Incomplete penetrance of a KANSL1 loss of function variant resolved by RNA analysis,  a case report

doi:10.21203/rs.3.rs-4710959/v1

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Case Report

Incomplete penetrance of a KANSL1 loss of function variant resolved by RNA analysis, a case report

https://doi.org/10.21203/rs.3.rs-4710959/v1

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Background

Epilepsy is a prevalent neurological disorder, affecting approximately 1% of the global population. The extensive clinical and genetic variability in epilepsy makes accurate diagnosis a significant challenge.

Case presentation

In this study, we describe a girl with a clinical presentation of developmental and epileptic encephalopathy (DEE). Using whole genome sequencing (WGS), we identified several candidate variants in the HNRNPU, NIPBL, and KANSL1 genes with partial overlap with the patient clinical presentation. Subsequent analysis revealed that only the variant in the HNRNPU gene arose de novo while the others were inherited from an unaffected parents. However, the previously reported pathogenic loss of function variant in the KANSL1 gene, inherited from a healthy mother, complicated comprehensive family counseling. A thorough investigation using RNA analysis showed that the variant in the KANSL1 gene is located in a duplicated locus, which is not functional, explaining the assumed incomplete penetrance.

Conclusions

This case illustrates the importance of integrating WGS with additional analyses to accurately diagnose and understand the molecular basis of incomplete penetrance in the KANSL1 gene.

KANSL1

HNRNPU

Incomplete penetrance

RNA analysis

Epilepsy is among the most prevalent neurological disorders, with an incidence rate of approximately 50 new cases per 100,000 people annually [1]. Genetic factors account for about 70–80% of epilepsy cases [2], and around 1% of the global population is affected by this condition. Approximately 75% of epilepsy cases manifest during childhood, highlighting the increased vulnerability of the developing brain to seizures [1]. Epilepsy encompasses various types and syndromes, classified based on clinical and EEG features, etiologies, and comorbidities. Developmental and epileptic encephalopathies (DEEs) represent the most severe spectrum of epilepsy disorders with a significant monogenic component [3], [4].

Due to the phenotypic and genetic heterogeneity of DEEs, a multigene screening approach is essential. Currently, 172 genes have been identified as responsible for DEEs [3]. Despite the routine use of panels and exome sequencing, the diagnostic efficiency for DEE remains limited and ranges from 18–48% [5]. WGS has the potential to enhance diagnostic outcomes by providing uniform coverage across the genome, detecting variants in noncoding regions and the mitochondrial genome, identifying repeat expansion variants, and offering more reliable detection of structural variants, especially those with breakpoints in repetitive regions and copy-neutral variants such as inversions and translocations [6].

In this study, we describe a female patient with early-onset epileptic encephalopathy. WGS identified a pathogenic variant in the HNRNPU gene. Additionally, a previously reported pathogenic variant in the KANSL1 gene, associated with Koolen-de Vries syndrome [7], was found to be inherited from the healthy mother. Through RNA analysis, we were able to establish the mechanism of incomplete penetrance of the disease and to explain the absence of a pathological phenotype in the proband and the proband's mother.

Subject

The proband, a two-year-old girl, and her parents underwent a detailed clinical examination at various medical institutions in Moscow (National Medical Research Center for Neurosurgery named after Academician N.N. Burdenko). DNA analysis and genetic counseling were performed at the Research Center for Medical Genetics, Russia. All research participants gave informed consent (or responsible consent form for the infant proband) for the clinical examination and publication of their anonymized data. The study was performed in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of the Research Center for Medical Genetics, Russia.

Whole genome sequencing

The whole genome sequencing of the proband's DNA sample, obtained from peripheral blood, was conducted in-house. DNA extraction from whole blood was carried out using the Quick-DNA Miniprep Kit (Zymo Research, California, USA), following the manufacturer's protocol. To assess DNA purity, absorbance measurements were taken at both 260/280 nm and 230/260 nm using a DS-11 FX + spectrophotometer/fluorometer (DeNovix).

