Novel frameshift variant in the PCNT gene associated with Microcephalic Osteodysplastic Primordial Dwarfism (MOPD) Type II and small kidneys.

BACKGROUND: Microcephalic Osteodysplastic Primordial Dwarfism (MOPD) Type II is an autosomal recessive condition encompassing a heterogeneous group of disorders characterized by symmetrical growth retardation leading to dwarfism, microcephaly, and a range of multiple medical complications including neurovascular diseases. Biallelic pathogenic variants in the pericentrin gene (PCNT) have been implicated in its pathogenesis. CASE
PRESENTATION: We performed whole-exome sequencing to ascertain the diagnosis of a 2 year and 6 months old boy who presented with severe failure to thrive, microcephaly, and facial gestalt suggestive of MOPD Type II which included features such as retrognathia, small ears, prominent nasal root with a large nose, microdontia, sparse scalp hair, bilateral fifth finger clinodactyly. He had a small ostium secundum atrial septal defect and bilaterally small kidneys. Microcephalic Osteodysplastic Primordial Dwarfism (MOPD) Type II was confirmed based on a pathogenic compound heterozygous frameshift variant in the PCNT gene c.5059_5060delAA | p. Asn1687fs (novel variant) and c.9535dup (p. Val3179fs). His parents were found to be heterozygous carriers for the variants.
CONCLUSION: We report a novel frameshift variant in the PCNT gene and a previously unreported phenotype for Microcephalic Osteodysplastic Primordial Dwarfism (MOPD) Type II.

Background

Microcephalic Osteodysplastic Primordial Dwarfism (MOPD) Type II (OMIM #210720) is a clinically heterogeneous group of conditions characterized by both pre and post-natal growth retardation together with microcephaly. This condition was first described in 1982 by Majewski Ranke, and Schinzel [1]. Described under the umbrella of Primordial dwarfism (PD) which comprises of several subtypes: Seckel syndrome, Russell Silver syndrome, Meier-Gorlin syndrome, and Majewski Osteodysplastic Primordial Dwarfism (MOPD) I/III and III. Currently, MOPD Type II is known to be the most common subtype. It is inherited as an autosomal recessive disorder caused by biallelic loss of function mutations in the pericentrin (PCNT) gene [2, 3]. Individuals who suffer from this condition have characteristic facies which include a prominent nose and disproportionate features, skeletal dysplasia, impaired growth persisting throughout the post-natal period reaching stunted adult size (average height of 40 cm post-pubertal and adult height of under 100 cm), abnormal dentition and insulin resistance [4, 5]. The care of these patients has now advanced owing to the increased accessibility of high throughput sequencing technologies such as Next Generation Sequencing. A more proactive approach can be taken to address their orthopedic manifestations, insulin resistance, hematological abnormalities, susceptibility to neurovascular diseases including systemic hypertension and renal complications. Thus, they should be encouraged to undergo regular screening to prevent cerebrovascular disease and growth monitoring [2, 6]. Even though MOPD Type II is associated with smaller brain sizes than average, their IQ is near normal [7]. The pericentrin (PCNT) gene located on chromosome 21q22.3 is implicated in mitotic spindle formation and chromosomal segregation [8]. The pericentrin protein (~ 370 kD), encoded by this gene is an anchoring protein that binds to calmodulin expressed in centrosomes. Additionally, it is a cell cycle regulator. The protein consists of a series of highly conserved coiled-coil domains. Thus far 41 pathogenic variants and 3 likely pathogenic variants are reported in Clinvar [9]. In this study, we describe a novel compound heterozygous variant in PCNT gene c.9535dup (p. Val3179fs), c.5059_5060delAA (p. Asn1687fs) giving rise to a new phenotype in a baby of Sri Lankan origin.

