2015] and four affected individuals with deafness-onychodystrophy (DDOD) syndrome [Yuan, et al., 2014 , Menendez, et al., 2017]. All of these cases were reported to have aplastic or hypoplastic nails, and most of the affected individuals also presented with hearing loss and aplastic or hypoplastic phalanges. Other features identified in previously reported individuals include gingival enlargement, coarse facies, hypertrichosis, triphalangeal thumb, scoliosis and intellectual disability. The clinical picture amongst these reported cases is highly variable, and all previously identified mutations appear de novo. Recently, an additional de novo variant in ATP6V1B2 was reported [Popp, et al., 2017] in an individual with severe ID, hypotonia, microcephaly and seizures. However, the authors inferred an uncertainty or “suspicion” of causality of the finding as the affected individual lacked some of the typical features of Zimmermann-Laband syndrome (hypertrichosis, gingival hyperplasia, and dystrophic nails). In this report, we describe a large, multigenerational Polish family with relatively mild (inconsistent) gingival and nail problems, no phalangeal hypoplasia, all with epilepsy, and exhibiting autosomal dominant inheritance of an ATP6V1B2 missense variant. This variant, identified by Whole Exome Sequencing (WES), further establishes evidence of the association of ATP6V1B2 with epilepsy without the typical features of ZLS, and exposes a novel inheritance mechanism for ATP6V1B2 mutation and disease.
3.
Clinical description
2
We have clinically examined 6 of 7 of the affected individuals from this family. We were unable to examine affected individual I:1 at this time. A three-generational family pedigree is presented in Figure 1, and a summary of the main clinical characteristics is presented in comparison to those cases reported thus far (Table 1). Affected individuals II:1, II:3, II:4 and III:3 present with nail morphology of ‘half-and-half nails’, typically associated with severe renal or liver insufficiency (Supplementary Figure 1(a+b)). Renal and liver function is normal however in all studied affected individuals. In two affected individuals (II:4 and III:3) we observed onychodystrophy of the thumb nails and fifth toe nails (Supplementary Figure 1(a+b)). X-ray of hands and feet showed no abnormalities in all affected individuals. Three out of the six affected individuals examined have mild gingival enlargement, whilst the remaining demonstrate only subtle signs of mild gingivitis (frequent bleeding, red rim surrounding dental crowns, aphtha) (Supplementary Figure 1(c)). In all cases examined, pregnancy, delivery, and early psychomotor development were described as normal. A detailed description of the neurological aspects in this family is presented in Table 2. Female affected individual I:1. Seizures developed in early childhood and remitted at 14 years of age. Treatment with valproic acid was successful. There was no EEG data and an epilepsy syndrome could not be determined. She no longer takes any antiepileptic drugs and remains seizure-free. Female affected individual II:1. Generalized, tonic-clonic seizures occurred at 14 years of age and were wellcontrolled with valproic acid, with a frequency of seizures of 1 every 3-4 years. The EEG background activity was normal, but typical generalized spike-and-wave discharges in wakefulness and sleep were observed; hyperventilation and intermittent photic stimulation did not activate the discharges. The electroclinical phenotype was of Epilepsy with generalized with tonic-clonic seizures alone [Berg, et al., 2010]. There were no dysmorphic features. Male affected individual II:3. Generalized tonic–clonic seizures began at 9 years of age and was wellcontrolled on valproic acid; the last attack was at age 44 years. The EEG showed normal background activity with generalized spike-and-wave at 2.5-3 Hz, not activated by photic stimulation or hyperventilation. The electroclinical phenotype was also of Epilepsy with generalized with tonic-clonic seizures alone. Dysmorphological examination revealed moderate hirsutism, thick eyebrows, and a thin upper lip. Male affected individual II:4. Convulsive seizures were observed in infancy. EEG testing was not obtainable, and a syndrome diagnosis could not be made. Currently, he is not prescribed any medication and is seizure-free. Dysmorphological examination showed thick eyebrows and a thin upper lip. Female affected individual III:1. She had her first generalized tonic–clonic seizure at age 16 years and is well controlled on valproic acid. Her EEG background was normal but generalized epileptiform spike-and-wave or polyspike-and-wave (3-3.5 Hz) lasting up to 3 seconds were observed without clinical signs. These were noted from age 15 when she was investigated for difficulties at school, when the epilepsy began and when last examined in middle life.