Library preparation (PCR-Free) was performed using MGI platforms following their respective protocols.. Subsequently, paired-end sequencing (2x150) was executed on the DNBSEQ-T7 sequencer from MGI with an average coverage of 30x. The data processing was carried out using the “NGSData-Genome” program (Beskorovainy N.S. Program “NGSData”//Certificate of State Registration of Computer Programs No. 2021662119.2021.). The reads were aligned to the reference genome hg19 using bwa v.0.7.17-r1188. Variant calling was performed with strelka2 v.2.9.10 and gatk v.4 algorithms. Copy number variations were assessed using cnvkit 0.9.9, while structural variants were detected with Manta v.1.6.0. Additionally, tandem repeats were analyzed using ExpansionHunterDenovo. Variant annotation was performed using SnpEff v5.0, annovar v.2017, and vep v.104.3. Splice predictors used included dbNSFP v.4, SPiP v.2.1, mmsplice v.2.3, spliceai v.1.3.1, and spidex v.1.

For annotation of identified variants, automatic pathogenicity prediction algorithms (SIFT, PolyPhen2-HDIV, PolyPhen2-HVAR, MutationTaster, LRT) were used, as well as evolutionary conservation position calculation algorithms (PhyloP, PhastCons). To assess the population frequencies of the identified variants, data from the “1000 Genomes” project, ESP6500, Exome Aggregation Consortium, and gnomAD were used.

Sanger sequencing

Validation of the variants identified through WGS was performed using Sanger sequencing. DNA samples from the proband's and his parents' peripheral blood were used as templates. The PCR was conducted using the ProFlex PCR System (Applied Biosystems). Visualization of the obtained PCR products was carried out using agarose gel electrophoresis. The PCR products were sequenced by Sanger sequencing in-house (Evrogen). For the validation and segregation of the NM_015443.4(KANSL1):c.727C > T variant, the following primers were used 5’-GGAGACAGCCAGTTTTGGAG-3’ and 5’-GGTCTCACTGGCAGCTTTTC-3’. For the validation and segregation of the NM_031844.3(HNRNPU):c.1665_1666del variant, the primers 5’-ATGGGAGGAAAGGCAAGATT-3’ and 5’-ATTTTGAGGACCGCATGTTC-3’ were used. For the validation and segregation of the NM_133433.4(NIPBL):c.8101A > G variant, the primers 5’-AAGAGTAAAGACACGGGTACAAA-3’ and 5’-TGAGAATCTGTTTCTGCTGCAC-3’ were used.

RNA analysis of a variant in KANSL1

Analysis of mRNA structure of the KANSL1 locus, containing the c.727C > T variant, was performed using RNA extracted from peripheral blood mononuclear cells (PBMC). PBMCs were extracted from the proband's and mother's peripheral blood by centrifugation in a Ficoll gradient. RNA was extracted using the Extract RNA reagent (Evrogen, Russia). Reverse transcription was carried out using the Reverse Transcription System (Dialat, Russia) according to the manufacturer's recommendations. The quality of the obtained cDNA was assessed by performing qPCR of housekeeping genes. To determine the presence of the c.727C > T variant in the KANSL1 mRNA, the target locus was amplified using the following priners: 5’-AGCACCTGTAAATGGGTTGG-3’ and 5’-AAGGGACTGTGTGGAGGATG-3’ with further Sanger sequencing

Clinical findings

The proband was first consulted by a geneticist at the Research Centre For Medical Genetics at the age of 1 year and 4 months due to seizures. The patient's medical history revealed that she was born from the first pregnancy, which included the use of Duphaston during the first trimester and maternal toxicosis. At 20 weeks gestation, fetal ultrasound indicated ventriculomegaly. Spontaneous natural delivery occurred at 40–41 weeks of gestation. At birth, her weight was 2960 grams, length 50 cm, and her Apgar scores were 8–9. Early development was delayed. Seizures debuted at 12 mounts and were characterized by brief periods of eye rolling lasting several seconds, with up to 14 episodes a day. Valproic acid therapy was unsuccessful in reducing seizure frequency. Video electroencephalography (EEG) monitoring showed epileptiform activity, and brain MRI confirmed ventriculomegaly.

The girl was also seen by a neurologist, who diagnosed her with delayed psychomotor and speech development, non-progressive internal hydrocephalus and generalized idiopathic epilepsy, as well as hypotonic-atactic syndrome. Physical examination by a geneticist noted plagiocephaly along with several mild dysmorphic features including, hypertelorism, long eyelashes, epicanthus, hypoplasia of the eyebrow ridges, a "wine" stain on the forehead, short neck, and an incomplete transverse palmar crease.

