Novel phenotype associated with homozygous likely pathogenic variant in the POP1 gene

The biallelic variants of the POP1 gene are associated with the anauxetic dysplasia (AAD OMIM 607095), a rare skeletal dysplasia, characterized by prenatal rhizomelic shortening of limbs and generalized joint hypermobility. Affected individuals usually have normal neurodevelopmental milestones. Here we present three cases from the same family with likely pathogenic homozygous POP1 variant and a completely novel phenotype: a girl with global developmental delay and autism, microcephaly, peculiar dysmorphic features and multiple congenital anomalies. Two subsequent pregnancies were terminated due to multiple congenital malformations. Fetal DNA samples revealed the same homozygous variant in the POP1 gene. Expression of the RMRP was reduced in the proband compared with control and slightly reduced in both heterozygous parents, carriers for this variant. To our knowledge, this is the first report of this new phenotype, associated with a novel likely pathogenic variant in POP1. Our findings expand the phenotypic spectrum of POP1‐related disorders.


| INTRODUCTION
The POP1 gene encodes the protein subunit of two different ribonucleoprotein complexes: the endoribonuclease for mitochondrial RNA processing complex and the ribonuclease P complex.This protein functions in the pre-RNA processing and maturation, which is essential in the formation of ribosomes. 1Disruption of this process can lead to a wide range of developmental abnormalities.
3][4][5] The phenotype comprises prenatal onset of severe short stature, vertebral anomalies, hypodontia, hypoplasia of the midface, femoral and iliac bones.Affected individuals typically exhibit normal neurodevelopment, with a single report of mild delay. 4 present three cases from the index family, affected with a homozygous likely pathogenic variant in POP1.The proband exhibited intellectual disability and autistic behavior, microcephaly, distinctive dysmorphic features, solitary kidney, severe eczematous dermatitis, olygodactyly and severe scoliosis.Two consecutive pregnancies were terminated due to multiple congenital anomalies and severe intrauterine growth retardation (IUGR).Whole exome sequencing of the proband and fetal DNA samples detected a homozygous likely pathogenic variant in the POP1 gene.Both parents were found to be heterozygous for this variant.Expression studies demonstrated a reduced expression of the RMRP in the proband compared to control and slightly reduced in both heterozygous parents, carriers of this variant.
Written informed consent was obtained from all family members, following the standards set by the Helsinki committee of the Institution.
Genomic DNA was extracted from peripheral blood by the QIAamp DNA Mini kit (QIAGEN), according to the manufactures' instructions.Data set files including the annotated information were analyzed with the following filtering steps: variants which were called less than nine times and synonymous variants were removed.Variants were filtered based on allele frequency less than 0.01 according to online databases; dbSNP, 1000G, ExAC and gnomAD.Likely pathogenicity was assessed if the variant was truncating (splicing or non-sense), missense or an in-frame indel.Missense and in-frame indels were considered if they were predicted to be pathogenic by online prediction tools, PolyPhen-2, SIFT and Mutation Taster.Conformation and familial segregation were performed using direct Sanger sequencing (3500 Genetic Analyzer Applied Biosystems).

| Exome sequencing
Chromosomal microarray: chromosomal microarray was performed on the patient DNA extracted from blood only, using Affymetrix ® CytoScan HD platform, the data files were analyzed using The Affymetrix Chromosome Analysis Suite 1.2.1 (genome build 37).
The study received approval from the institutional Research Ethics Committee.

| Clinical characterization
A healthy, non-consanguineous couple of Bukharin Jewish origin presented with a severely affected daughter and two consecutive terminated pregnancies due to similar multiple congenital anomalies (Figure 1).Chromosomal microarray: chromosomal microarray showed normal female karyotype without evidence for deletions or insertion in known regions of genetic syndromes.Several large (more than 3000 kbp) homozygous regions were found.

| Exome sequencing
Trio Whole exome sequencing performed for the patient and parents revealed that the patient carried a homozygous likely pathogenic variant in the POP1 gene c.449G > T; p.Arg150Leu (NM_001145860.1), both parents were heterozygous carriers.This variant is very rare, was reported in gnomAD only in one heterozygous carrier, it is fully conserved among different species and predicted deleterious by in silico prediction tools.According to ACMG/AMP criteria (PP3 + PM2 + BP1) the variant is classified as variant of uncertain significance (VUS), after corroboration with the expression study, the variant was reclassified as likely pathogenic.The variant was validated by Sanger sequencing performed at our clinical lab.Further Familial segregation revealed that the two fetuses were homozygous and two healthy elder siblings were heterozygous for this variant (Figure 2).No other suspected variants of uncertain significance were found in the analysis also in genes which are not yet known to be related to disease, especially in the homozygous regions that were discovered in the microarray.

| Expression studies
To further validate the pathogenicity of the POP1 variant, we analyzed the abundance of two transcripts that were previously shown to be affected by POP1 deficiency 1-3 -RMRP, RNA component of the mitochondrial RNA processing endoribonuclease, and Pre-5.8S, a target of said endoribonuclease complex.In accordance with the overall trend displayed in previous studies, the p.Arg150Leu variant causes a decrease in RMRP and increase in Pre-5.8Stranscript levels (Figure 3).

