Pathogenic variants in KPTN gene identified by clinical whole-genome sequencing


Pathogenic variants in KPTN gene
identified by clinical whole-genome
sequencing
Isabelle Thiffault,1,2,3 Andrea Atherton,4 Bryce A. Heese,4 Ahmed T. Abdelmoity,4

Kailash Pawar,4 Emily Farrow,1,3,4 Lee Zellmer,1 Neil Miller,1 Sarah Soden,1,3,4

and Carol Saunders1,2,3

1Center for Pediatric Genomic Medicine, Children’s Mercy Hospital, Kansas City, Missouri 64108, USA;
2Department of Pathology and Laboratory Medicine, Children’s Mercy Hospitals, Kansas City, Missouri 64108,
USA; 3University of Missouri–Kansas City School of Medicine, Kansas City, Missouri 64108, USA; 4Department
of Pediatrics, Children’s Mercy Hospitals, Kansas City, Missouri 64108, USA

Abstract Status epilepticus is not rare in critically ill intensive care unit patients, but its diag-
nosis is often delayed or missed. The mortality for convulsive status epilepticus is depen-
dent on the underlying aetiologies and the age of the patients and thus varies from study
to study. In this context, effective molecular diagnosis in a pediatric patient with a geneti-
cally heterogeneous phenotype is essential. Homozygous or compound heterozygous var-
iants in KPTN have been recently associated with a syndrome typified by macrocephaly,
neurodevelopmental delay, and seizures. We describe a comprehensive investigation of
a 9-yr-old male patient who was admitted to the intensive care unit, with focal epilepsy, stat-
ic encephalopathy, autism spectrum disorder, and macrocephaly of unknown etiology, who
died of status epilepticus. Clinical whole-genome sequencing revealed compound het-
erozygous variants in the KPTN gene. The first variant is a previously characterized 18-bp
in-frame duplication (c.714_731dup) in exon 8, resulting in the protein change
p.Met241_Gln246dup. The second variant, c.394 + 1G > A, affects the splice junction of
exon 3. These results are consistent with a diagnosis of autosomal recessive KPTN-related
disease. This is the fourth clinical report for KPTN deficiency, providing further evidence of a
wider range of severity.

[Supplemental material is available for this article.]

INTRODUCTION

In recent years, next-generation sequencing (NGS) technologies have revolutionized ap-
proaches in clinical genetics. Whole-exome sequencing (WES) or whole-genome sequenc-
ing (WGS) allows diagnoses in many patients with complex phenotypes and unusual clinical
presentations. As the cost of NGS falls, it has become feasible to use this powerful technol-
ogy in clinical care, simultaneously unraveling variations in about 19,000 genes.
Technological advances have led to the ability to sequence, analyze, and interpret entire ge-
nome data in a timely manner, clearly changing the diagnostic paradigm and proving to be
cost-effective in many cases (Soden et al. 2014). Clinical diagnostic sequencing currently fo-
cuses on identifying causal mutations in the exome (∼1% of the genome), where most dis-
ease-causing mutations are known to occur. WGS permits analysis of coding regions as
well as regulatory elements that control gene expression; however, noncoding variants

Corresponding author:
csaunders@cmh.edu

© 2020 Thiffault et al. This article
is distributed under the terms of
the Creative Commons
Attribution-NonCommercial
License, which permits reuse and
redistribution, except for
commercial purposes, provided
that the original author and
source are credited.

Ontology terms: complex febrile
seizures; delayed fine motor
development; delayed gross
motor development; epileptic
encephalopathy; intellectual
disability, severe; macrocephaly
at birth; polymorphic focal
epileptiform discharges

Published by Cold Spring Harbor
Laboratory Press

doi:10.1101/mcs.a003970

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remained largely unexplored in clinical diagnostics because of the interpretive challenges
(Warman Chardon et al. 2015). Although it lacks the depth of coverage of an exome,
WGS can have more reliable and uniform sequence coverage, particularly in regions of
the genome with low sequence complexity or high GC-rich content. Both WGS and WES ap-
proaches have benefits and limitations, but the limitation of NGS gene panels and WES
should be considered prior to clinical testing (Williams et al. 2008; Soden et al. 2014;
Ankala et al. 2015; Warman Chardon et al. 2015). In this context, the present work demon-
strates the application of clinical WGS in the pediatric population in which we provided a fast,
accurate, and cost-effective molecular diagnosis in a pediatric patient with a genetically het-
erogeneous phenotype.

