Identification of Osteogenesis Imperfecta Type VI: A First Case Report from a Pakistani Family

Background: Osteogenesis imperfecta type VI (OI type VI) is a rare autosomal recessive disease of bone mineralization characterized by multiple bone fractures after six months of age, without a history of other extra-skeletal complications. SERPINF1 (serpin inhibitor clade F1) is the causative gene for this abnormality, having a chromosomal location 17p13. Many cases have been reported from different populations of the world. No case has been reported from the population of Pakistan related to this deformity. Case Presentation: In the current study, we presented a case of Osteogenesis imperfecta type VI. The patient's clinical findings indicated her with short stature and progressive distortion of the skeleton, without the record of other complications like hearing problems, dental anomalies, and abnormal vision. She was 16 years old, could not walk due to deformation and weakness of lower limbs. At the time of Patient’s radiological examination, history of multiple fractures of long bones was reported. The radiological findings showed the condition of kyphoscoliotic impairment in the cervicodorsal spine. Long bones showed bowing and relatively decreased bone mineralization. Patients' sequencing data indicated a new homozygous frameshift mutation Case Study Parveen et al.; JPRI, 33(60B): 2532-2539, 2021; Article no.JPRI.79265 2533 c.262_263insCCCTCTC (p. Ala91Profs*23) in SERPINF1 responsible for splice site changes in PEDF protein. Conclusion: This identified mutation was the first report from Pakistan, and an increase in the pathogenic variants in SERPINF1 caused OI type VI.


INTRODUCTION
Osteogenesis imperfecta (OI) is a heterogeneous defect of bone fragility characterized by significant skeletal abnormalities like short stature and susceptibility to multiple fractures. Extra-skeletal complications like blue sclera, hypermobility of joints, renal dysfunction, and dentinogenesis imperfecta appeared in some cases depending upon the involvement of a specific gene [1,2,3,4]. It is estimated that 6-7 individuals were affected in 10,000 live births with varying severity ratios from frequent fracture to intrauterine fracture and perinatal mortality (Minillo et al., 2014). In OI type VI patients, multiple fractures were reported after six months, while other extra-skeletal complications were absent. Histological examination of bone depicted improper bone mineralization, accumulation of unmineralized osteoid, which gives a unique lamellar demonstration to bone (Zhang et al., 2018; [5] [6]. SERPINF1, a specific gene responsible for osteogenesis imperfecta type VI is localized on chromosome 17p13. 3. PEDF (pigment epithelium-derived factor) is a 50-kDa secreted glycoprotein of the serpin superfamily. The various functionality of PEDF in cells is its involvement in multiple mechanisms as lipid metabolism, neuroprotection, anti-angiogenesis, and tumorigenesis. In bone, PEDF shows a crucial involvement in mineralization, osteoblast differentiation, and expression of osteocyterelated factors (Wang et al., 2017; Trejo et al., 2017; [7,6]. Up till now, 62 pathogenic variants in SERPINF1 were detected, which affected about 58 individuals. These mutations were reported from different populations of the world with diverse geography (Table 1). However, detailed investigations of OI type VI in the Pakistani population regarding phenotypes and gene mutations are still needed. In the present study, we analyzed phenotypic traits and pathogenicity of variants in Pakistani patients born due to an inter-family relationship between mother and father. Molecular analysis of coding regions of SERPINF1 has indicated a nonreported homozygous in-frame insertion mutation c.262_263insCCCTCTC (p. Ala91Profs*23) in the enrolled patient.