| DISCUSSION
3][4][5] Clinical manifestations include prenatal rhizomelic shortening of limbs, severe growth failure leading to dwarfism and generalized joint hypermobility.While most affected individuals achieve normal neurodevelopmental milestones, mild developmental delay was reported in one case 4 (Table S1).
This study presents three cases from the same family, all homozygous for a likely pathogenic variant in the POP1 gene, displaying a distinctive and previously unreported phenotype.
The female proband, exhibits growth retardation, severe neurodevelopmental disorder dysmorphic features and multiple congenital F I G U R E 2 Sanger sequencing of the c.449G > T; p.Arg150Leu variant in the POP1(NM_001145860.1)gene.Carrier heterozygosity in the parents and homozygosity in the patient and the two fetuses were confirmed, the two healthy siblings were heterozygous like the parents.[Colour figure can be viewed at wileyonlinelibrary.com] anomalies.Notably, two consecutive pregnancies were terminated due to similar presentations (Table S1).Intriguingly, all cases manifested oligodactyly, the most severe skeletal anomaly.Moreover, dysmorphic features and microcephaly, presented in our case, have never been described before.
The POP1 gene encodes the protein, known as processing of precursor RNA1 (POP1), shared by two highly essential ribonucleoprotein complexes, RNAse MRP and RNAse P. RNAse MRP (RMRP) is a highly conserved ribonucleoprotein complex, responsible for processing pre-RNA in the mitochondria.RNAse P is found both in the cytoplasm and mitochondria and functions in maturation of tRNA molecules. 6,7Both ribonucleoproteins are evolutionary and structurally related.POP1, is one of 11 interacting proteins of these complexes, composed of a short N-terminal domain, internal motif and a large C-terminal motif.
[8] Previous studies have shown that mutations in POP1, lead to RMRP depletion, suggesting that alterations in ribosomal biogenesis are a major pathogenic mechanism for severe short stature and skeletal dysplasia. 9,10r patient's RMRP level is lower than in reported cases, the same holds for the increase in Pre-5.8S.In a milder phenotype with severe short stature, Pre-58.S did not accumulate. 5Accumulation of the Pre-58S may contribute to a novel severe phenotype.POP1, as a component of the RNA processing complex, plays essential role in RNA metabolism.Pathogenic variants in POP1 may lead to a disruption of this complex, causing accumulation of abnormal RNA molecules, and interfere in developmental pathways. 11Interestingly, the novel likely pathogenic variant found in the presented cases is located in the N-domain of POP1.Another variant at the same position c.449G > A; p.Arg150His (NM_001145860.2) is submitted as a VUS in ClinVar.This domain is common to both RNase MRP and RNase P ribonucloproteins and plays a crucial role in pre-tRNA substrate recognition, [6][7][8][9][10][11][12] which is essential for the precise translation process.Disturbances in tRNA recognition may result in irreversible consequences and have been proposed to contribute to various anomalies and neurodevelopmental disorders. 13sturbances in ribosome biogenesis, and tRNA recognition could lead to abnormal protein synthesis in various tissues including the central nervous system, contributing to multiple anomalies during early development.
However, the precise role of POP1 in determination of the severe phenotype remains unknown, emphasizing the necessity for further investigations.
Trio whole exome sequencing was performed on the patient, and the parents DNA.The samples were enriched with Sureselect Human All Exome v.5 kit 50 Mb (Agilent, Santa Clara, CA, USA).Sequencing was carried out on HiSeq 2500 (Illumina, San Diego, CA, USA) as 100-bp paired-end runs.Reads were aligned with the human reference genome (assembly GRCh37/hg19).Pipeline was performed using the Genoox platform based on BWA (version 0.7.16) for read alignment and GATK HaplotypeCaller (version 3.7) and FreeBayes (version 1.1.0)for variant calling.

The 3 -
year-old girl initially attended the genetic clinic due to global developmental delay, microcephaly, multiple congenital anomalies and distinctive dysmorphic features.The family history is negative for neurological disorders or congenital anomalies.Two elder siblings are healthy.Prenatal sonographic anatomical surveys revealed multicystic kidney and IUGR.She was born at 38th gestational week, birth weight was 2200 g, birth length was 42 cm, and head circumference was 30 cm.In the neonatal period she exhibited moderate hypotonia, feeding difficulties and failure to thrive, necessitating percutaneous endoscopic gastrostomy during the first year.She had global developmental delay, failing to attain ageappropriate milestones.Neurological follow-up examinations revealed intellectual disability, progressive microcephaly and autistic features.She suffered from severe eczematous dermatitis from the age of 10 months.She was followed by pediatric nephrologist due to solitary kidney.Skeletal anomalies included severe scoliosis, bilateral 5th digit clinodactyly, dysgenesis of carpal bones and four toes in both feet.At the age 5 years, she comprehended simple commands, had not developed language; her communication was limited to inarticulate mumbling.Her developmental quotient (DQ) was measured at 30.F I G U R E 1 Family Pedigree: Squares, male; Circles, female; filled symbols, clinically affected subjects.The arrow indicates the proband.Medical interpretations of pregnancies are crossed out.Genotype is indicated on the pedigree.Height was 81 cm and weight 11 kg (À4SDS for age), head circumference was 40 cm (À5SDS).She exhibited proportionate short stature with normal upper-to lower-ratio and did not display any signs of micromelia, rzhizomelia, mesomelia, acromelia or brachydactyly.Dysmorphic features included a low hairline, arched eyebrows, small, down-slanted palpebral features, thin upper vermilion, a tented mouth, prominent lower lip and low-set ears (Figures S1, S2).Brain MRI at the age of three showed nonspecific changes (Figures S3, S4 ).She experienced severe disability and passed away at the age of 8 years.Two consecutive pregnancies revealed similar findings on sonographic anatomical survey.The first fetus presented at 16 weeks gestation with cystic hygroma, bilateral hand syndactyly, feet olygodactyly and severe IUGR.The pregnancy was terminated at 18th gestational week.The subsequent fetus showed severe IUGR, microcephaly, similar dysmorphic features and a tented mouth, clenched hands, hypoplastic fingers and feet olygodactyly.The pregnancy was terminated.