- Email: [email protected]
Novel variant in the KCNK9 gene in a girl with Birk Barel syndrome
Accepted Manuscript Novel variant in the KCNK9 gene in a girl with Birk Barel syndrome Marie Šedivá, Petra Laššuthová, Josef Zámečník, Lucie Sedláčková, Pavel Seeman, Jana Haberlová PII:
S1769-7212(18)30486-5
DOI:
https://doi.org/10.1016/j.ejmg.2019.01.009
Reference:
EJMG 3619
To appear in:
European Journal of Medical Genetics
Received Date: 2 July 2018 Revised Date:
2 January 2019
Accepted Date: 19 January 2019
Please cite this article as: M. Šedivá, P. Laššuthová, J. Zámečník, L. Sedláčková, P. Seeman, J. Haberlová, Novel variant in the KCNK9 gene in a girl with Birk Barel syndrome, European Journal of Medical Genetics (2019), doi: https://doi.org/10.1016/j.ejmg.2019.01.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
Page 1 of 10
ACCEPTED MANUSCRIPT Novel Variant in the KCNK9 Gene in a Girl with Birk Barel Syndrome Marie Šediváa, Petra Laššuthová a, Josef Zámečníkb, Lucie Sedláčkováa, Pavel Seemana, Jana Haberlováa
University in Prague and Motol University Hospital
Department of Pathology and Molecular Medicine, 2nd Faculty of Medicine, Charles
M AN U
University in Prague and Motol University Hospital
SC
b
RI PT
Affiliations: a Department of Paediatric Neurology, 2nd Faculty of Medicine, Charles
Corresponding author: MUDr. Marie Šedivá, KDN FNM a 2LF UK, V Úvalu 84, Praha 5, 15006, the Czech Republic. Tel: +420 224 433 301, Fax: +420 224 433 322. Email:
TE D
[email protected]
Supported by: MH CR AZV 16-30206 and DRO 00064203 University Hospital Motol, Prague,
EP
Czech Republic
AC C
Disclosure: Nothing to disclose
Page 2 of 10
ACCEPTED MANUSCRIPT Abstract Birk Barel syndrome also known as KCNK9 imprinting syndrome is a rare developmental disorder associated with a loss-of-function variant in KCNK9, an imprinted gene with maternal expression on the 8th chromosome encoding the TASK3 (TWIK-related acidity
RI PT
inhibited K+-channel 3). Only two variants of KCNK9 have been associated with this condition before, both of them leading to the same amino-acid exchange p.Gly236Arg (Barel ´ 2008, Graham ´ 2016). We describe a case of a 17-year-old girl presenting with very similar
SC
phenotype and pure motor neuropathy with a novel variant c.710C>A: p.Ala237Asn
(NM_001282534.1) in KCNK9 found by whole exome sequencing. Our case suggests that Birk
M AN U
Barel syndrome may not be caused only by variants leading to amino-acid exchange p.Gly236Arg but also by other missense variant in this gene and that peripheral motor neuropathy might be a feature of this syndrome.
