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Mutation of COL2A1 in a Chinese Family with Presentations of Legg-Calvé-Perthes Disease

Denglu Yan1*, Zhaojie Wang2, Zhi Zhang3

1 Department of Orthopedics, First People's Hospital of Zhaoqing, Guangzhou, China

2 Department of Orthopaedics, Twelve People’s Hospital of Guangzhou, Guangzhou, China

3 Department of Orthopedics, Third Hospital of Guangzhou Medical University, Guangzhou, China

*Corresponding Author:
Denglu Yan
Department of Orthopedics,
First People's Hospital of Zhaoqing,
Guangzhou,
China
E-mail:
[email protected]

Received: 22-Jul-2022, Manuscript No. JOB-22-69914; Editor assigned: 26-Jul-2022, PreQC No. JOB-22-69914 (PQ); Reviewed: 09-Aug-2022, QC No. JOB-22-69914; Revised: 16-Aug-2022, Manuscript No. JOB-22-69914 (R); Published: 26-Aug-2022, DOI: 10.4172/2322-0066.10.6.004.

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Abstract

Background: The aim of this study was to identify genetic factors and chromosomal regions contributing to Osteonecrosis of the Femoral Head (ONFH) in a Chinese family with presentations of Legg-Calvé-Perthes Disease (LCPD).

Methods: In this study, we performed whole exon sequencing of a Chinese family with LCPD for mutation detection. Ten members had ONFH in twenty-seven family members in four generations family, 5 unaffected members of the studied family and 5 normal peoples as control were underwent whole exome sequencing for mutation detection. Structural modeling test was applied to analyze the potential structural changes caused by the missense substitution.

Results: In this Chinese family affected by LCPD, the mutation (c.3508 G>A, p. Gly1170Ser) in exon 50 of COL2A1 in the Gly–X–Y domain was present in 10 patients but absent in 5 unaffected members of the studied family and in 5 control chromosomes from unaffected individuals of matched geographical ancestry. The COL2A1 gene mutation was further validated by Sanger sequencing, confirmed that were heterozygous for the mutation. Then, we identified the p.Gly1170Ser mutation in exon 50 of COL2A1 in a Chinese family with LCPD.

Conclusions: This study maps the mutation of mutation (c.3508 G>A, p. Gly1170Ser) in exon 50 of COL2A1 in the Gly–X–Y domain in a Chinese family of LCPD, which causes osteonecrosis of femoral head.

Keywords

Genetics; Osteonecrosis; Femoral Head; COL2A1

Abbrevations

ONFH: Osteonecrosis of the Femoral Head; LCPD: Legg-Calvé-Perthes Disease; HD: Hip Dysplasia; SNP: Single Nucleotide Polymorphism

Introduction

Osteonecrosis of the Femoral Head (ONFH) was seen in adult and children, and the osteonecrosis of the femoral head that occurs in children was called Legg-Calvé-Perthes Disease (LCPD). Although LCPD was described over 100 years ago, its etiology is still controversial. LCPD can be associated with abnormalities in factor V Leiden mutation [1].The Factor V Leiden gene mutation in children with LCPD. Described a family with three-generation transmission of factor V Leiden and LCPD developed in three siblings in this family [2]. Report heterozygosity for factor V Leiden was the only inherited risk factor associated with the development of LCPD [3]. Suggest that the homozygous form of Factor V Leiden mutation has some role in the clinical course of LCPD [4,5].

Although it has recently been postulated that thrombophilia may have a role in the aetiology of LCPD, it remain conflicting [6].Report no association between factor V Leiden and LCPD[7]. Report thrombotic component in the etiology of LCPD [8]. Study did not find an increased rate of factor V or prothrombin mutations in children with en with LCPD compared to the natural incidence [9]. Found no genetic association between LCPD and increased genetic thrombophilia among patients compared with the control group[10]. The prothrombin II mutation is located in 11p11–q12 at position G20210A and activates the coagulation cascade which increases the risk of thrombosis. Report methylenetetrahydrofolate not associated with LCPD[11-13].