Case presentation

The proband is 2 years and 6 months old male. He is the only child to healthy nonconsanguineous parents of Sri Lankan origin. He was delivered when his mother was 24 years old, at 35 weeks of gestation via an emergency lower segment cesarean section due to severe oligohydramnios and marked fetal growth restriction. There was no history of antenatal bleeding, fetal decelerations, or any miscarriages before this pregnancy. His Apgar scores at 1 min and 5 min were 8 and 10 respectively. At birth, his anthropometric parameters were as follows; birth weight—1080 g, birth length—30 cm, and occipitofrontal circumference—24 cm. All parameters were well below—3SD in the standard WHO growth charts. He was admitted to the Special Care Baby Unit during the 1st week of life due to very low birth weight. From birth to 6 months, he had been extensively evaluated for poor weight gain. All recorded biochemical investigations were within the normal range. However, endocrine assessments related to growth were not performed due to financial constraints. His current growth parameters are shown in Fig. 1A–C which are also below—3SD. His developmental milestones remained age-appropriate since birth and there aren’t any symptoms suggestive of any food intolerances, metabolic or malabsorption syndromes. Despite optimal calorie intake, his growth velocity is constant. On examination, the following dysmorphisms were noted retrognathia, small ears, prominent nasal root with a large nose, microdontia, sparse scalp hair (Fig. 2A, B). He also has a characteristic high-pitched voice. Hypopigmented patches were noted in the upper limbs, but they did not follow the Blaschko lines. Limb examination revealed bilateral fifth finger clinodactyly. During other systems screening, echocardiography revealed a small ostium secundum atrial septal defect. An ultrasound scan of the abdomen revealed that the proband had markedly smaller normally functioning kidneys. His right and left kidneys measured 4.5 cm and 3.8 cm respectively. The normal range is 7.1 (6.8–7.4) cm for the right kidney and 7.0 (6.7–7.2) cm for the left kidney [9]. Therefore, the probands kidney is below the 1st percentile for the kidney length in cm according to age. The hip radiograph showed a poorly formed narrow pelvis with a flat acetabulum.

Fig. 1

A Birth weight marked in WHO growth charts, B current weight marked in WHO growth charts, C length and OFC (birth and current measurements) marked in WHO growth charts

Fig. 2

A frontal view of the face, B lateral view of the face

Whole exome sequencing and bioinformatic analysis

Prior to performing Whole Exome Sequencing we obtained written informed consent from the proband’s parents under a protocol approved by the Ethics Review Committee of the Faculty of Medicine University of Colombo. Extraction of the genomic DNA from the blood leukocytes was done using the QIAamp DNA Mini Kit according to the manufacturer’s protocol. The SureSelectXT® Human(Mouse) All Exon V6 5190-886 kit was used in Illumina® NovaSeq® 6000 Next Generation Sequencer for Whole Exome Sequencing. An in-house bioinformatics pipeline was used to analyze the generated data. Aligning the paired-end sequencing data to GrCh37 human reference genome and variant calling was performed using the BWA-mem algorithm and Genome Analysis Tool Kit (GATK). Annotation of the generated variants calling format file was performed using SNP‐eff with the help of Refseq, clinical, and population frequency databases. Then a virtual gene panel consisting of genes that were known to cause skeletal dysplasia (Table 1) was used to filter out the variants relevant to the proband's phenotype. According to the standard ACMG guidelines (https://www.acgs.uk.com/media/11631/uk-practice-guidelines-for-variant-classification-v4-01-2020.pdf), benign variants were filtered out. In silico functional prediction tools (Mutation Taster, SIFT, PolyPhen2, and Provean) were used to predict the functional significance of the detected variants. Functional impact on the protein structure and conservation of the resided region were used to further scrutinize the variants. After filtration, results revealed a pathogenic compound heterozygous frameshift variant in the PCNT gene c.5059_5060delAA | p. Asn1687fs (novel variant) and c.9535dup (p. Val3179fs). On screening his parents, his mother and father were found to be heterozygous for the variants (Fig. 3).