TE D
Key words: KCNK9 imprinting syndrome, Birk Barel syndrome, peripheral motor neuropathy,
EP
developmental disorder, TASK3
List of abbreviations:, TASK3 –TWIK-related acidity inhibited K+-channel 3, EEG –
AC C
electroencephalography, EMG – electromyography, SMA – spinal muscular atrophy, SMN1 – survival motor neuron gene 1, WES – whole exome sequencing
Introduction Birk Barel syndrome also known as KCNK9 imprinting syndrome is a very rare disease with only six families mentioned in literature so far (Barel ´ 2008, Graham ´ 2016). The condition is caused by variants in the KCNK9 gene which is located in an imprinted region on the 8th
Page 3 of 10
ACCEPTED MANUSCRIPT chromosome. The gene encodes a two pore-domain potassium channel TASK3 (TWIK-related acidity inhibited K+-channel 3) and is silenced by genomic imprinting on paternally inherited chromosome. Thus far only two nucleotide variants have been described in all yet reported patients with this disease (c.706G>C and c.706G>A in exon 2), both leading to the same
RI PT
amino-acid exchange p.Gly236Arg (Barel ´ 2008, Graham ´ 2016). Birk Barel syndrome is characterised by congenital hypotonia, generalised or proximal weakness, intellectual
disability of variable degree and craniofacial stigmatisation, including elongated face with bi-
SC
temporal narrowing, retromicrognathia, palatoschisis, tented upper-lip, short philtrum,
atypically-shaped ears, flared eyebrows arched upwards and long eyelashes. Among other
M AN U
features hypomimia, lagophthalmos, decreased tearing, dysphagia, gastro-esophageal reflux, neonatal hypoglycemia and myoclonic jerks have also been described. In most patients there is no severe progression of the symptoms during time (Barel ´ 2008, Graham ´ 2016). TASK3 potassium channels are ubiquitous but their concentration is high in neurons,
TE D
especially neurons of the cerebellum (Veale´ 2014). They belong to the family of always open or ´leak´ potassium channels and are responsible for a permanent efflux of potassium out of
EP
cells. Their activity is inhibited by lower pH and other agents, such as zinc or ruthenium red (Veale´ 2014). It has been proven in cell cultures in vitro, that the efflux of potassium
AC C
through these channels is increased by fenamic acid derivates (Graham´ 2016, Veale´ 2014). TASK3 channels modulate the excitability of cells and also play an important role during the development of central nervous system. Their role has been shown in regulating the migration of cortical neuronal cells in mice (Bando´ 2014) and also in regulating the apoptosis of cultured granular cerebellar neurons in vitro (Lauritzen´ 2003). KCNK9 is an imprinted gene with maternal expression, which means it is silenced by methylation on the promoter region of the gene in the paternally inherited allele.
Page 4 of 10
ACCEPTED MANUSCRIPT The dysregulation of imprinted genes has been described in connection with developmental disorders (e.g. Prader-Willi´s and Angelmann´s syndrome or Beckwith Wiedemann syndrome) or dysregulation of cell proliferation (e.g. gestational trophoblastic disease,
RI PT
somatic tumors) (Ruf´2007).
Clinical report
We present a 17-year-old girl who was firstly examined in our clinic at the age of 11 months
SC
for hypotonia and delayed psychomotor development. The patient had a negative family history in respect of similar problems, and she had one older healthy half-brother from her
M AN U
mother´s side. She was born to a 30-year-old mother and a 22-year-old father at term, from 2nd pregnancy, during which her mother was treated by magnesium for irritable uterus and hypertension from the 16th gestational week. The birth weight and length (3210 g (+0,43 SD)/50 cm (+0,08 SD)) were normal, with a good postpartum adaptation. Since birth there
TE D
was apparent hypotonia and congenital malformations, including a cleft palate, micrognathia, tented upper lip, short philtrum, low-positioned ears and slightly elongated
EP
face. She had a weak suck and episodes of hypoxia during feeding. Cranial ultrasound showed a normal finding and she was discharged the 8th day after birth. Her psychomotor
AC C
development was delayed, corresponding to the 6th month of age at the age of 11 months. At that time she was not able to roll, nor sit, and concerning vocalization she was only able to whoop. (Figure 1) Neurological examination showed hypotonia with areflexia and mild bilateral contractures of the biceps femoris and triceps surae muscles. There was no elevation of the muscle-associated enzymes in serum, and also the lactate/pyruvate ratio in serum was normal. Similarly, the EEG was within the normal range. EMG revealed signs of primarily axonal peripheral pure motor neuropathy. The pattern in EMG was similar to the
Page 5 of 10