Previous studies showed that LCPD aggregates strongly in families, and the genetic predisposition to osteonecrosis is well documented in COL2A1 [14-18]. Report the cases of two children who presented with abnormal development of both hips and in whom novel mutations in the COL2A1 gene were found. Report 2 generations of 4 male family members with LCPD-like features and mutation of the COL2A1 gene of the 12q13 chromosome. Identify a heterozygous mutation (c.1888 G>A, p. Gly630Ser) in exon 29 of COL2A1 in the Gly–X–Y domain, in a Chinese family affected by LCPD and ANFH. An understanding of the causes of LCPD is important for its prevention, diagnosis, and treatment. Although its obscure etiology is still under discussion, recent studies have focused on the importance of genetic factors in the development of LCPD because genetic factors can provide insight into the pathophysiology of ONFH and point to new treatments. We evaluated a large family with a dominantly inherited of ONFH in Chinese family with LCPD.

Materials and Methods

Patient recruitment/sample collection

Patients of a Chinese family with LCPD suffered severe hip pain were diagnosed ONFH in the Zhaoqing first people’s hospital. The study protocol was approved by the Committee on Clinical Investigation at the Zhaoqing first people’s hospital. Informed written consent was obtained from all study participants and the study was conducted in accordance with the Declaration of Helsinki II and approved by the local Ethical Committees. Twenty-seven members of this Chinese family with LCPD participated in the study, of which ten showed clinical symptoms of ONFH shown in Figure 1.

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Figure 1: Pedigrees of the four generation LCPD family. Patients with LCPD are shown by darkened symbols. An arrow indicates the proband.

Clinical examination showed that none of these patients has any other associated bone and joint signs, visual dysfunction or diabetes mellitus. All patients complained of pain in the affected hip with limited range of motion and walking distance, and the first symptoms of hip pain were self-reported at ages ranging from 1 to 13 years old (Table 1).

Patients Age Gender Height Dwarfism Myopia Hearing Spinal Skeletal
- (years) - (cm) - - loose changes ONFH HD Other abnormal
II:1 59 F 156 N N N N Y N N
II:3 57 F 157 N N N N Y N N
II:5 53 M 163 N N N N Y Y N
III:1 34 F 160 N N N N Y N N
III:3 32 F 158 N N N N Y N N
III:5 30 M 164 N N N N Y N N
III:7 36 F 160 N N N N - N N
III:9 31 F 159 N N N N Y N N
III:11 29 M 165 N N N N Y N N
III:13 26 F 161 N N N N - Y N
III:15 13 M 159 N N N N Y Y N
IV:1 9 M 138 N N N - - -
IV:2 12 F 145 N N N N - - N
IV:3 3 M 89 N N N Y - -
IV:4 14 F 153 N N N N - - N
IV:5 8 F 132 N N N - - -
IV:6 4 M 97 N N N - - -

Table 1: Summarization of clinical manifestations of the affected family members.

Whole exome Sequencing

There were ten members had ONFH in twenty-seven family members in four generations family. The ten family members of ONFH, 5 unaffected members of the studied family and 5 normal peoples as control were underwent whole exome sequencing for mutation detection. The DNA samples were extracted from peripheral blood leukocytes using QIAamp Blood Mini DNA kit (Qiagen, Santa Clara, CA, USA). Before analysis of the samples, DNA aliquots were re-precipitated to remove proteins and fragments. A whole exome–enriched library was prepared from 3 μg of genomic DNA from the proband (II:6) using Agilent’s SureSelect Human All Exon 50 Mb solution-based capture reagent. Exome capture was performed according to the manufacturer’s protocol (Agilent, USA). The captured DNA was then sequenced using the Illumina HiSeq2000 platform. Raw image files were processed by Illumina Basecaller Software 1.7 (San Diego, CA, USA) for base calling with default parameters.

Variant annotation

The obtained sequence reads were aligned to the human genome (hg19) using the SOAP2 and BWA tools for Single Nucleotide Polymorphism (SNP) and insertion/deletion (indel), respectively. The percentages of read alignment to both the reference genome and the targeted exome were calculated using Perl scripts. Similarly, Perl scripts were used for the detection of mismatch frequencies and error positions. SNP calling was done with SOAPsnp, and indels were identified through the alignment result with GATK. Detailed annotation information was obtained from dbSNP, CCDS, UCSC Genome Browser, Ensembl, and Encode databases. Using these annotations, we screened the novel and likely deleterious variants for further study.

Sanger sequencing

Sanger sequencing was then conducted for mutation verification and prevalence test in 10 unrelated controls using a previously defined protocol. Specific primers were designed for the target region, and the PCR products were sequenced on an ABI 3730 DNA analyzer following standard procedures (Life Technologies, USA). The sequence reads were analyzed using the Sequencher software package (GeneCodes Inc, USA). The sequencing traces were visually inspected in Finch TV v1.4 (Geospiza Inc, USA).