Molecular findings

At the age of 1.5 years, due to the onset of seizures, developmental delay, and dysmorphisms, a genetic cause of the disease was suspected, and whole-genome sequencing was prescribed for the proband.

WGS revealed three candidate variants: HNRNPU, NIPBL, and KANSL1 (Fig. 1A, 1B) (Table 1).

Figure 1. A. Pedigree of the reported family with genotypes. B. Validation of variants identified in the proband through WGS using Sanger sequencing. C. Presence of the c.717C > T variant in the KANSL1 gene in DNA samples from blood and buccal epithelial tissues of available family members.

Table 1

Variants table.
Gene	Chromosome	HGVS DNA reference	HGVS protein reference	Variant type	Predicted effect	Genotype
HNRNPU	1	NM_031844.2:c.1665_1666del	L556Afs*12	del	Loss-of-function	wt/del
KANSL1	17	NM_015443.3: c.727C > T	Q243*	noncense	Loss-of-function	G/A
NIPBL	5	NM_133433.3: c.8101A > G	M2701V	missense	?	A/G
N/A – not available

A novel heterozygous missense variant in exon 47 of the NIPBL gene, NM_133433.4:c.8101A > G, resulting in an amino acid substitution at position 2701 of the protein (p.(Met2701Val)), was identified (Fig. 1B). Heterozygous pathogenic variants, including missense, in the NIPBL gene are known to cause Cornelia de Lange syndrome type 1 (OMIM: 122470). This syndrome is characterized by intellectual disability, distinctive facial features, prenatal and postnatal growth retardation, and hirsutism. Congenital anomalies include malformations of the upper limbs, gastrointestinal malformations/rotations, pyloric stenosis, diaphragmatic hernia, heart defects, and genitourinary malformations (Boyle et al., 2015). The neurological symptoms and facial dysmorphism of Cornelia de Lange syndrome partially matched the proband's phenotype (Table 2). Segregation analysis indicated that the variant was inherited from the proband’s heatlhy father.

The second heterozygous variant in exon 9 of the HNRNPU gene, NM_031844.3:c.1665_1666del, was also identified, resulting in a frameshift and formation of a premature termination codon (p.(Leu556Alafs*12)) (Fig. 1B). This substitution wasn’t described before. Heterozygous loss-of-function (LoF) variants in the HNRNPU gene have been reported in patients with developmental and epileptic encephalopathy type 54 (OMIM: 617391), often accompanied by variable craniofacial and skeletal anomalies [8] (Table 2). Segregation analysis confirmed the de novo status of the variant.

Finally, WGS revealed a nonsense variant previously described as pathogenic in exon 2 of the KANSL1 gene, NM_015443.4:c.727C > T (p.(Gln243Ter)) (clinvar accession number SCV004703389) (Fig. 1B). Loss-of-function variants in the KANSL1 gene are associated with Koolen-de Vries syndrome (OMIM: 610443), characterized by congenital malformations, developmental delay/intellectual disability, neonatal/childhood hypotonia, epilepsy, dysmorphisms, and behavioral features. Psychomotor developmental delay is noted in all individuals from an early age [7, 8]. It is important to note that the variant was detected in 18 out of 53 reads (mutant allele fraction of 34%). Segregation analysis showed that this variant was inherited from the healthy mother. The clinical presentation of Koolen-de Vries syndrome only partially matched the proband's clinical picture (Table 2), which may be a consequence of the incomplete penetrance of this syndrome.

Table 2

Frequency of select features in patients afflicted by HNRNPU-related neurodevelopmental syndrome, Cornelia de Lange syndrome, Koolen-de Vries syndrome. The presence of these features in the proband.
Symptom	HNRNPU-related neurodevelopmental syndrome*	Cornelia de Lange syndrome**	Koolen-de Vries syndrome***	Proband’s symptoms
Developmental delay	100%	> 95%	> 75%	+
Dysmorphic craniofacial features	97%	98%	75%	+
Seizure disorder	95%	25%	25%-49%	+
Intellectual disability	84%	> 95%	75%	+
Speech delay	80%	> 95%	75%	+
Hypotonia	79%	25%-49%	50%-75%	+
Feeding difficulties	57%	> 95%	50%-75%	+
Behavioral issues	50%	25%-49%	50%-75%	+
Cardiac abnormalities	30%	30%	50%-75%	+

* [8]