ACCEPTED MANUSCRIPT pattern seen in spinal muscular atrophy. A muscle biopsy performed at the age of 18 months showed signs of neurogenic transformation with mosaic selective atrophy of type 2 fibers and hypertrophy of type 1 fibers (Figure 2). Based on these findings, spinal muscular atrophy was considered. SMN1 gene was therefore examined and a normal copy number (2) was
RI PT
detected. Brain MRI at the age of 11 years was normal. In further course of the disease the psychomotor development progressed slowly, the patient was able to walk independently by the age of 4 years and to say first words by the age of 5 years. The psychological tests
SC
performed at the patient at the age of 5 years indicated that her intellectual development corresponded to a 3-year-old. However, in further development there was amelioration and
M AN U
only a border-line intellectual deficit had been described by adolescence. The cleft palate was surgically corrected at the age of 5 years. Nowadays the patient attends school for children with physical disabilities, where she has a personal assistant. The patient is not selfsufficient and requires help with clothing, food preparation and hygiene due to problems
TE D
with both fine and gross motor skills. At the age of 17 years she weighs 70 kg (+1,4 SD), her height is 160 cm (-1,1 SD). The head circumference was measured only once at the age of 13
EP
years and was normal, 53,5 cm (-0,5 SD) and at that time her height was -2 SD, weight +0,9 SD, BMI being constantly +2 SD in both 13 and 17 years of age. In neurological examination
AC C
there is hypomimia, bilateral lagophthalmos, tongue fasciculations, dysartria, dysphonia and dysphagia of liquids with a well evocable pharyngeal reflex (Figure 1). A mild cerebellar syndrome and a peripheral motoneuron syndrome with areflexia are observed as well with generalised mild muscle weakness (3 - 4/5). The patient falls frequently and has impairment of fine motor skills in hands. There are mild contractures of triceps surae muscles and decreased physical endurance. Although her muscle weakness is rather stable, she has been
Page 6 of 10
ACCEPTED MANUSCRIPT using mechanical wheelchair for longer distances since the age of 12 years, which is most likely caused by the body growth. The patient was classified as atypical SMA based on findings in EMG and muscle biopsy, further genetic testing was performed in search for another explication of her phenotype.
RI PT
Massively parallel sequencing (MPS) of a custom gene panel for hereditary neuropathies was performed but no variants of likely significance that could explain the cause of the disease
with Agilent Sure Select Human all Exon v.6 kit
SC
were found. We continued testing by whole exome sequencing (WES) which was performed
(https://www.agilent.com/en/promotions/sureselect-human-all-exon-v6, Agilent, Santa
M AN U
Clara, CA, USA ). The obtained data were evaluated according to GATK Best practices (https://software.broadinstitute.org/gatk/best-practices/). In the first step, only variants in genes known to be associated with human diseases (genes with OMIM number) were evaluated. A novel variant c.710C>A (NM_001282534.1) in a heterozygous state in the 2nd
TE D
exon of the KCNK9 gene was found. This variant leads to an amino-acid change p.Ala237Asn. The amino-acid at this position is highly conserved and the substitution is predicted disease-
EP
causing. The healthy father of the patient has also been tested and is not a carrier of this variant. The DNA of the patient's mother was not available for testing, as she is deceased,
AC C
but we tested both maternal grandparents of the patient and have not detected the variant in either of them.
Discussion We report a novel variant c.710C>A (p.A237N) in KCNK9 gene associated with the phenotype of KCNK9 imprinting syndrome (Birk Barel syndrome) in a 17-year-old girl from the Czech Republic. Although no functional studies of this variant were performed, the authors
Page 7 of 10
ACCEPTED MANUSCRIPT assume that the similarity of the phenotype of our patient with the previously described patients with KCNK9 imprinting syndrome strongly supports the causality of the variant in KCNK9 detected by WES. This hypothesis is further supported by in-silico models, by highly
variant in healthy relatives of the patient.
RI PT
conserved status of alanine at position 237 in KCNK9 gene and by the absence of the same
As the patient's mother was not available for analysis, we performed the testing in both of her unaffected maternal grandparents. Given the information about the expression of the
SC
variant only from maternal allele and the fact that the patient's mother was clinically
unaffected, we conclude that the mutation originated probably de novo or in the meiosis of
M AN U
the maternal grandfather or in the mother.