CASE PRESENTATION
A consanguineous Pakistani couple (IV-1, IV-2) ( Fig. 1) had a 16 years old female as 1st baby in their family. She was born as a result of a normal pregnancy by vaginal delivery. She was referred to University Hospital, The University of Lahore, for the initial clinical findings of OI by following criteria: frequent repetitive fractures under minor injuries with inevitable extra-skeletal distractions such as mild impairment of hearing and uncontrolled movements of joints but dentinogenetic imperfecta and blue sclera not observed in this patient. Her parameters at the time of birth, such as height, weight, and skull circumference, were recorded as usual. The family history indicted that one male in siblings was affected while others were healthy. The normal growth and development were recorded by parents, starting from her birth, still six months of age. After that, parents reported a history of arm and leg bone fractures with minor trauma. It was observed that patient had no free movements of the neck like normal individuals. Phenotypic appearance of facial deformity, teeth anomalies, and corneal defect were absent, but lower limbs showed a curvature shape ( Fig. 2: a and b). The patient was bearing normal mental health with excellent cooperative behavior. The patient's clinical examination history, radiological investigations, and pedigree analysis had confirmed the OI type VI rather than other types of OI. Bone deformities, hypermobility, and multiple fractures history differentiated it from different types. Radiological examination revealed a diffuse decreased density of the visualized bones and irregular articular surface. X-ray films of the upper and lower extremities and thoracolumbar vertebrae were also examined. During examination, marked kyphoscoliotic impairment in the cervicodorsal spine was observed. The extremities of long bones were frequently affected by fractures. The moderate-to-severe bone fragility resulted in short stature, decreased movements and bone deformities (such as curvature of long bones, severe type of scoliosis, and kyphosis). Furthermore, the major recorded observations of skinny long bone and compressions in vertebrae and infinite osteoporosis were observed ( Fig. 3: a-d). Long bones showed bowing, thinning of cortices, metaphyseal flaring, and relatively decreased bone mineralization. The collected family samples were processed for sequencing of SERPINF1 to identify the genetic mutation of this abnormity and for possible preventive measures for its management.
Sanger sequencing technique was used for the identification of a pathogenic variant in SERPINF1 and co-segregation analysis in the family. SERPINF1 genomic transcript (NM_002615.5) and the mutation nomenclature of the gene have been extracted from the genome database. An updated software "AmplifX v1.5.4" was used for primers designing, and PCR was carried out for the amplification of coding regions. ExoSap protocol was performed for the Cleanup of PCR products. An ABI 3730 genetic analyzer with BigDye chemistry v3.1 was used for sequencing. The obtained chromatograms were altered with the reference sequence based on SeqMan Pro (DNASTAR, Inc., Madison, WI, UK) software. Analysis has confirmed the allelic frequency of the identified pathogenic variant by taking the help of the Genome aggregation database.
The coding sequences (eight exons) of SERPINF1 were PCR amplified for the patient (V-1) DNA samples. The sequence analysis results indicated a novel homozygous 7bp inframe insertion c.262_263insCCCTCTC (p. Ala91Profs*23) in the SERPINF1 gene (Fig. 4). The sequencing of identified pathogenic variants was carried out for both parents (IV-1, IV-2).

DISCUSSION AND CONCLUSION
In this study, we reported a consanguineous Pakistani family with one affected member (V-1) (Fig. 1) with a history of frequent fractures under mild trauma and certain other skeletal deformities on later stages of life presented as a patient of OI type VI. The clinical laboratory findings coincided with the apparent phenotypic appearance of a female patient. The thorough examination of parents (IV-1, IV-2) and two unaffected brothers (V-3 and V-4) revealed the absence of major symptoms of Osteogenesis Imperfecta (OI  (Rauch and Glorieux, 2004;[6,7]. As described in previous studies, clinical examination of the current case showed no corneal defect and teeth anomalies in the affected individual [6,7], Minillo, 2014). Previous in vitro studies demonstrated that the density of bone is defected by PEDF inhibition of osteoclasts [8]. The phenotypical appearance of frequent bone fractures after six and absence of extra-skeletal defects differentiate it from other types of OI [6,7].
The primary causative gene (SERPINF1) for this specific type of osteogenesis has a chromosomal location 17p13.3, which translates into a unique (PEDF) glycoprotein containing 418 amino acids [6,2,7,9]. It has a diverse functionality with expression in vast tissues such as the adult brain, spinal cord, plasma, lung, eye, heart, and bone [5].
In the knockout mouse model (Serpinf1-/-) presence of vast deposits of unmineralized bone, the matrix built a resemblance with phenotypes of OI type VI (Wang et al., 2017).
Some frame insertion and deletion mutations have also been published along with previous ones responsible for premature termination codons [9].
Most of the reported pathogenic variants in SERPINF1 are frameshift and nonsense mutations that cause defects in the normal functionality of PEDF by altering the protein network (Wang, 2017). The current study described a case of osteogenesis imperfecta OI. The molecular clinical study of a Pakistani patient born due to an inter-family union confirmed pathogenic mutation causing type VI of OI bone disorder. The current study's findings are novel and considered an addition to the mutational spectrum of the SERPINF1 gene.

DISCLAIMER
The products used for this research are commonly and predominantly used products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by any funding agency rather it was funded by personal efforts of the authors.

CONSENT
As per international standard or university standard, patients' written consent has been collected and preserved by the author(s).

ETHICAL APPROVAL
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