Spike-wave discharges were activated by photic stimulation. but not by hyperventilation. The
3
electroclinical phenotype was Epilepsy with generalized with tonic-clonic seizures alone.
She has mild
hypertelorism, posteriorly rotated ears, a pit on ear helix, wide nasal bridge, and mildly hypoplastic nostrils. Female affected individual III:2. She developed typical absence seizures at the age of 3. At 5 years old, myoclonic and atonic seizures appeared. Interictal EEG background was normal with generalized spike-and-wave and polyspike-and-wave discharges. Hyperventilation did not provoke EEG abnormality. A clinical absence with upper limb myoclonus was recorded and showed generalized 2.5-3Hz spike-and-wave and polyspike-and-wave, lasting 20 seconds. She was treated using valproic acid without satisfactory control of seizures. After 8 years without seizures, valproic acid was ceased. The electroclinical phenotype was consistent with Epilepsy with myoclonic atonic seizures. She has a wide nasal bridge, thick eyebrows, posteriorly rotated ears, right facial palsy, short philtrum. Female affected individual III:3. She underwent neurological assessment due to behavioural issues at 6 years of age. An EEG showed normal background with brief bursts of fast (3-6 Hz) generalized polyspike-andwave and spike-and-wave discharges . Absence seizures with eyelid myoclonia were recorded (Supplementary Figure 1d). She was refractory to treatment with valproic acid, topiramate and lamotrigine. At 9 years of age, ethosuximide led to some improvement with shortening of absence attacks. Behavioural disturbances increased and in teenage, generalized tonic-clonic seizures occurred and absence seizures intensified. At the age of 16, non-convulsive status epilepticus of absence seizures occurred. Her EEG showed active generalized epileptiform discharges, with some focal discharges, without activation by hyperventilation or intermittent photic stimulation. The electroclinical phenotype was Absences with eyelid myoclonias. She has no dysmorphic features.
4.
Methods.
Genetic studies were approved by all local human research ethics committees and written informed consent was obtained from all participating individuals. DNA from II:3 and III:1 (Figure. 1(a)) was extracted from whole-blood (QIAamp DNA blood maxi kit; Qiagen, Limburg, Netherlands) and screened by WES. WES (Agilent SureSelect v6) was performed on a HiSeq2500 (Illumina) by the Australian Genome Research Facility. Reads were mapped to the human genome (Hg19) using BWA-MEM [Li and Durbin, 2009] and mapping refined using Genome Analysis Toolkit (GATK) version 3.5 [DePristo et al., 2011]. Mapping achieved a minimum median target coverage depth of 39 reads/sample and covered 94% of intended targets with at least 10 reads. Single nucleotide variants and small insertions and deletions were called by the GATK haplotype caller version 3.5 [DePristo et al., 2011]. Larger copy number and structural variants were analysed by CoNIFER [Krumm et al., 2012]; however, no segregating, rare copy number variants were detected. All variants were annotated for allele frequency, clinical significance, locus identity, and likely pathogenicity using ANNOVAR [Wang et al., 2010]. Novel genotypes shared between affected individuals,
4
but absent from a control set of 5 exomes, were separated using the vcf-contrast module from VCFtools [Danecek et al., 2011]. Amino acid sequences were aligned using ClustalW (MegAlign; DNAStar, Wisconsin, USA). Sanger sequencing using standard methods was used to confirm and segregate the ATP6V1B2 variant in genomic DNA through the family, using primers flanking the ATP6V1B2 gene variant: F - 5’- GAC AGT AGG ATT CAT CTA GGA G -3’ and R - 5’- ATA GCA TTA GAC ATG GGT TTC -3’.