KCNK9 gene has an important role in embryogenesis, regulation of cell proliferation and development of central and possibly peripheral nervous system. Axonal polyneuropathy is a prevailing feature in our patient and there were certain signs of impairment of peripheral
TE D
nerve in the patients described in the previous studies as well. The muscle weakness was described in most patients from the previous two studies (Barel´ 2004, Graham´ 2016). There
EP
was a notion of electromyography only in two patients from the original studies, one of them showing a denervation-type pattern (as our patient did), the other one suggesting a
AC C
generalised myopathy. However, in two patients, who had a muscle biopsy, there were findings compatible with SMA. (Barel´2004). Given these findings suggesting a denervation pattern of skeletal muscles, we speculate that there could be a defect not only in the development of the cortical neurons but also in the development of alpha motor neurons of the spine. It is apparent that these hypotheses require further investigation to be elucidated. The clinical course of Birk Barel syndrome, as well as the above-described roles of KCNK9
Page 8 of 10
ACCEPTED MANUSCRIPT gene product TASK3, show that Birk Barel syndrome is most probably a developmental disorder with only very mild progression of neurological findings during the life-span. There have been efforts to influence the course of the disease by fenamic acid derivates, which was proven to significantly increase the efflux of potassium ions through the mutated
RI PT
channels in vitro (Graham´ 2016, Veale´ 2014). Graham´ (2016) administered this treatment to two patients in his study and there were no serious adverse events described and even some positive effects were noted, especially acceleration of development, better caloric
SC
intake and decreased fatigue. However, it is very challenging to measure the effect due to the limited number of patients and the variability of the phenotype severity in each
M AN U
individual. Moreover, a higher incidence of gastro-esophageal reflux has been described in children with this condition, which could be aggravated by acidification of gastric fluids. As neither the effect of these agents nor their possible adverse effects have been fully mapped in this condition and mainly given the fact that the condition is not progressive and probably
TE D
developmental, the therapy was not indicated. Our case supports the previously described association of variants in the KCNK9 gene and
EP
Birk Barel syndrome and suggests that other missense variant of this gene than the previously published ones - namely c.710C>A: p.Ala237Asn (NM_001282534.1) - could be
AC C
causal for this disorder.
Acknowledgments: We thank the family for cooperation.
Page 9 of 10
ACCEPTED MANUSCRIPT Figures Figure 1: Our patient at the age of 11 months (left) and 17 years (middle and right). Notice the typical cranio-facial stigmatisation with elongated face, microretrognathia, lowpositioned ears, tented upper lip, broad nasal tip, flared eyebrows, hypomimia and
RI PT
lagophthalmos
Figure 2: Muscle biopsy from m. tibialis anterior: diffuse selective atrophy of type 2 fibers
SC
and hypertrophy of type 1 fibers. A finding corresponding to neurogenic transformation due to a motoneuron disese (hematoxylin-eosin, left; myofibrillar ATPase staining at pH 9.4,
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EP
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right).
Page 10 of 10
ACCEPTED MANUSCRIPT References:
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(1) Barel O, Shalev SA, Ofir R, Cohen A, Zlotogora J, Shorer Z, et al. Maternally Inherited Birk Barel Mental Retardation Dysmorphism Syndrome Caused by a Mutation in the Genomically Imprinted Potassium Channel KCNK9. Am J Hum Genet (2008). doi: 10.1016/j.ajhg.2008.07.010. (2) Graham JM, Zadeh N, Kelley M, Tan ES, Liew W, Tan V, et al. KCNK9 imprinting syndrome-further delineation of a possible treatable disorder. Am J Med Genet (2016). doi: 10.1002/ajmg.a.37740. (3) Veale EL, Hassan M, Walsh Y, Al-Moubarak E, Mathie A. Recovery of Current through Mutated TASK3 Potassium Channels Underlying Birk Barel Syndrome. Mol Pharmacol (2014), doi: 10.1124/mol.113.090530. (4) Bando Y, Hirano T, Tagawa Y. Dysfunction of KCNK Potassium Channels Impairs Neuronal Migration in the Developing Mouse Cerebral Cortex. Cereb Cortex (2014). doi: 10.1093/cercor/bhs387. (5) Lauritzen I, Zanzouri M, Honoré E, Duprat F, Ehrengruber MU, Lazdunski M, et al. K + dependent Cerebellar Granule Neuron Apoptosis. J Biol Chem (2003). Doi: 10.1074/jbc.M302631200. (6) Ruf N, Bähring S, Galetzka D, Pliushch G, Luft FC, Nürnberg P, et al. Sequence-based bioinformatic prediction and QUASEP identify genomic imprinting of the KCNK9 potassium channel gene in mouse and human. Hum Mol Genet (2007). doi: 10.1093/hmg/ddm216
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ACCEPTED MANUSCRIPT
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ACCEPTED MANUSCRIPT
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ACCEPTED MANUSCRIPT
S1769-7212(18)30486-5
DOI:
https://doi.org/10.1016/j.ejmg.2019.01.009
Reference:
EJMG 3619
To appear in:
European Journal of Medical Genetics
Received Date: 2 July 2018 Revised Date:
2 January 2019
Accepted Date: 19 January 2019
Please cite this article as: M. Šedivá, P. Laššuthová, J. Zámečník, L. Sedláčková, P. Seeman, J. Haberlová, Novel variant in the KCNK9 gene in a girl with Birk Barel syndrome, European Journal of Medical Genetics (2019), doi: https://doi.org/10.1016/j.ejmg.2019.01.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
Page 1 of 10
ACCEPTED MANUSCRIPT Novel Variant in the KCNK9 Gene in a Girl with Birk Barel Syndrome Marie Šediváa, Petra Laššuthová a, Josef Zámečníkb, Lucie Sedláčkováa, Pavel Seemana, Jana Haberlováa
University in Prague and Motol University Hospital
Department of Pathology and Molecular Medicine, 2nd Faculty of Medicine, Charles
M AN U
University in Prague and Motol University Hospital
SC
b
RI PT
Affiliations: a Department of Paediatric Neurology, 2nd Faculty of Medicine, Charles
Corresponding author: MUDr. Marie Šedivá, KDN FNM a 2LF UK, V Úvalu 84, Praha 5, 15006, the Czech Republic. Tel: +420 224 433 301, Fax: +420 224 433 322. Email:
TE D
[email protected]
Supported by: MH CR AZV 16-30206 and DRO 00064203 University Hospital Motol, Prague,
EP
Czech Republic
AC C
Disclosure: Nothing to disclose
Page 2 of 10
ACCEPTED MANUSCRIPT Abstract Birk Barel syndrome also known as KCNK9 imprinting syndrome is a rare developmental disorder associated with a loss-of-function variant in KCNK9, an imprinted gene with maternal expression on the 8th chromosome encoding the TASK3 (TWIK-related acidity
RI PT
inhibited K+-channel 3). Only two variants of KCNK9 have been associated with this condition before, both of them leading to the same amino-acid exchange p.Gly236Arg (Barel ´ 2008, Graham ´ 2016). We describe a case of a 17-year-old girl presenting with very similar
SC
phenotype and pure motor neuropathy with a novel variant c.710C>A: p.Ala237Asn
(NM_001282534.1) in KCNK9 found by whole exome sequencing. Our case suggests that Birk
M AN U
Barel syndrome may not be caused only by variants leading to amino-acid exchange p.Gly236Arg but also by other missense variant in this gene and that peripheral motor neuropathy might be a feature of this syndrome.