RESULTS

Clinical Presentation
A 9-yr-old Caucasian male was admitted to the Children’s Mercy Hospital pediatric intensive
care unit (PICU) for status epilepticus and further evaluation of a suspected underlying genet-
ic condition because of his history of macrocephaly, intractable epilepsy, autism, severe
developmental delays, hypotonia, and hypoglycemia. Dysmorphic features include frontal
bossing, sunken eye sockets, downslanting palpebral fissures, small ears, thin upper lip,
and small nose (Fig. 1). The prenatal history was unremarkable: Delivery was at full term,
and neonatal development was normal. He was the third child for his parents together,
born to a 26-yr-old mother and 30-yr-old father. Family history was noncontributory. At birth,
he weighed 2.71 kilograms and was 49.5 cm long. His head circumference was noted to be

BA

C

Figure 1. Our patient was a 9-yr-old male with epilepsy, static encephalopathy, autism spectrum disorder, and
(A) macrocephaly of unknown etiology who died of status epilepticus. (B) Several facial dysmorphisms were
found, including frontal bossing, sunken eye sockets, downslanting palpebral fissures, small ears, thin upper
lip, and small nose. (C) At 4 yr old, he was normal height and weight, but his head circumference measured
57 cm (>98th percentile).

KPTN deficiency identified by WGS

C O L D S P R I N G H A R B O R

Molecular Case Studies

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greater than the 99th percentile at birth. At 4 yr old, he was of average height and weight, but
his head circumference measured 57 cm (>98th percentile, +4SD). He had poor tracking and
eye contact but a normal ophthalmologic exam. A head CT was completed at 2 mo of age
and reported to be normal. At 3 mo of age, he started to have partial seizures but EEG was
normal. At 5 mo of age he was noted to have hepatosplenomegaly accompanied by hypo-
glycemic episodes. Developmentally, he smiled socially at 2 mo, rolled at 8 mo, sat without
assistance at 10–11 mo, crawled at 14 mo, and walked at 2.5 yr. A brain MRI was normal at
4 mo of age; however a second, performed at 9 yr of age, was concerning for a possible ar-
teriovenous malformation, with abnormal hyperintense T2/FLAIR signals in the subcortical
white matter of the right anteromedial temporal lobe. An extensive etiologic workup was
normal, including high-resolution karyotype, CGH microarray, PTEN sequencing, fragile X,
enzyme analysis for Gaucher, mucopolysaccharidosis I and II, plasma amino acids, pristanic
and phytanic acids, very long chain fatty acid (VLCFA), and urine organic acids. Mild eleva-
tions of C14 species on an acylcarnitine profile in 2010 were detected in conjunction with
ketotic hypoglycemia and were likely associated with physiologic response to fasting.
Previously, the seizures occurred at a frequency of 1–2 per hour, consisting of focal tonic-
clonic seizures involving his left or right side of the body but occasionally progressed to
status epilepticus, and a pentobarbital infusion was started with the goal to acquire burst
suppression. Unfortunately, this was unsuccessful, and he passed away at age 9.