Bioinformatics analysis

Microsatellite markers were selected based on ABI PRISM Linkage Mapping Set and the UCSC database. PCR products were electrophoresed on a 96-capillary automated DNA sequencer (MegaBACE 1000, Amersham, Germany) and were analyzed with Genetic Profiler software (version 1.5. Two-point LOD scores were calculated using MLINK from the LINKAGE package. Autosomal dominant inheritance, disease-gene frequency of 0.0001, and 95% penetrance were assumed. Haplotyping was constructed using Cyrillic.

Results

All patients were contacted for clinical and radiological follow-up and had standard AP pelvic and a lateral hip radiographs. Skeletal radiographs including full spine, double hip, and full length of distal laxity x-ray film were reviewed, and no other abnormal skeletal disorders could be found. Overall, the abnormalities noted in the affected family members included nine patients had osteonecrosis of the femoral heads, one had ONFH combine hip dysplasia, and one had hip dysplasia roofs in Figure 2.

biology-anteroposteriorhip

Figure 2: The Pedigrees anteroposterior hip radiograph of patients with LCPD, who had osteonecrosis of femoral head (small white arrow) and hip dysplasia (big white arrow).

The average exome sequencing generated 7.42 Gb sequence data per sample as 90-bp paired-end reads. After quality control, 92.8% of sequence data was aligned to the UCSC human reference genome (build 37.1) and 62.6% of sequence data was mapped to target regions. The distribution of per-base sequencing depth in target regions approximated a Poisson distribution, which showed that the captured exome region was evenly sampled.

Mean depth per base within the target regions was 111.85-fold, and 97.7% of these regions were covered by four or more reads (94.5% by 10 or more reads) by paired-end sequencing. A total of 1934 genetic variations, including non-synonymous mutations, splice site variations, and indels, were identified from the proband shown in Table 2.

Mutation type Number
SNP analysis Missense 1382
- Nonsense 53
- Splice site 215
- Readthrough 8
Indel analysis indel 39
- Splice site 42
- Frameshift 53
- 5-UTR 89
- 3-UTR 46
- Promoter 7
Total 1934

Table 2: Variations identified by whole exome sequencing.

Because numerous mutations were detected, we combined whole exome sequencing and linkage analysis to sift through the potential causative mutations. According to the manifestation of ONFH of the femoral heads, most genetic skeletal disorders could be ruled out, sequence analysis of the candidate gene was subsequently carried out to further validate the underlying disorder of this family. A mutation of COL2A1, a known gene causing congenital skeletal disorders, was included in these mutations. In this Chinese family affected by LCPD, we identify heterozygous mutation (c.3301 G>A, p. Gly1101Ser) in exon 49 of COL2A1 in the Gly–X–Y domain, mutation (c.3508 G>A, p. Gly1170Ser) in exon 50, mutation (c.4006 G>A, p. Gly1336Ser) in exon 52, and mutation (c.4213 G>A, p. Gly1405Ser) in exon 53. The mutation (c.3508 G>A, p. Gly1170Ser) in exon 50 of COL2A1 in the Gly–X–Y domain was present in 10 patients but absent in 5 unaffected members of the studied family and in 5 control chromosomes from unaffected individuals of matched geographical ancestry. The COL2A1 gene mutation was further validated by Sanger sequencing, confirmed that were heterozygous for the mutation. Then, we identified the p.Gly1170Ser mutation in exon 50 of COL2A1 in a Chinese family with LCPD shown in Table 3.

Patients Age Gender Height Skeletal Mutation COL2A1
- (years) - (cm) ONFH HD exon49 exon50 exon52 exon53
II:1 58 F 156 Y N Y Y N N
II:3 56 F 157 Y N N Y Y N
II:5 50 M 163 Y Y N Y N N
III:1 34 F 160 Y N Y Y N N
III:3 32 F 158 Y N N Y N Y
III:5 30 M 164 Y N N Y Y N
III:9 31 F 159 Y N N Y N Y
III:11 29 M 165 Y N Y Y N N
III:15 13 F 143 Y Y N Y Y N
IV:5 3 M 85 Y - N Y N -

Table 3: The mutation COL2A1 in the Chinese family with LCPD.