TE D
Key words: KCNK9 imprinting syndrome, Birk Barel syndrome, peripheral motor neuropathy,
EP
developmental disorder, TASK3
List of abbreviations:, TASK3 –TWIK-related acidity inhibited K+-channel 3, EEG –
AC C
electroencephalography, EMG – electromyography, SMA – spinal muscular atrophy, SMN1 – survival motor neuron gene 1, WES – whole exome sequencing
Introduction Birk Barel syndrome also known as KCNK9 imprinting syndrome is a very rare disease with only six families mentioned in literature so far (Barel ´ 2008, Graham ´ 2016). The condition is caused by variants in the KCNK9 gene which is located in an imprinted region on the 8th
Page 3 of 10
ACCEPTED MANUSCRIPT chromosome. The gene encodes a two pore-domain potassium channel TASK3 (TWIK-related acidity inhibited K+-channel 3) and is silenced by genomic imprinting on paternally inherited chromosome. Thus far only two nucleotide variants have been described in all yet reported patients with this disease (c.706G>C and c.706G>A in exon 2), both leading to the same
RI PT
amino-acid exchange p.Gly236Arg (Barel ´ 2008, Graham ´ 2016). Birk Barel syndrome is characterised by congenital hypotonia, generalised or proximal weakness, intellectual
disability of variable degree and craniofacial stigmatisation, including elongated face with bi-
SC
temporal narrowing, retromicrognathia, palatoschisis, tented upper-lip, short philtrum,
atypically-shaped ears, flared eyebrows arched upwards and long eyelashes. Among other
M AN U
features hypomimia, lagophthalmos, decreased tearing, dysphagia, gastro-esophageal reflux, neonatal hypoglycemia and myoclonic jerks have also been described. In most patients there is no severe progression of the symptoms during time (Barel ´ 2008, Graham ´ 2016). TASK3 potassium channels are ubiquitous but their concentration is high in neurons,
TE D
especially neurons of the cerebellum (Veale´ 2014). They belong to the family of always open or ´leak´ potassium channels and are responsible for a permanent efflux of potassium out of
EP
cells. Their activity is inhibited by lower pH and other agents, such as zinc or ruthenium red (Veale´ 2014). It has been proven in cell cultures in vitro, that the efflux of potassium
AC C
through these channels is increased by fenamic acid derivates (Graham´ 2016, Veale´ 2014). TASK3 channels modulate the excitability of cells and also play an important role during the development of central nervous system. Their role has been shown in regulating the migration of cortical neuronal cells in mice (Bando´ 2014) and also in regulating the apoptosis of cultured granular cerebellar neurons in vitro (Lauritzen´ 2003). KCNK9 is an imprinted gene with maternal expression, which means it is silenced by methylation on the promoter region of the gene in the paternally inherited allele.
Page 4 of 10
ACCEPTED MANUSCRIPT The dysregulation of imprinted genes has been described in connection with developmental disorders (e.g. Prader-Willi´s and Angelmann´s syndrome or Beckwith Wiedemann syndrome) or dysregulation of cell proliferation (e.g. gestational trophoblastic disease,
RI PT
somatic tumors) (Ruf´2007).
Clinical report
We present a 17-year-old girl who was firstly examined in our clinic at the age of 11 months
SC
for hypotonia and delayed psychomotor development. The patient had a negative family history in respect of similar problems, and she had one older healthy half-brother from her
M AN U
mother´s side. She was born to a 30-year-old mother and a 22-year-old father at term, from 2nd pregnancy, during which her mother was treated by magnesium for irritable uterus and hypertension from the 16th gestational week. The birth weight and length (3210 g (+0,43 SD)/50 cm (+0,08 SD)) were normal, with a good postpartum adaptation. Since birth there
TE D
was apparent hypotonia and congenital malformations, including a cleft palate, micrognathia, tented upper lip, short philtrum, low-positioned ears and slightly elongated
EP
face. She had a weak suck and episodes of hypoxia during feeding. Cranial ultrasound showed a normal finding and she was discharged the 8th day after birth. Her psychomotor
AC C
development was delayed, corresponding to the 6th month of age at the age of 11 months. At that time she was not able to roll, nor sit, and concerning vocalization she was only able to whoop. (Figure 1) Neurological examination showed hypotonia with areflexia and mild bilateral contractures of the biceps femoris and triceps surae muscles. There was no elevation of the muscle-associated enzymes in serum, and also the lactate/pyruvate ratio in serum was normal. Similarly, the EEG was within the normal range. EMG revealed signs of primarily axonal peripheral pure motor neuropathy. The pattern in EMG was similar to the
Page 5 of 10