5.
Results
Variants remaining after filtering (Supplementary Table 1) were manually assessed based on frequency / presence in known SNP databases, any previous association with disease, predicted functional impact, any nucleotide / amino acid conservation, and the potential detrimental biochemistry of any amino acid substitution observed (CADD>25). Only one variant remained after the filtering process, ATP6V1B2. Alignment
and
coverage
statistics
are
in
Supplementary
Table
2.
The
ATP6V1B2:
NG_047013.1(NM_001693):c.1192C>G variant was confirmed by Sanger sequencing (Figure 1 (b)) and segregated with the phenotype in this family. The leucine (Leu) change to valine (Val) at position 398 in the protein occurs at a base which is completely conserved in available vertebrate species, and is predicted to be “damaging” (score 0: SIFT, http://blocks.fhcrc.org/sift/SIFT.html) and “probably damaging” (score 0.999: PolyPhen2: http://genetics.bwh.harvard.edu/pph2/) with a CADD [Kircher et al., 2014] score prediction of “deleterious”, at 25.2 This variant affects a conserved residue in the V-type (H+) ATP-ase domain. There are no missense
variants
at
this
position
currently
(http://exac.broadinstitute.org/gene/ENSG00000147416)
reported or
in
ExAC gnomAD
(http://gnomad.broadinstitute.org/gene/ENSG00000147416). Information regarding this variant has been submitted to the LOVD (Leiden Open Variation Database), genomic variant #0000473757. Amino acid sequences from both ATP6V1B2 orthologs and paralogs were aligned using ClustalW method. Alignment of orthologs showed that the mutated ATP6V1B2 amino acid residue at position 398 is in fact invariable across all species, however only selected representative species were shown (Figure 1(c)). Upon aligning the paralogous ATP6V1B2 gene family, some variation was observed at position 398. Upon closer examination of phylogeny though (Figure 1(d)), it was clear that the ATP6V1B2 and ATP6V1B1 proteins cluster together evolutionarily and share the same residue at this position, whilst it is only those less-closely related proteins which vary at residue 398 as well as the surrounding amino acids. A summary of the main clinical characteristics (Table 1), in comparison to previously reported cases indicates that there are three major phenotypic subgroupings of patient with variants in ATP6V1B2. Those individuals with autosomal dominant congenital deafness with onychodystrophy (DODD) and Zimmermann-Laband syndrome 2
5
(ZLS2) have variants clustering towards the carboxy terminal of the ATP6V1B2 protein / V-ATPase_V1_B Domain (Figure 2). Conversely, individuals reported to have intellectual disability, epilepsy and milder ZLS2-type characteristics, cluster together further towards the middle of the ATP6V1B2 protein / V-ATPase_V1_B Domain.
6.
Discussion.
Using WES, we have identified a novel missense variant in ATP16V1B2 in a family with epilepsy, intellectual disability and mild gingival and nail abnormalities. In silico analyses, segregation testing and the online resources Polyphen2, Sift and CADD provide additional evidence of this variant’s pathogenicity. Mutations in ATP6V1B2 are a well-established cause of autosomal dominant Zimmermann-Laband syndrome 2, as well as autosomal dominant congenital deafness with onychodystrophy [Castori, et al., 2013, Yuan, et al., 2014, Kortum, et al., 2015, Menendez, et al., 2017]. The typical symptoms of ZLS2 include significant gingival hyperplasia, hypoplasia of terminal phalanges and nails, whilst DDOD is associated with deafness, anonychia, hypoplasia of nails, dystrophic nails, phalangeal hypoplasia and/or other hand and feet malformations [Castori, et al., 2013, Yuan, et al., 2014, Kortum, et al., 2015, Menendez, et al., 2017]. We have reported here a family where 3 out of 7 affected individuals exhibit mild gingival enlargement. Other gingival and teeth abnormalities such as severe caries, early loss of teeth, gingivitis and aphtha were detected in 5 cases. None of our family members had either hypoplastic/aplastic nails nor hypoplastic/aplastic phalanges, the latter was confirmed by radiograms of hands and feet (data not shown). We have observed mild nail involvement in 4 out of 7 affected individuals from the studied family. We frequently saw whitening of almost the entire nail bed in these individuals, or half-and-half nails (Lindsay`s nails) which