Genetic Analysis
Clinical WGS was performed on the patient and WES was performed on his healthy mother,
following informed consent. The patient was compound heterozygous for two pathogenic
variants in the KPTN gene (Table 1). This gene was ranked 351st in the Phenomizer gene
list. The first variant identified was an 18-bp in-frame duplication (c.714_731dup
ACCGACCACATCTGCAGA; rs587777148) in exon 8, resulting in the protein change
p.Met241_Gln246dup. This variant has been previously reported in trans with a second trun-
cating variant in multiple affected individuals from two Amish families (Baple et al. 2014).
Prior in vitro transfection studies indicated the mutant protein is mislocalized and accumu-
lates in neurons of affected individuals, leading to a dominant negative effect (Baple et al.
2014). The p.Met241_Gln246dup variant is not found in population databases such as
dbSNP, Exome Variant Database, or ExAC but was found in 1 of 560 Amish control chromo-
somes (Baple et al. 2014); 136/275574 in gnomAD data set (0.05%). The second variant,
c.394 + 1G > A, affects the splice junction of exon 3. This variant has not been reported in
affected individuals but is predicted to cause aberrant splicing. This variant was observed
in ∼0.01% individuals of European ancestry in the NHLBI Exome Sequencing Project and
Exome Aggregation Consortium (ExAC); 19/277090 in gnomAD data set (0.007%). The
c.394 + 1G > A variant, but not the c.714_731dup variant, was inherited from the mother,
suggesting these variants are in trans. This genotype was confirmed by Sanger sequencing
and is consistent with a diagnosis of autosomal recessive KPTN-related disease. In addition,
two variants of unknown significance were reported ((CHD2-NM_001271.3:c.5268G > C
(p.Gln1756His); CTDP1-NM_004715.4:c.1219T > C (p.Trp407Arg)). No incidental findings
were reported for this case. The turnaround time was 33 d.

DISCUSSION

The use of NGS techniques by clinical laboratories has risen tremendously and has greatly
facilitated the elucidation of the etiologic diagnosis in patients suspected of having a genetic
disease. The various approaches, including WES, WGS, and targeted panels, all have ben-
efits and limitations. Targeted panels offer a specific list of genes relevant to the clinical

KPTN deficiency identified by WGS

C O L D S P R I N G H A R B O R

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phenotype in question and often boast higher coverage than WES/WGS. However, whether
this improves sensitivity, particularly in Mendelian disorders, is not clear. In addition, a wide
range of interlaboratory variability exists for gene lists offered for the same condition. For in-
stance, commercial clinical NGS panels for macrocephaly include anywhere from 12 to 44
genes. Although targeted panels may appear to be a more economical approach to WES/
WGS, this is only the case if the gene relevant to the patient being tested is present on
the panel, which is often difficult to know a priori, particularly in patients with nonspecific
symptoms such as intellectual disability or seizures. In many cases, serial testing of additional
genes and panels quickly surpasses the expense of WES/WGS (Soden et al. 2014). In addi-
tion, targeted panels may fail to incorporate newly discovered disease-associated genes. For
example, new genes associated with both syndromic and nonsyndromic macrocephaly have
been identified, further expanding the genetic heterogeneity to more than 165 conditions,
including 17 metabolic disorders, associated with macrocephaly (Williams et al. 2008;
Keppler-Noreuil et al. 2014; Koh et al. 2014; Marchese et al. 2014; Sugathan et al. 2014;
Nevado et al. 2015; Nguyen et al. 2015). One such gene, KPTN, encoding kaptin, was first
reported in January of 2014 in patients with a syndrome typified by macrocephaly, neurode-
velopmental delay, and seizures (MRT41; OMIM# 615637) (Baple et al. 2014; Pajusalu et al.
2015). KPTN is not currently offered in any clinical panels for macrocephaly in the United
States. Of the clinical laboratories listed in the NextGxDx website, only two of 16 offer
KPTN as part of an autism/intellectual disability panel, and only two of 17 include it as
part of an epilepsy panel. The comparison of such gene lists is difficult for clinicians and
the curation is onerous for the clinical laboratory to manage. Clinical WES/WGS removes
the guesswork as far as which gene to include, as all genes relevant to the patient’s
phenotype are queried in the analysis process. In the current case, by using clinical WGS
in a 9-yr-old Caucasian male admitted in pediatric intensive care unit for status epilepticus,
a diagnosis of KPTN-related disease was made.