ACCEPTED MANUSCRIPT pattern seen in spinal muscular atrophy. A muscle biopsy performed at the age of 18 months showed signs of neurogenic transformation with mosaic selective atrophy of type 2 fibers and hypertrophy of type 1 fibers (Figure 2). Based on these findings, spinal muscular atrophy was considered. SMN1 gene was therefore examined and a normal copy number (2) was
RI PT
detected. Brain MRI at the age of 11 years was normal. In further course of the disease the psychomotor development progressed slowly, the patient was able to walk independently by the age of 4 years and to say first words by the age of 5 years. The psychological tests
SC
performed at the patient at the age of 5 years indicated that her intellectual development corresponded to a 3-year-old. However, in further development there was amelioration and
M AN U
only a border-line intellectual deficit had been described by adolescence. The cleft palate was surgically corrected at the age of 5 years. Nowadays the patient attends school for children with physical disabilities, where she has a personal assistant. The patient is not selfsufficient and requires help with clothing, food preparation and hygiene due to problems
TE D
with both fine and gross motor skills. At the age of 17 years she weighs 70 kg (+1,4 SD), her height is 160 cm (-1,1 SD). The head circumference was measured only once at the age of 13
EP
years and was normal, 53,5 cm (-0,5 SD) and at that time her height was -2 SD, weight +0,9 SD, BMI being constantly +2 SD in both 13 and 17 years of age. In neurological examination
AC C
there is hypomimia, bilateral lagophthalmos, tongue fasciculations, dysartria, dysphonia and dysphagia of liquids with a well evocable pharyngeal reflex (Figure 1). A mild cerebellar syndrome and a peripheral motoneuron syndrome with areflexia are observed as well with generalised mild muscle weakness (3 - 4/5). The patient falls frequently and has impairment of fine motor skills in hands. There are mild contractures of triceps surae muscles and decreased physical endurance. Although her muscle weakness is rather stable, she has been
Page 6 of 10
ACCEPTED MANUSCRIPT using mechanical wheelchair for longer distances since the age of 12 years, which is most likely caused by the body growth. The patient was classified as atypical SMA based on findings in EMG and muscle biopsy, further genetic testing was performed in search for another explication of her phenotype.
RI PT
Massively parallel sequencing (MPS) of a custom gene panel for hereditary neuropathies was performed but no variants of likely significance that could explain the cause of the disease
with Agilent Sure Select Human all Exon v.6 kit
SC
were found. We continued testing by whole exome sequencing (WES) which was performed
(https://www.agilent.com/en/promotions/sureselect-human-all-exon-v6, Agilent, Santa
M AN U
Clara, CA, USA ). The obtained data were evaluated according to GATK Best practices (https://software.broadinstitute.org/gatk/best-practices/). In the first step, only variants in genes known to be associated with human diseases (genes with OMIM number) were evaluated. A novel variant c.710C>A (NM_001282534.1) in a heterozygous state in the 2nd
TE D
exon of the KCNK9 gene was found. This variant leads to an amino-acid change p.Ala237Asn. The amino-acid at this position is highly conserved and the substitution is predicted disease-
EP
causing. The healthy father of the patient has also been tested and is not a carrier of this variant. The DNA of the patient's mother was not available for testing, as she is deceased,
AC C
but we tested both maternal grandparents of the patient and have not detected the variant in either of them.
Discussion We report a novel variant c.710C>A (p.A237N) in KCNK9 gene associated with the phenotype of KCNK9 imprinting syndrome (Birk Barel syndrome) in a 17-year-old girl from the Czech Republic. Although no functional studies of this variant were performed, the authors
Page 7 of 10
ACCEPTED MANUSCRIPT assume that the similarity of the phenotype of our patient with the previously described patients with KCNK9 imprinting syndrome strongly supports the causality of the variant in KCNK9 detected by WES. This hypothesis is further supported by in-silico models, by highly
variant in healthy relatives of the patient.
RI PT
conserved status of alanine at position 237 in KCNK9 gene and by the absence of the same
As the patient's mother was not available for analysis, we performed the testing in both of her unaffected maternal grandparents. Given the information about the expression of the
SC
variant only from maternal allele and the fact that the patient's mother was clinically
unaffected, we conclude that the mutation originated probably de novo or in the meiosis of
M AN U
the maternal grandfather or in the mother.