is traditionally observed in chronic kidney disease or Terry`s nails, which is observed in affected individuals with chronic congestive heart failure, cirrhosis or diabetes [Holzberg, 1984, Lindsay, 2015]. Two of our affected individuals also presented with onychodystrophy of single toes. Dysmorphic features have been previously described in affected individuals with ZLS including soft, thick ear helices and lobules, thick laterally flared eyebrows, long eyelashes, bulbous, fleshy nose with soft nose cartilage, broad nasal bridge, prominent nasal septum or even bifid nasal tip or cutaneous-cartilaginous ridging on nasal tip [Castori, et al., 2013]. In this family however, dysmorphism was present in some but not all affected persons and only comprised thick eyebrows, thin upper lip, posteriorly rotated ears, ear pits, prominent nose with wide nasal bridge. In our opinion these dysmorphic features are too subtle to inspire clinical geneticists to suspect diagnosis of ZLS in the absence of severe gingival component. In this family, all 7 affected subjects had epilepsy as the main symptom. All affected individuals described have experienced or still experience epileptic seizures, with the first seizure appearing between early childhood and adolescence (16 years). In 5/7 the epilepsy type was proven generalized epilepsy based on the seizure semiology and EEG; in the other two generalized epilepsy was suspected but not proven. In 3 cases the specific
6
syndrome was Epilepsy with generalized tonic-clonic seizures, with single cases of Epilepsy with myoclonic-atonic seizures. and Absences with eyelid myoclonia. EEG examination, in 5 studied individuals showed normal background activity with interictal generalised discharges (spike-and- wave and polyspike-and-wave) lasting 1-2 seconds. In one affected individual focal discharges were also detected. In families with generalized epilepsy heterogeneous syndromes are often seen [Epi4K Consortium, 2017] as shown here. In most such families the genetic architecture is believed to be oligogenic or polygenic, but rare families segregate an autosomal dominant gene. These include SLC2A1, GABRG2 and GABRA1 [Mullen, et al., 2018]. ATP6V1B2 can now be added to this list and should be suspected in families where intellectual disability, and dysmorphic features especially of the gums and nails are observed in some affected subjects. In addition to intellectual disability, observed in 4 affected individuals, neurological examination of this family revealed a postural tremor, seen in 4 out of 7 affected individuals. It may also be a side effect of treatment with antiepileptic drugs, especially valproic acid. MRI examination was not characteristic, two pineal cysts and enlargement of subarachnoid space in frontal spaces were visualised. All affected members had epilepsy. The only other individual reported thus far, with a likely pathogenic variant in ATP6V1B2 and seizure comorbidity, was recently published by Popp et al. [2017]. This affected individual was described as having severe intellectual disability, hypotonia, microcephaly and three seizures. No further seizure detail was provided. It is also worth noting that, Baulac et al. [2008] identified a 13-Mb interval by linkage analysis on chromosome 8p23-p21 in a large family with generalised epilepsy with febrile seizures plus (GEFS+). The members of this family presented with febrile seizures but also afebrile generalized tonic-clonic seizures or absence epilepsy, however, sequencing of the coding sequence of the ATP6V1B2 gene revealed no pathogenic variant [Baulac, et al., 2008]. Review of the current literature, together with this data, suggests that DNA variation in the ATP6V1B2 gene is associated with variable, inter- and intra- familial clinical outcomes. Pathogenic variants in other genes have previously been shown to cause similar variability in clinical expressivity involving nail / orodental abnormalities and epilepsy / intellectual disability. A prime example is the TBC1D24 gene, known to cause DOORS syndrome, with onychodystrophy, deafness, intellectual disability and seizures, but also familial infantile myoclonic epilepsy (FIME), progressive myoclonus epilepsy (PME), early-infantile epileptic encephalopathy 16 (EIEE16), autosomal recessive nonsyndromic hearing loss, DFNB86, and autosomal dominant nonsyndromic hearing loss [Corbett, et al., 2010, Campeau, et al., 2014(1), Campeau, et al., 2014(2)]. The vacuolar ATPase (V-ATPase) family are membrane-bound protein complexes which use the free energy released when ATP is hydrolysed, to drive the transmembrane movement of protons [Harrison, et al., 2018] and are critical to normal cell functioning. The V-ATPases have been implicated in a number of human genetic diseases, including distal renal tubule acidosis and deafness [Karet, et al., 1999], osteoporosis [Frattini, et al., 2000] and of course DDOD and ZLS2 [Castori, et al., 2013, Yuan, et al., 2014, Kortum, et al., 2015, Menendez,