Table 1

Skeletal dysplasia virtual gene panel

ACAN	ACP5	ACVR1	ADAMTS10	ADAMTS17	AFF4	AGA	AGPS
AIFM1	ALPL	AMER1	ANKH	ANO5	ARCN1	ARSB	ARSE
ARSL	ASCC1	ASPM	ATR	B3GALT6	B3GAT3	B4GALT7	BGN
BMP1	BMP2	BMPER	BMPR1B	BPNT2	C2CD3	CA2	CANT1
CASR	CCDC8	CCN6	CDC45	CDC6	CDKN1C	CDT1	CENPJ
CEP120	CEP135	CEP152	CEP63	CFAP410	CHST14	CHST3	CHUK
CILK1	CLCN7	COG1	COL10A1	COL11A1	COL11A2	COL1A1	COL1A2
COL27A1	COL2A1	COL9A1	COL9A2	COL9A3	COMP	CREB3L1	CRTAP
CSF1R	CSGALNACT1	CSPP1	CTSA	CTSK	CUL7	CWC27	DDR2
DDRGK1	DHCR24	DIP2C	DLL3	DLX3	DMRT2	DNA2	DONSON
DVL1	DVL3	DYM	DYNC2H1	DYNC2LI1	EBP	EIF2AK3	ESCO2
EVC	EVC2	EXOC6B	EXOSC2	EXT1	EXT2	EXTL3	FAM20C
FAM46A	FAR1	FBN1	FGF23	FGF9	FGFR1	FGFR2	FGFR3
FIG4	FKBP10	FLNA	FLNB	FN1	FTO	FUCA1	FZD2
GALNS	GALNT3	GDF5	GDF6	GHR	GHRHR	GHSR	GJA1
GLB1	GMNN	GNAS	GNE	GNPAT	GNPTAB	GNPTG	GNS
GORAB	GPC6	GPX4	GSC	GUSB	GZF1	HES7	HGSNAT
HPGD	HSPG2	HYAL1	IARS2	ICK	IDS	IDUA	IFITM5
IFT122	IFT140	IFT172	IFT43	IFT52	IFT57	IFT74	IFT80
IFT81	IGF1	IGF2	IHH	IMPAD1	INPPL1	JAG1	KAT6B
KIAA0586	KIAA0753	KIF22	KL	KMT2A	LARP7	LBR	LEMD3
LFNG	LIFR	LIG4	LMNA	LMX1B	LONP1	LOXL3	LRP4
LRP5	LRRK1	LTBP2	LTBP3	MAFB	MAN2B1	MANBA	MAP3K7
MATN3	MBTPS2	MCM5	MCPH1	MEOX1	MESP2	MGP	MMP13
MMP14	MMP2	MMP9	MNX1	MSX2	MYH3	MYO18B	NAGLU
NANS	NBAS	NEK1	NEU1	NKX3-2	NOG	NOTCH2	NPPC
NPR2	NPR3	NSDHL	NSMCE2	NXN	OBSL1	OCRL	ORC1
ORC4	ORC6	OSTM1	P3H1	P4HB	PAM16	PAPSS2	PCGF2
PCNT	PCYT1A	PDE4D	PEX5	PEX7	PGM3	PISD	PKDCC
PLK4	PLOD2	PLS3	POC1A	POLR1A	POP1	POR	PPIB
PPP3CA	PRKAR1A	PTDSS1	PTH1R	PTHLH	PTPN11	PYCR1	RAB33B
RBBP8	RECQL4	RIPPLY2	RMRP	RNU4ATAC	ROR2	RSPRY1	RTTN
RUNX2	SC5D	SEC24D	SERPINF1	SERPINH1	SETBP1	SFRP4	SGSH
SH3PXD2B	SLC17A5	SLC26A2	SLC35D1	SLC39A13	SLCO2A1	SLCO5A1	SMAD4
SMARCAL1	SNRPB	SNX10	SOX9	SP7	SPARC	SQSTM1	SRCAP
SUCO	SULF1	TAB2	TAPT1	TBCE	TBX15	TBX3	TBX5
TBX6	TBXAS1	TCIRG1	TCTEX1D2	TCTN3	TGFB1	TMEM165	TMEM38B
TNFRSF11A	TNFRSF11B	TNFSF11	TRAPPC2	TREM2	TRIM37	TRIP11	TRMT10A
TRPS1	TRPV4	TTC21B	TUBGCP6	TYROBP	VAC14	VPS33A	WDR19
WDR34	WDR35	WDR60	WISP3	WNT1	WNT3	WNT3A	WNT5A
XRCC4	XYLT1	XYLT2	ZMPST

Fig. 3

Sanger chromatograms of the PCNT gene variants in the proband (compound heterozygous) mother (heterozygous) and father (heterozygous) and the corresponding pedigree chart