KCNK9 gene has an important role in embryogenesis, regulation of cell proliferation and development of central and possibly peripheral nervous system. Axonal polyneuropathy is a prevailing feature in our patient and there were certain signs of impairment of peripheral
TE D
nerve in the patients described in the previous studies as well. The muscle weakness was described in most patients from the previous two studies (Barel´ 2004, Graham´ 2016). There
EP
was a notion of electromyography only in two patients from the original studies, one of them showing a denervation-type pattern (as our patient did), the other one suggesting a
AC C
generalised myopathy. However, in two patients, who had a muscle biopsy, there were findings compatible with SMA. (Barel´2004). Given these findings suggesting a denervation pattern of skeletal muscles, we speculate that there could be a defect not only in the development of the cortical neurons but also in the development of alpha motor neurons of the spine. It is apparent that these hypotheses require further investigation to be elucidated. The clinical course of Birk Barel syndrome, as well as the above-described roles of KCNK9
Page 8 of 10
ACCEPTED MANUSCRIPT gene product TASK3, show that Birk Barel syndrome is most probably a developmental disorder with only very mild progression of neurological findings during the life-span. There have been efforts to influence the course of the disease by fenamic acid derivates, which was proven to significantly increase the efflux of potassium ions through the mutated
RI PT
channels in vitro (Graham´ 2016, Veale´ 2014). Graham´ (2016) administered this treatment to two patients in his study and there were no serious adverse events described and even some positive effects were noted, especially acceleration of development, better caloric
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intake and decreased fatigue. However, it is very challenging to measure the effect due to the limited number of patients and the variability of the phenotype severity in each
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individual. Moreover, a higher incidence of gastro-esophageal reflux has been described in children with this condition, which could be aggravated by acidification of gastric fluids. As neither the effect of these agents nor their possible adverse effects have been fully mapped in this condition and mainly given the fact that the condition is not progressive and probably
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developmental, the therapy was not indicated. Our case supports the previously described association of variants in the KCNK9 gene and
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Birk Barel syndrome and suggests that other missense variant of this gene than the previously published ones - namely c.710C>A: p.Ala237Asn (NM_001282534.1) - could be
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causal for this disorder.
Acknowledgments: We thank the family for cooperation.
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ACCEPTED MANUSCRIPT Figures Figure 1: Our patient at the age of 11 months (left) and 17 years (middle and right). Notice the typical cranio-facial stigmatisation with elongated face, microretrognathia, lowpositioned ears, tented upper lip, broad nasal tip, flared eyebrows, hypomimia and
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lagophthalmos
Figure 2: Muscle biopsy from m. tibialis anterior: diffuse selective atrophy of type 2 fibers
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and hypertrophy of type 1 fibers. A finding corresponding to neurogenic transformation due to a motoneuron disese (hematoxylin-eosin, left; myofibrillar ATPase staining at pH 9.4,
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right).
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ACCEPTED MANUSCRIPT References:
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(1) Barel O, Shalev SA, Ofir R, Cohen A, Zlotogora J, Shorer Z, et al. Maternally Inherited Birk Barel Mental Retardation Dysmorphism Syndrome Caused by a Mutation in the Genomically Imprinted Potassium Channel KCNK9. Am J Hum Genet (2008). doi: 10.1016/j.ajhg.2008.07.010. (2) Graham JM, Zadeh N, Kelley M, Tan ES, Liew W, Tan V, et al. KCNK9 imprinting syndrome-further delineation of a possible treatable disorder. Am J Med Genet (2016). doi: 10.1002/ajmg.a.37740. (3) Veale EL, Hassan M, Walsh Y, Al-Moubarak E, Mathie A. Recovery of Current through Mutated TASK3 Potassium Channels Underlying Birk Barel Syndrome. Mol Pharmacol (2014), doi: 10.1124/mol.113.090530. (4) Bando Y, Hirano T, Tagawa Y. Dysfunction of KCNK Potassium Channels Impairs Neuronal Migration in the Developing Mouse Cerebral Cortex. Cereb Cortex (2014). doi: 10.1093/cercor/bhs387. (5) Lauritzen I, Zanzouri M, Honoré E, Duprat F, Ehrengruber MU, Lazdunski M, et al. K + dependent Cerebellar Granule Neuron Apoptosis. J Biol Chem (2003). Doi: 10.1074/jbc.M302631200. (6) Ruf N, Bähring S, Galetzka D, Pliushch G, Luft FC, Nürnberg P, et al. Sequence-based bioinformatic prediction and QUASEP identify genomic imprinting of the KCNK9 potassium channel gene in mouse and human. Hum Mol Genet (2007). doi: 10.1093/hmg/ddm216
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