7
et al., 2017], and more recently de novo ID with hypotonia and epilepsy [Popp et al., 2017]. All variants in ATP6V1B2 reported thus far, associated with disease, have arisen de novo. Our multigenerational family is the first example of an autosomal dominant inheritance of epilepsy and ID due to a mutation in ATP6V1B2. This mutation occurs in an amino residue which is invariable across all orthologs, as well as the only closely related member of the human gene family. Interestingly, we also examined that within the ATP6V1B2 protein, variants giving rise to DDOS / ZLS2 cluster towards the carboxy terminal of the ATP6V1B2 protein / V-ATPase_V1_B Domain, whilst variants associated with ID and epilepsy (and in our case milder ZLS2-llike characteristics) reside further central to the protein. However, no major conclusions about genotype/phenotype correlation can yet be made due to the scarcity of ATP6V1B2 variants reported so far. Diagnosis, and in particular prognosis prediction, in affected individuals with mutation in the ATP6V1B2 gene remains a challenge. In light of our findings, we propose that ATP6V1B2 gene variation should certainly be considered in singletons or families with autosomal dominant epilepsy with or without intellectual disability. The presence of subtle gingival and nail problems in the affected individuals might be another useful clinical sign of the ATP6V1B2 gene associated disorder.
7.
Facultative sections.
7.1. Acknowledgements. We thank the members of the families studied for their participation. This work was supported by the Australian National Health and Medical Research Council grants APP101593 and APP1041920, and a statutory grant of Chair and Department of Medical Genetics of PUMS, Poznan, Poland.
7.2. Conflict of Interest statement. Authors do not have any conflict of interest. 7.3. Appendix A. Supplementary data Supplementary data related to this article can be found below.
8.
Figure Headings
Figure 1. (a) Pedigree of the Polish family with variant in ATP6V1B2. Whole exome sequenced individuals are indicated by red asterisks. ATP6V1B2 genotype is indicated where DNA analysis was carried out. (b) ATP6V1B2 Sanger sequencing from family members and wild-type control (WT). Arrow indicates nucleotide variant site (c.1192C>G). (c) Amino acid sequence alignments flanking the p.(Leu398Val) mutated residue (red box) against selected orthologs (top panel) and all paralogs (bottom panel). Sequence alignment of amino acids was performed using CLUSTALW. (d) Phylogenetic tree displaying amino acid substitution per 100 residues.
8
Figure 2. All currently known mutations annotated on a schematic of the ATP6V1B2 protein, associated phenotypes are indicated. A total of 4 different mutations have been reported, including the one presented in this report (in bold). Asterisks denote mutations identified more than once. Supplementary Figure 1. Clinical photographs of affected individuals with mutation in the ATP6V1B2. (a) Halfand-half nails in affected individuals III:3, II:1, II:3 and II:4 with mutation in ATP6V1B2 gene. Onychodystrophy of great toes and 5th toes in affected individuals III:3 and II:4. (b) Half-and-half nails in hands of affected individual III:3 and II:4. (c) Gingivas and teeth of affected individuals with ATP6V1B2 mutation. Mild gingival enlargement and severe caries in affected individuals II:1, II:3, II:4. Signs of mild gingivitis without gingival enlargement in affected individuals III:1, III:2 and III:3. (d) Affected individual III:3 (12 years old at the moment of EEG examination) awake EEG examination. At normal background activity, generalized high amplitude spike and polyspike wave discharges recorded during two subsequent absence episodes with myoclonus of eyelids.