A comparison of the individual clinical signs in patients reported with KPTN-related dis-
ease are shown in Table 2. The most unifying features are macrocephaly (92%), developmen-
tal delay (100%), and intellectual disability (100%). Recurrent dysmorphic features included
frontal bossing, abnormal head shape, and prominent chin. With the exception of the
p.Met241_Gln246dup, which is predicted to be nonfunctional by producing a misfolded al-
tered protein, all patients have truncating variants (p.S259∗; p.S223Qfs∗50; p.S200Ifs∗55); no
patients were homozygous for the p.Met241_Gln246dup. RT-PCR or western blot experi-
ments were not performed to assess if those variants result in nonsense-mediated mRNA de-
cay or whether a truncated protein (lacking the carboxy-terminal amino acids) is produced.
The second variant uncovered in our patient, c.394 + 1G > A, is located upstream of the
two previously reported truncating variants. This is the first splicing variant reported in
KPTN, it is likely to result in nonsense-mediated mRNA decay, which may explain the
more severe outcome. Additional external factors such as modifier genes could also influ-
ence the phenotype. Of the 12 previous patients described, seven have survived into their
second and third decades, with two having expired at ages 29 and 30 to a head injury and
pneumonia (Table 2; Baple et al. 2014; Pajusalu et al. 2015; Lucena et al. 2020). At this time,
limited data exist for genotype–phenotype correlations; further phenotyping and natural his-
tory of disease study are needed to understand the clinical spectrum and prognosis of KPTN-
related syndrome.

This is the fourth clinical report for KPTN-deficiency, the 13th patient but the first to
die in childhood with status epilepticus, providing further evidence of a wider range of
severity.

Several studies have shed new light on the molecular and cellular processes that
orchestrate the human neuronal circuitry and is defective in neurological disorders (Ropers
et al. 2011; Riviere et al. 2012; Baple et al. 2014; Koh et al. 2014; Sugathan et al. 2014).

KPTN deficiency identified by WGS

C O L D S P R I N G H A R B O R

Molecular Case Studies

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.

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Evidence implicating defects in proteins involved in actin dynamics, microtubule homeosta-
sis, and vesicle trafficking in developmental disorders via effects on neuronal development
and migration and brain architecture has accumulated over the past decade. The role of
microtubule homeostasis (LIS1 and DCX) or tubulin (TUBA1A, TUBA8, TUBB2A,
TUBB4A, TUBB2B, TUBB3, and TUBB and TBCE) genes has been established in several
brain disorders. More recently, the actin-encoding genes ACTB and ACTG1 have been
shown to be involved in brain malformations causing Baraitser–Winter syndrome (OMIM
# 243310) (Riviere et al. 2012). Additional intellectual disability genes involved in actin dy-
namics and vesicle trafficking include STXBP1 and SYP (Barcia et al. 2013; Kato 2015).
Patients with KPTN deficiency consistently exhibit macrocephaly, intellectual disability,
and developmental delay. Furthermore, KPTN is a novel actin binding protein, enriched
in neuronal growth cones and cortical sites of neurons at early developmental stages, likely
playing a central role in modulating neuron morphology and growth. Although genes af-
fecting many of these pathways are associated with hearing loss, to date, no patients
with KTPN-related disease, including our patient, have been reported with deafness. As
with several other published studies (Riviere et al. 2012; Soden et al. 2014; Ankala et al.
2015; Warman Chardon et al. 2015; Willig et al. 2015), WGS was found to be a rapid
and cost-efficient approach for molecular diagnostic of a genetically heterogeneous condi-
tion (Thiffault et al. 2019). Thus, this report confirms the occurrence of KPTN-related syn-
drome outside of the Amish population and demonstrates the variability in the
phenotypic spectrum and severity. Pathological mechanisms of abnormal neuronal actin
cytoskeleton and discrepancy between the underlying phenotypes caused by KPTN defi-
ciency remain to be elucidated.