In-silico analysis

For genomic analysis of the present case, the FASTA sequence of the homo sapiens pericentrin B (PCNT2) mRNA, complete cds with GenBank ID: AF515282.1 of 10020 bp was downloaded from the NCBI nucleotide database (https://www.ncbi.nlm.nih.gov/nuccore/ AF515282). In the wild-type sequence, there were two adenine nucleotides at cDNA positions 5059 and 5060. Manual deletion of this nucleotide pair was carried out to generate the variant corresponding to the proband c.5059_5060delAA. Both mRNA sequences were submitted to the ORF tool to access the differences in the coding amino acids. We observed a change in the amino acid sequence owing to a shift in the reading frame. Thus, in the mutated protein at the 1687th position amino acid asparagine "N" was replaced by glutamine "Q". Furthermore, there was an appearance of TGA (i.e., termination codon) succeeding 10 triplet codons after the initial amino acid change. This resulted in premature termination of protein synthesis. The downloaded FASTA sequence of the wild-type protein (UniProtKB ID-O95613, PCNT_HUMAN) had 3336 amino acid residues and 3D modeling was carried out using the SPDBV offline software. Modeling of the wild type and mutated PCNT was done by homology approach by taking 1JQN as a template structure. According to the 3D models of the two proteins, the amino acid numbers were 643 and 615 respectively. We compared the number of non-glycine and non-proline residues which were 600 and 576 respectively. It was also observed that in the mutated protein one amino acid was found in the disallowed region thus contributing to an unstable tertiary structure (Fig. 4). There was also an increase in the number of cavities from 4 in the wildtype to 5 in the mutant protein with altered amino acid configurations around these cavities (Fig. 5).

Fig. 4

Ramachandran Plot showing the empirical distribution of amino acids observed in a A normal and B mutated PCNT protien structure for structure validation

Fig. 5

Amino acids present around the cavities of wild type (A–D) and mutated (E–I) PCNT protein

Discussion and conclusion

Herein we described the first genetically confirmed case of Microcephalic Osteodysplastic Primordial Dwarfism (MOPD) Type II in a patient of Sri Lankan origin. Based on the genetic diagnosis the parents were counselled and multidisciplinary shared care was arranged locally.

Growth monitoring was carried out with specific growth curves designed for this condition [10]. Thus, growth parameters of the proband were plotted between median and − 1 SD in MOPD Type II specific growth curves. A study conducted on 47 individuals with this condition revealed 64% of the study population was diagnosed with a vascular condition such as moyamoya and intracranial aneurysms or both. Additionally, vascular complications involving the renal arteries, coronaries, and external carotids were also reported in this group [2]. The proband had normal findings in the baseline ultrasound KUB and MRI brain. However, structural heart defects were reported which is a rare phenotype [11]. Due to the high risk of neurovascular manifestations in later life, routine surveillance was arranged as recommended in the literature. Proband will be followed up with multidisciplinary inputs by a cardiologist and neurologist. Furthermore, we observed bilaterally small kidneys in the proband, which was not described with MOPD Type II before. Yearly skeletal surveillance to identify hip pathologies such as hip dislocations and scoliosis is also essential, a narrow dysplastic hip with a flat acetabulum is a cardinal feature [12]. Oro dental findings such as microdontia, malformed teeth, short roots especially of the molars, tooth agenesis, and enamel hypoplasia are commonplace. Hence proper dental care and regular follow-up with a dentist are essential. Their also prone to endocrine abnormalities, mainly insulin resistance which should be anticipated after 5 years of age. Growth hormone therapy is not usually recommended considering the risk of scoliosis and the lack of evidence in the outcome.

MOPD Type II affects multiple organ systems this could reflect the spindle dysfunction caused by PCNT variants. Thus, loss of function of pericentrin is implicated in mislocalization of cellular proteins due to mitotic spindle defects causing missegregation of chromosomes, mitotic failure with eventual cell arrest, and cell death [13]. Pericentrin binds calmodulin expressed in the centrosomes, it contains a series of coiled-coil domains that interact with the microtubule nucleation component gamma-tubulin which is essential throughout the cell cycle. As confirmed by protein modeling approaches defects in the protein–protein interaction domains of PCNT could have contributed to the phenotype of MOPD Type II in our patient. According to the literature, there was a high incidence of cerebrovascular malformations when the last exons from 30 to 43 were involved [12]. The two variants identified in the proband c.9535dup (p.Val3179fs) resides in exon 43 and c.5059_5060delAA (p. Asn1687fs) resides in exon 27. The first variant is reported to cause non-sense-mediated decay of mRNA. It is present in population databases (rs747058622) at a very low frequency (G = 0.00002/5 (GnomAD_exomes) and G = 0.000033/4 (ExAC)) and is recorded as a likely pathogenic variant in the Clinvar database (Variation ID: 264920). The second variant has not previously been reported in population databases or clinical databases and represents a null allele in the PCNT gene for which loss-of-function is a known mechanism of MPOD II disease. In this study, the detection of biallelic variants in the PCNT gene confirmed the diagnosis of MOPD Type II and presented a new phenotype, thus expanding the phenotypic spectrum of PCNT variants associated with this condition.