9
9.
Tables
Table 1. Clinical characteristics of all reported affected individuals with ATP6V1B2 variants. AD- autosomal dominant, ID- intellectual disability, ND- no data. Affected individual S7
Affected individual S8
3 affected individuals
Affected individual
Affected individual S_065
c.1454G>C
c.1454G>C
c.1516C>T
c.1516C>T
c.1120G>C
p.(Arg485Pro)
p.(Arg485Pro)
p.(Arg506*)
p.(Arg506*)
p.(Glu374Gln)
[Kortum F. et al., 2015]
[Kortum F. et al., 2015]
[Yuan Y. et al., 2014]
[Menendez I. et al., 2017]
[Popp B. et al., 2017]
II:1
II:3
II:4
III:1
III:2
III:3
This study
This study
This study
This study
This study
This study
Features already described in affected individuals with ATP6V1B2 gene variants ID
+
+
-
-
severe
-
moderate
borderline
-
moderate
mild
Coarse face
+
+
ND
-
ND
-
-
-
-
-
-
Gingival enlargement
+
+
ND
-
-
mild
mild
mild
-
-
-
Hearing loss
+
-
+
+
-
-
-
-
-
-
-
Aplastic or hypoplastic nails
+
+
+
+
-
-
-
-
-
-
-
Aplastic or hypoplastic phalanges
+
+
+
(triphalangeal thumb bilaterally)
-
-
-
-
-
-
-
Scoliosis
+
ND
ND
ND
ND
-
-
-
-
-
-
Hypertrichosis
+
+
ND
-
ND
-
+
+
-
-
-
Features not described so far in affected individuals with ATP6V1B2 gene variants Seizures
-
-
-
EEG normal
+
+
+
+
+
+
+
Half-and-half nails
-
-
-
-
-
+ (feet)
+ (feet)
+ (feet and hands)
-
-
+ (feet)
10
Onychodystrophy
-
-
-
-
-
-
-
+ (1st and 5th toenail)
-
-
+ (1st and 5th toenail)
severe caries, early loss of teeth
gingivitis, aphtha
gingivitis
gingivitis
Other gingival and teeth abnormalities
-
-
-
-
-
-
severe caries, early loss of teeth
Height (cm)
-
-
-
-
-
ND
168
ND
170
172
160
BMI (Z-score)
-
-
-
-
-
ND
1.47
ND
0.50
-0.84
2.68
Birth Weight (Z-score)
-
-
-
-
-
ND
ND
ND
-1.0 > 0.0
-1.0 > 0.0
0.0 > 1.0
Head Circumference (centile)
-
-
-
-
-
ND
>97
ND
75
25-50
25-50
Other features
-
-
-
-
microcephaly, hypotonia
ND
lipoma on right shoulder, lip hematoma
ND
higharched palate
-
brachydactyly
Inheritance
De novo
De novo
De novo
De novo
De novo
AD
AD
AD
AD
AD
AD
Table 2. Detailed presentation of neurological findings in analysed affected individuals. GTCS – generalized tonic-clonic seizures, M – myoclonus , AB – generalized absence, typical, AEM –absence with eyelid myoclonia, BA – background activity, GD – generalised discharges, SWD – spike-and-wave discharges, PSWD – polyspike-and-wave discharges, FD – focal discharges, EEG – electroencephalography, AED – antiepileptic drugs, VPA – valproic acid, TPM – topiramate, LMT – lamotrigine, ETS – ethosuximide, CLB – clobazam, NE – neurological examination, N – normal, T- positional tremor, A- absence, ID- intellectual disability, ND- no data. Affected individual, this study I:1 II:1 II:3 II:4 III:1 III:2
Age of first seizures (in years) ? 14 9 ? 16 3
Type of first seizures ND GTCS GTCS GTCS GTCS AB
III:3
6
AEM
Other seizures M AT GTCS
BA
+
+
+ + + + + +
ND + + ND + +
+
-
+
+
EEG GD SWD PSWD ND ND + + + + ND ND + + + +