** [9]

*** [10]

Study of the pathogenicity of the KANSL1 variant

Since a pathogenic causative variant in the HNRNPU gene was identified in the patient, the presence of another variant described as pathogenic in the KANSL1 gene in both the patient and his healthy mother complicated the diagnosis and counseling of the patient. Since incomplete penetrance, as the most obvious cause, is not described for Koolen-de Vries syndrome, we began by investigating the likelihood of mosaicism for the variant. De novo mosaic variant has been previously described in the patient with atypical phenotype presentation [7, 8, 11]. The ratio of peaks of the heterozygous variant c.727C > T in the proband (wt/mut = 2/1) suggested the possibility of mosaicism. To test this hypothesis, we searched for the variant in genomic DNA samples extracted from the peripheral blood and buccal epithelial cells of the proband and the proband's mother. The presence of the variant with the same ratio of peaks was confirmed in all DNA samples tested, which ruled out mosaicism.

Additionally, the variant was sought in all available family members (Fig. 1C). Analysis revealed that the proband's mother and maternal grandmother is also a heterozygous carrier of the variant. In DNA samples from all family members carrying the variant, an unusual ratio of the heterozygous variant peaks c.727C > T (wt/mut = 2/1) was observed. Thus, the mosaic state of the c.727C > T variant was not confirmed

The allelic imbalance observed in variants detected during WGS and Sanger sequencing in the proband prompted us to consider the presence of a duplication spanning the KANSL1 locus. Subsequent copy number variation (CNV) reanalysis of the WGS data confirmed the duplication of this region, delineated by approximate boundaries of chr17:44,165,260_44,651,550dup (156 kb). According to GnomAD, such a duplication is common and has a population frequency of approximately 20%. This common duplication encompasses exons 1 and 2 of the KANSL1 gene, including the region where the c.727C > T variant was identified. We hypothesized that the c.727C > T variant listed in the patient's WGS report is located not in the KANSL1 gene, but in its duplicated locus. This duplicated region of the gene is highly likely to be non-functional. To determine the expression of the duplicated KANSL1 locus, we designed a set of primers to amplify the mRNA locus of the KANSL1 gene spanning exons 1 to 3.

Figure 2. Diagram showing the layout of the KANSL1 gene, with the duplication region marked in blue. Primer locations for RNA analysis are indicated as F2-R3 and F2-R7. Sequencing chromatograms of the RT-PCR products are also provided.

The boundaries of the locus were selected to ensure amplification occurs only from the main gene and not from the gene region included in the tandem duplication (Fig. 2). Sequencing of the RT-PCR product revealed that this mRNA locus is transcribed and contains the heterozygous c.727C > T variant.

A subsequent review of the duplication boundaries in the patient’s genome data indicated that the patient's duplication differs from the common duplication registered in gnomAD, as it includes exon 3 and part of intron 3. We developed a new PCR system to amplify the KANSL1 mRNA from exon 2 to exon 7. The analysis did not show the presence of the c.727C > T variant in the mRNA.

Due to the complexity of the region, we were unable to determine the exact boundaries of the duplication on the DNA level. Based on coverage and allelic imbalance, we estimate its approximate boundaries to be chr17:44165365–44375856 with a size of 210 kb.

Thus, we demonstrated that the variant c.727C > T in the KANSL1 gene resides within a duplication of this gene and is non-functional, enabling us to classify it as benign. The variant was submitted in ClinVar (accession number SCV005049516).

Among hereditary diseases, a significant portion is represented by conditions with broad clinical presentations and variable expressivity. Clinical heterogeneity makes it impossible to diagnose these patients based solely on clinical symptoms. In such cases, an accurate diagnosis can only be made based on the identification of the causative genetic variant.

Finding the molecular cause is often challenging, as many pathologies have similar clinical manifestations but result from disruptions in the expression of different genes. Whole genome sequencing is the method of choice for these patients. For a number of genetically heterogeneous conditions, WGS has dramatically improved the diagnostic yield [12].

However, WGS, like any diagnostic method, has its nuances and limitations [13]. In particular, the interpretation of WGS data often reveals multiple candidate variants, raising the question of how to prioritize them. Segregation analysis is commonly used for this purpose. This quick and straightforward approach determines whether a variant is inherited from one of the parents or arose de novo. However, segregation analysis is not an absolutely universal method, as many inheritance features, such as incomplete penetrance or mosaic inheritance, can complicate the interpretation.

