10.5061/DRYAD.8PK0P2NNJ
Neocleous, Vassos
0000-0002-6890-8395
Cyprus Institute of Neurology and Genetics
Fanis, Pavlos
Cyprus Institute of Neurology and Genetics
Toumba, Meropi
Cyprus Institute of Neurology and Genetics
Gorka, Barbara
Cyprus Institute of Neurology and Genetics
Kousiappa, Ioanna
Cyprus Institute of Neurology and Genetics
Tanteles, George
0000-0002-8117-9852
Cyprus Institute of Neurology and Genetics
Iasonides, Michalis
Iliaktida Pediatric & Adolescent Medical Centre
Nicolaides, Nicolas
National and Kapodistrian University of Athens
Christou, Yiolanda
Cyprus Institute of Neurology and Genetics
Michailidou, Kyriaki
Cyprus Institute of Neurology and Genetics
Nicolaou, Stella
Archbishop Makarios III Hospital
Papacostas, Savvas
Cyprus Institute of Neurology and Genetics
Christoforides, Athanasios
0000-0003-4964-5726
Aristotle University of Thessaloniki
Kyriakou, Andreas
Archbishop Makarios III Hospital
Vlachakis, Dimitrios
Agricultural University of Athens
Skordis, Nicos
University of Nicosia
Phylactou, Leonidas
Cyprus Institute of Neurology and Genetics
Pathogenic and low frequency variants in children with central precocious
puberty
Dryad
dataset
2021
Pediatric endocrinology
A G Leventis Foundation
https://ror.org/01qkhz224
RCB Bank Ltd.*
RCB Bank Ltd.
2021-09-16T00:00:00Z
2021-09-16T00:00:00Z
en
https://www.frontiersin.org/submission/submit?status=p&id=2756356
61829 bytes
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CC0 1.0 Universal (CC0 1.0) Public Domain Dedication
Background Central precocious puberty (CPP) due to premature activation of
GnRH secretion results in early epiphyseal fusion and to a significant
compromise in the achieved final adult height. CPP is usually idiopathic
and is disproportionally observed in girls compared to boys. Currently,
only few genetic determinants of children with CPP have been described and
the role they exert on the development of the disorder. In this original
study rare variants in MKRN3, DLK1, KISS1, KISS1R and MAGEL2 genes are
reported in patients with CPP. Methods Fifty-four index females and 2
index males with CPP underwent whole exome sequencing (WES) by Next
Generation Sequencing (NGS). The identified rare variants were initially
examined by in silico computational algorithms and confirmed by Sanger
sequencing. Additionally, a genetic network for the MKRN3 gene mimicking a
holistic regulatory depiction of the crosstalk between MKRN3 and other
genes is designed. Results Three previously described pathogenic MKRN3
variants in the coding region of the gene occurred in 12 index females
with CPP. With the p.Gly312Asp pathogenic variant of the MKRN3 gene being
the most prevalent and exclusively found among the Cypriot CPP cohort, it
is projected to be the result of founder effect phenomenon. In seven
additional CPP patients from the same cohort several other likely and rare
pathogenic upstream variants in the MKRN3 gene were also observed. In
addition to the MKRN3 variants, a total of 16 other rare variants in DLK1,
KISS1 and MAGEL2 were also identified in other CPP patients from the same
cohort. Interestingly, the frequent variant rs10407968 (p.Gly8Ter) of the
KISS1R gene appeared to be less frequent in the cohort of patients with
CPP. Conclusion The results of the present study denote the key role of
the imprinted MKRN3 gene in puberty. Additionally, pathogenic variants can
also exist in the noncoding region of the MKRN3 gene such as the proximal
promoter and 5’-UTR region and which can also be considered as
contributing factors to CPP. Overall, the results of present study have
emphasised the necessity of the allied genetic and clinical approach which
is necessary for the management and treatment of CPP.
Genetic Analysis Genomic DNA was extracted from peripheral blood using the
Gentra Puregene Kit (Qiagen, Valencia, CA, USA) according to the
manufacturer’s instructions. The DNA purity was measured using the
Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE,
USA). Prior to library preparation for whole exome sequencing (WES)
genomic DNA was quantified using the Qubit dsDNA BR Assay Kit (Invitrogen,
Life Technologies, Eugene, OR, USA) on a Qubit® 2.0 Fluorometer
(Invitrogen, Life Technologies, Eugene, OR, USA). WES was performed by
using the TruSeq Exome Kit (Illumina Inc., San Diego, CA, USA) with
paired-end 150 bp reads. NGS was performed using the NextSeq 500/550 High
Output Kit v2.5 (150 Cycles) on an NextSeq500 system (Illumina Inc., San
Diego, CA, USA). The FastQC quality control tool
(http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) was used to
evaluate the quality of the WES procedure. The mean target coverage of the
whole exome was 62.13X. Specifically, 10X coverage was reached for 92.34%
of the nucleotides, 20X coverage for 86.03% of the nucleotides and 30X
coverage for 76.96% of the nucleotides, indicating that the WES reaction
was of sufficiently high quality for subsequent analysis. Variant Analysis
The fastq data obtained by WES were processed using an in-house
bioinformatics pipeline. Briefly, all variants were inputted into the
VarApp Browser and filtered. VarApp is a graphical user interface, which
supports GEMINI (18). Variants in selected genes involved in pubertal
onset and were mutations have been reported for precocious puberty were
further analyzed using the Qualimap v2.2.1 tool to calculate the target
coverage. Mean target coverage was 60X of the selected genes
(Supplementary Table 1). Variants in these genes were additionally
filtered using the VarApp Browser for minor allele frequencies of less
than 1% in public databases such as 1000 genomes, ExAC browser and Exome
Sequencing Project (ESP). Moreover, variants were filtered and selected
according to their impact such as frameshift, splice acceptor, splice
donor, start lost, stop gained, stop lost, inframe deletion, inframe
insertion, missense, protein altering and splice region. In addition,
variants were filtered by the VarApp Browser for their pathogenicity by
two in silico tools, SIFT and Polyphen2. Population-specific data from an
in-house WES library composed of 43 randomly selected samples of Cypriot
origin were used to evaluate the potential disease-causing variants. All
variants identified were confirmed by Sanger sequencing. Finally, the
variants were categorized for their pathogenicity using the standards and
guidelines of the American College of Medical Genetics and Genomics and
the Association for Molecular Pathology.