+
+
FD
VPA
TPM
AED LMT
ETS
CLB
ID
NE
Brain MRI
Without AED
ND ND -
+ + + + + +
-
+
+
-
+ + +
ND normal ND ND small pineal cysts normal
+ + -
+
+
N N T N T T, facial nerve paresis (since birth) T
+
+
+
+
+
enlargement of subarachnoid spaces in frontal regions, small pineal cyst
-
11
Supplementary Table 1. Summary of variants identified (after filtering) by whole exome sequencing. P – Present. A -Absent. E – Whole Exome Sequencing. S - Sanger Sequencing. Chromosome
Chr8
Chr14
ChrX
Chr12
Chr6
Chr3
Chr2
Chr19
Position
20074761
92537353
135333480
2622093
42932606
4718380
24916179
42880522
Gene name
ATP6V1B2
ATXN3
MAP7D3
CACNA1C
PEX6
ITPR1
NCOA1
MEGF8
refseq
NM_001693
NM_001164782
NM_001173517
NM_000719
NM_000287
NM_001168272
NM_003743
NM_001410
Reference
C
-
C
G
T
G
G
G
50
31
0
35
41
37
36
24
G
G
-
A
C
T
A
A
31
7
13
31
35
33
36
29
Other relative
Sister: P-S
-
-
-
-
-
-
-
tested (NGS or
Brother: P-S
-
-
-
-
-
-
-
Sanger) with the
Sister: A-S
-
-
-
-
-
-
-
result
Daughter: P-E-S
-
-
-
-
-
-
-
Daughter: P-S
-
-
-
-
-
-
-
sequence PROBAND: number of reads with reference PROBAND : alternative PROBAND: num ber of reads with alternative
Mutation type
Niece: P-S
-
-
-
-
-
-
-
Nonsynonymous
Frameshift
Frameshift
Nonsynonymous
Nonsynonymous
Nonsynonymous
Synonymous
Synonymous
SNV
insertion
deletion
SNV
SNV
SNV
SNV
SNV
Mutation : DNA
c.1192C>G
c.68_69insC
c.38delG
c.1333G>A
c.2728A>G
c.2817G>T
c.771G>A
c.7932G>A
Mutation : protein
p.(Leu398Val)
p.(Pro25Thrfs*24)
p.(Ser13Thrfs*4)
p.(Glu445Lys)
p.(Thr910Ala)
p.(Met939Ile)
p.(Thr257Thr)
p.(Pro2644Pro)
Prediction < SIFT
Damaging
-
-
Deleterious
Deleterious
Tolerated
-
-
Prediction <
Probably
-
-
Deleterious
Deleterious
Benign
-
-
PolyPhen-2
Damaging
Prediction <
Deleterious
-
-
Deleterious
Deleterious
Not Deleterious
-
-
-
-
-
-
-
-
-
rs370578585
CADD dbSNP
12
Supplementary Table 2. Alignment Duplicate and Coverage Statistics. Description Reads Total Read pairs mapped Unmapped reads Read length (bp) Duplicate reads Mapped reads singletons Cover over 10x Cover over 50x Number of reads with < q30 bases
II:3
III:1
74728047 74459940 268107 100 24780674 123023 98.84 86.48 5014204
81664087 81454198 209889 100 27560000 112623 98.87 89.2 5314868
13
8. References. Baulac, S., et al. A novel locus for generalized epilepsy with febrile seizures plus in French families. Arch Neurol, 2008. 65(7): p. 943-51. Berg, AT., et al. Revised terminology and concepts for organization of seizures and epilepsies: report of the ILAE Commission on Classification and Terminology, 2005-2009. Epilepsia, 2010. 51(4): p. 676-85. Campeau (1), P.M., et al. DOORS syndrome: phenotype, genotype and comparison with Coffin-Siris syndrome. Am J Med Genet C Semin Med Genet, 2014. 166C(3): p. 327-32. Campeau (2), P.M., et al. The genetic basis of DOORS syndrome: an exome-sequencing study. Lancet Neurol, 2014. 