METHODS

Genomic DNA was extracted from peripheral blood mononuclear cells using a Chemagen
MSM1 robot (PerkinElmer). WGS was prepared using the Kapa Hyper library prep omitting
PCR. Sequencing was completed on an Illumina HiSeq 2500 instrument utilizing paired end
2 × 125 base pair reads with v4 Chemistry (Illumina). The proband’s sample was sequenced
to a depth of 115.16 Gb for a mean coverage of ∼37× (Supplemental Table S1). Bidirectional
sequence was assembled, aligned to reference gene sequences based on human genome
build GRCh37/UCSC hg19, and analyzed using custom-developed software, RUNES, and
VIKING (Saunders et al. 2012; Soden et al. 2014; Willig et al. 2015). Variants were filtered
to a 1% minor allele frequency and then prioritized by the American College of Medical
Genetics (ACMG) categorization (Richards et al. 2015), OMIM identity, and phenotypic as-
sessment. Alignments were viewed using Integrative Genomic Viewer software version
2.3.8 (IGV; Broad Institute). A candidate gene list was generated by Phenomizer using
Human Phenotype Ontology (Kohler et al. 2014) terms: Macrocephaly (HPO:0000256),
Muscular hypotonia (HPO:0001252), Autism (HPO:0000717), Seizures (HPO:0001250),
Global developmental delay (HPO:0001263), and Abnormal facial shape (HPO: 0001999),
with a cutoff at P-value of 0.5. This gene list contained approximately 700 genes and was im-
ported into VIKING to guide the analysis; however, a separate analysis, not limited by this
gene list, was performed in parallel to look for relevant pathogenic genotypes in genes
not included in the HPO gene list. Pathogenic, likely pathogenic, and variants of unknown
significance in HPO genes were reported; likely benign and benign variants are not reported
but are made available upon request. Incidental findings in the 56 genes recommended
by ACMG were requested by the family and analyzed for pathogenic and likely pathogenic
variants only.

KPTN deficiency identified by WGS

C O L D S P R I N G H A R B O R

Molecular Case Studies

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ADDITIONAL INFORMATION

Data Deposition and Access
Our patient consent does not permit patient sequence data to be uploaded to a data repos-
itory. The variants reported have been deposited in the ClinVar database (https://www.ncbi
.nlm.nih.gov/clinvar/) and can be found under accession numbers VCV000100680.3,
VCV000499654.1, and VCV000377653.5.

Ethics Statement
Written informed consent was obtained from the patient’s legal guardians for publication of
this case report and governed by the Institutional Review Board of Children’s Mercy
Hospitals and Clinics. A copy of the written consent is available for review by the Editor-
in-Chief of this journal.

Acknowledgments
We are very grateful to this family for allowing this case to be published. We thank our col-
leagues in the Center for Pediatric Genomic Medicine and Children’s Mercy Hospital in
Kansas City. This work was not supported by a grant.

Author Contributions
C.J.S., E.F., I.T., L.Z., and S.S. conceived and designed the experiments; C.J.S., E.F., and I.T.
performed the experiments; N.M. and E.F. contributed reagents/materials/analysis tools; I.T.
and C.J.S. wrote the paper; E.F., S.S., L.Z., B.H., A.A., and N.M. reviewed the manuscript;
K.P. reviewed the manuscript and contributed to the clinical investigation of the patient;
and A.A., B.H., and A.T.A. contributed to the recruitment and clinical investigations of the
patient for the study.

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Competing Interest Statement

The authors have declared no
competing interest.

Referees

Sander Pajusalu
Anonymous

Received January 7, 2019;
accepted in revised form
April 16, 2020.

KPTN deficiency identified by WGS

C O L D S P R I N G H A R B O R

Molecular Case Studies

Thiffault et al. 2020 Cold Spring Harb Mol Case Stud 6: a003970 9 of 10

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KPTN deficiency identified by WGS

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