In our study, segregation analysis did not allow us to exclude the LoF variant in the KANSL1 gene from the list of candidate variants. This variant, in addition to the proband, was found in healthy family members across two generations. This circumstance and the unusual peak ratio of the heterozygous variant led to the hypothesis of mosaicism of the variant. One individual with a de novo mosaic nonsense KANSL1 variant (p.(Gln243ter)) was described recently [11, 13]. In our case the hypothesis of mosaicism was not confirmed, as the variant was found in all DNA samples taken from different tissues of the proband.

Furthermore, segregation analysis cannot be relied upon if candidate genes exhibit incomplete penetrance. Although incomplete penetrance has not been described for KANSL1, in the family we studied, two healthy members are carriers of a heterozygous nonsense variant. Loss-of-function variants in KANSL1 are a known mechanism for Koolen-de Vries syndrome [14]. Additionally, according to the population database GnomAD, KANSL1 has a pLI score of 1. These factors combined prevent the exclusion of the c.727C > T variant from the list of candidate genes and necessitate further analysis of the variant.

Structural rearrangements present a particular challenge in the interpretation and further validation of WGS data [15],[16]. In addition to the obvious difficulties associated with detecting and annotating rearrangements, the presence of frequent population rearrangements significantly contributes to the search for the causative variant. Frequent duplications may contain transcriptionally inactive copies of genes or gene segments. Variants can accumulate in such loci and be annotated by bioinformatics algorithms as rare and pathogenic. One method for investigating the pathogenicity of such variants is RNA analysis. In our study, RNA analysis revealed that the variant in the KANSL1 gene is located within a duplicated region, which is non-functional and therefore cannot contribute to the development of a pathological phenotype.

Using a comprehensive approach that included segregation and RNA analysis, we demonstrated that variants in the NIPBL (ClinVar accession number VCV002920673) and KANSL1 genes are not pathogenic and excluded them from the list of candidate genes. Given the clinical presentation of the proband and the de novo status of the variant in HNRNPU (ClinVar accession number VCV001343373), it was determined to be pathogenic and causative.

Whole genome sequencing is a powerful modern diagnostic tool that has greatly advanced the diagnosis of numerous genetic disorders. However, it does not always provide an exact diagnosis for patients, emphasizing the need for a comprehensive approach to accurately identify the molecular causes of complex genetic conditions.

In our work, we used a combination of whole-genome sequencing methods, segregation analysis, and RNA analysis to diagnose the patient and describe the molecular mechanism of incomplete penetrance for the nonsense variant in the KANSL1 gene.

DEE

developmental and epileptic encephalopathy

WGS

whole genome sequencing

PBMC

peripheral blood mononuclear cells

EEG

electroencephalography

CNV

copy number variation

LoF

loss-of-function

Ethics approval and consent to participate -

This study was approved by the Institutional Review Board of the Research Centre for Medical Genetics, Russia and was performed in accordance with the Declaration of Helsinki. For this work written informed consent was obtained from all participants. For participants under 16 years old written informed consent was obtained from the participant’s parent.

Consent for publication -

All research participants gave written informed consent (or responsible consent form for infant proband) to the clinical examination and the publication of their anonymized data.

Competing interests -

The authors declare that they have no competing interests.

Funding -

The research was carried out within the state assignment of the Ministry of Science and Higher Education of the Russian Federation for RCMG.

Author Contribution

D.A. performed the segregation studies, functional analysis and wrote the manuscript. D.G. -wrote and revised the manuscript and performed the clinical examination of the patient. M.N. – analysed the NGS data. M.S. – wrote and revised the manuscript. All authors read and approved the final manuscript.

Acknowledgement

We thank Peter Sparber for his invaluable contribution for the quality of the manuscript

Availability of data and materials -

Data that support the findings of this study are available from the corresponding authors upon reasonable request

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You are reading this latest preprint version

Incomplete penetrance of a KANSL1 loss of function variant resolved by RNA analysis, a case report

Status:

Version 1

Abstract

Background

Case presentation

Conclusions

Figures

Introduction

Materials and methods

Subject

Whole genome sequencing

Sanger sequencing

Results

Clinical findings

Molecular findings

Discussion

Conclusions

Abbreviations

Declarations

Competing interests -

Funding -

Author Contribution

Acknowledgement

Availability of data and materials -

References

Additional Declarations

Supplementary Files

Status:

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