13(1): p. 44-58. Castori, M., et al. Clinical and genetic study of two patients with Zimmermann-Laband syndrome and literature review. Eur J Med Genet, 2013. 56(10): p. 570-6. Corbett MA., et al. A focal epilepsy and intellectual disability syndrome is due to a mutation in TBC1D24. Am J Hum Genet. 2010. 87: p. 371–5. Danecek, et al. The variant call format and VCF tools. Bioinformatics. 2011. 27(15): p. 2156-8. DePristo, et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet. 2011. 43(5): p. 491-8. The Epi4K Consortium. Phenotypic Analysis of 303 Multiplex Families with Common Epilepsies. Brain 2017. 140: p. 2144-2156 Frattini A, et al. Defects in TCIRG1 subunit of the vacuolar proton pump are responsible for a subset of human autosomal recessive osteopetrosis. Nat Genet. 2000. 25(3): p. 343-6. Harrison M.A. Muench S.P. The Vacuolar ATPase – A Nano-scale Motor That Drives Cell Biology. In: Harris J., Boekema E. (eds) Membrane Protein Complexes: Structure and Function. Subcellular Biochemistry. 2018. p. 87. Holzberg M, Walker HK. Terry’s nails: revised definition and new correlations. Lancet. 1984. 1(8382): p. 896–899. Karet F.E., et al. Mutations in the gene encoding B1 subunit of H+-ATPase cause renal tubular acidosis with sensorineural deafness. Nat Genet. 1999. (1): p. 84-90. Kircher, et al. A general framework for estimating the relative pathogenicity of human genetic variants. Nat Genet. 2014. 46(3): p. 310-5. Kortṻm F., et al. Mutations in KCNH1 and ATP6V1B2 cause Zimmermann‐Laband syndrome. Nat. Genet. 2015. 47: p. 661–667. Krumm, et al. Copy number variation detection and genotyping from exome sequence data. Genome Res. 2012. 22(8): p. 1525-32. Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics, 2009. 25(14): p. 1754-60. Lindsay PG. The half-and-half nail. Arch Intern Med. Dermatological Manifestations of Kidney Disease. Place of Publication Not Identified: Springer, 2015. 1967;119(6): p. 583–587. Menendez, I. et al., Dominant deafness-onychodystrophy syndrome caused by an ATP6V1B2 mutation. Clin Case Rep, 2017. 5(4): p. 376-379. Mullen SA, Berkovic SF. ILAE Genetics Commission. Genetic generalized epilepsies. Epilepsia, 2018. 59: p. 1148-1153. Popp, B., et al. Exome Pool-Seq in neurodevelopmental disorders. Eur J Hum Genet, 2017. 25(12): p. 1364-1376. Wang, et al. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 2010. 38(16). e164.
14
Yuan Y., et al. De novo mutation in ATP6V1B2 impairs lysosome acidification and causes dominant deafness‐ onychodystrophy syndrome. Cell Res. 2014. 24: p. 1370–1373.
15
(a)
∂
∂
*
C/G
C/G
C/G
C/C
∂
*
C/G
C/G
C/G
(b)
(c)
(d)
236.8 200
150
100
50
0
1
Exons Amino Acids
1
2 45
3
4
5
6
7
8
9
10
11
12
13
V-ATPase_V1_B Domain
14 509
DEAFNESSONYCHODYSTROPHY SYNDROME
R506X**** (de novo)
R485P** (de novo)
ZIMERMANN-LABAND SYNDROME 2
ID, EPILEPSY, (MILD ZLS FEATURES)
511
E374Q (de novo)
L398V (AD inherited)