Discussion

Genetic association studies suggest that mutations involved in cartilage constitution, bone metabolism, detoxification, coagulation system or vascular endothelium function might participate in the aetiology of osteonecrosis [19-22]. Based on an initial screening of exome sequencing and the subsequent confirmation of Sanger sequencing of this study, pathogenic mutations were identified in COL2A1 in a Chinese family with LCPD. The COL2A1 gene is localized at 12q13.11-q13.2 and about 30 kb in length with 54 exons, which provides a key element for the production of type II collagen [23,24]. Three a1 chains encoded by the COL2A1 gene are folded together in a triple-helical configuration to form the procollagen homotrimer [25,26]. The triple helical domain, containing about 300 amino acids, exists in the limited space of the procollagen, and is restricted so that every three amino acids is a glycine, generating a repeating (Gly–X–Y)n sequence pattern. If the Gly in this specific region is replaced by other amino acids, the structure of type II collagen will be destroyed [27,28]. Our findings indicate that the osteonecrosis of femoral head in a Chinese family with LCPD showed an inheritance pattern.

COL2A1 gene encodes a homotrimer of three identical a-chains that composes type II collagen. Mutations in COL2A1 gene can lead to a wide spectrum of skeletal disorders designated as type II collagenopathies. Recent studies showed that the genetic predisposition to osteonecrosis is well documented in COL2A1 [29,30].Study showed alert clinicians to the possibility that children who present with bilateral Perthes-like disease of the hip might have an underlying mutation in the gene encoding type II collagen. Have located a missense mutation (p.G1170S) in the type II collagen gene (COL2A1) in a Japanese family with an autosomal dominant hip disorder manifesting as LCPD and showing considerable intra-familial phenotypic variation.

LCPD may result from a variety of mutations in the COL2A1 gene, most of which are located within the triple-helical domain of the protein [31-33]. It has been postulated that mutations in the triple-helical domain of procollagen a chains can delay the folding of the protein into the triple-helical conformation, thus leading to posttranslational over modification of the protein [34,35]. In this study, all affected individuals were found to carry p.Gly1170Ser mutation in exon 50 of COL2A1 gene, which resulted in a change from glycine to serine at codon 1170 (Gly1170Ser). A Gly1170Ser mutation potentially interrupts the mandatory Gly-X-Y triplet sequence required for the normal formation of stable triple-helical type II collagen molecules, which could further lead to the degradation of premature collagen molecules, or to the production of overmodified type II collagen [36,37]. Earlier molecular analysis has demonstrated that specific sites located within a triple-helical region of the collagen molecule serve as important binding sites of telopeptides in the early stages of fibril formation.

Variation in the phenotypic expression of different individuals suggests that some other genetic and environmental effects may play a role in the final clinical expression [38-40]. To date, the knowledge on the development of LCPD in humans is limited because of the vast number of mutations found in the COL2A1 gene and the variability in the clinical phenotype [41,42]. It is apparent that the process in which mutations in collagens alter connective tissues is complicated and cannot be described by one single pathway. In the future study, this pedigree can be extended by including the distal relatives of the family, which we believe can probably shed some light into the delineation of the pathological phenotype heterogeneity of this disease.

Conclusion

This study maps the mutation of mutation (c.3508 G>A, p.Gly1170Ser) in exon 50 of COL2A1 in the Gly–X–Y domain in a Chinese family of LCPD, which causes osteonecrosis of femoral head. Reported that the p.Gly1170Ser mutation of COL2A1 in the family described is responsible for pathology confined to the hip joint. As the previous study, the p.Gly1170Ser mutation in exon 50 of COL2A1 was identified in this study. However, this study has a large Chinese family of femoral head osteonecrosis compare to the previous reports

Availability of Data and Materials

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Ethical Approval and Consent to Participate

Not applicable

Competing Interests

The authors declare that they have no competing interests.

Funding

This research was funded by Science and Technology Department of Guangdong Province (2014A020212041) and Shenzhen science and Technology Innovation Committee (JCYJ20140411091151442).

Author's Contributions

Conceived and designed the experiments: Denglu Yan, Performed the experiments: Zhaojie Wang, Analyzed the data and wrote the paper: Zhi Zhang.

Acknowledgments

We sincerely appreciated the genial help from all scholars who contributed to our research and the support from Science and Technology Department of Guangdong Province (2014A020212041) and Shenzhen science and Technology Innovation Committee (JCYJ20140411091151442).

References

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