Exploring the Genetic Landscape of Knobloch Syndrome: Novel Variant Identification and Literature Review

Mohammad-Reza Ghasemi ^1,2, Maryam Mirahmadi ^3,4, Hadi Bayat ⁵, Morteza Sheikhi Nooshabadi ^6,7, Sanaz Jamshidi ², Shadab Salehpour ⁸, Reza Mirfakhraie ¹, Fatemeh Fazeli ², Mohammad Miryounesi ^1,* , Milad Gholami ^9,* 

1. Department of Medical Genetics, Shahid Beheshti University of Medical Sciences, Tehran 1516745811, Iran

2. Center for Comprehensive Genetic Services, Shahid Beheshti University of Medical Sciences, Tehran 1516745811, Iran

3. Department of Medical Genetics, Faculty of Medicine, Tarbiat Modares University, Tehran 14117-1316, Iran

4. Department of Exomine, PardisGene Company, Tehran 1917635816, Iran

5. Biochemical Neuroendocrinology, Montreal Clinical Research Institute (IRCM), affiliated to McGill University, Montreal, Quebec H2W 1R7, Canada

6. School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran 1516745811, Iran

7. Autoimmune Disease Research Center, Kashan University of Medical Sciences, Kashan 3715835155, Iran

8. Department of Pediatrics, Clinical Research Development Unit, Loghman Hakim Hospital, Shahid Beheshti University of Medical Sciences, Tehran 1516745811, Iran

9. Department of Biochemistry and Genetics, School of Medicine, Arak University of Medical Sciences, Arak 3819693345, Iran

* Correspondences: Mohammad Miryounesi  and Milad Gholami 

Academic Editor: Fabrizio Stasolla

Received: November 22, 2024 | Accepted: April 02, 2025 | Published: April 14, 2025

OBM Genetics 2025, Volume 9, Issue 2, doi:10.21926/obm.genet.2502291

Recommended citation: Ghasemi M, Mirahmadi M, Bayat H, Nooshabadi MS, Jamshidi S, Salehpour S, Mirfakhraie R, Fazeli F, Miryounesi M, Gholami M. Exploring the Genetic Landscape of Knobloch Syndrome: Novel Variant Identification and Literature Review. OBM Genetics 2025; 9(2): 291; doi:10.21926/obm.genet.2502291.

© 2025 by the authors. This is an open access article distributed under the conditions of the Creative Commons by Attribution License, which permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is correctly cited.

1. Introduction

Defects in collagen genes can cause various collagenopathies. Collagen is essential for establishing connective tissues and acts as a signaling molecule. So far, 28 different types of collagen have been identified in the human body [1]. Therefore, the pathogenic variants of these genes can affect multiple organs.

Knobloch Syndrome (KS) (OMIM: 267750) is a rare collagenopathy that was first reported by Knobloch and Layer. It is mainly caused by a biallelic pathogenic variant of COL18A1, which is associated with retinal detachment and encephalocele [2]. Ocular manifestations in the posterior and anterior segments can be irregular, including lens subluxation, high myopia, smooth irides (iridodonesis), early-onset cataracts, retinal detachment, vitreoretinal degeneration, and retinal degeneration with severe cone-rod dystrophy [3,4]. Patients also commonly experience non-ophthalmic symptoms such as brain defects (polymicrogyria and Dandy-Walker Malformation), mid-face hypoplasia, occipital changes (encephalocele, cutis aplasia, bone defects), micrognathia, flat nasal bridge, hyperextensible joints, early onset renal failure, associated with increased creatinine levels and ultrastructural changes in the basement membrane of the kidney, as observed in COL18A1-deficient mice [5,6], lung hypoplasia, epilepsy, high arched palate, dental abnormalities [7], and delayed skin healing due to dysfunction of the dermal-epidermal junction during wound healing [8].

The COL18A1 gene, found on chromosome 21q22.3, plays a key role in connecting the eye's inner limiting membrane to the vitreous fibrils. It encodes the alpha chain of collagen type XVIII, a proteoglycan with multiple functions commonly found in the basement membrane of human tissues [9]. Furthermore, it has been identified that the COL18A1 protein, upon proteolytic cleavage, generates endostatin, which serves as an inhibitor of endothelial cell proliferation, migration, and angiogenesis [10]. COL18A1 comprises three distinct isoforms, which vary in their N-terminal non-collagenous domain, length, and tissue distribution. These isoforms are produced through alternative promoter usage and splicing mechanisms [11]. The long isoform is involved in maintaining the structure of the human eye. In contrast, the short isoform appears to play a crucial role in neural migration, eye structure, and neural tube closure during embryogenesis [12]. A ‘delay in ocular developmental regression’ refers to the prolonged retention of transient embryonic ocular structures, such as the hyaloid vasculature, which typically dissolve during postnatal maturation. For instance, in COL18A1-deficient mice, this delay disrupts the timely remodeling of blood vessels, which is essential for normal visual development [13].

This study aimed to examine a patient experiencing vision problems through the application of whole-exome sequencing (WES) to identify underlying genetic pathogenic variants. This study aimed to establish a comprehensive understanding of the genotypic and phenotypic spectrum associated with KS, with a particular emphasis on ocular manifestations and non-ophthalmic symptoms, through a standardized literature review. The findings of this study will contribute to expanding our knowledge about KS and its underlying genetic mechanisms.

2. Materials and Methods

2.1 Clinical Report

The patient was a 10-year-old female who presented with symptoms of horizontal nystagmus, strabismus, and suspected vision loss. The patient’s parents were non-consanguineous, and she was the only known individual with this condition in her family, with no similar clinical signs observed in other family members (Figure 1a).

Figure 1 The schematic pedigree of the proband’s family and Sanger Sequencing findings. a) The pedigree was depicted by using a user-friendly online Invitae family history tool (https://familyhistory.invitae.com/). The red color indicates KS. b) Frameshift pathogenic variant in the exon 7; and c) Premature stop codon in the exon 16.

2.1.1 Prenatal and Birth History

The patient was delivered via cesarean section at 38 weeks of gestation, with a birth weight of 2800 grams and a length of 50 cm, both within the normal range for the gestational age. Her head circumference was normal. The patient's mother was hospitalized for one week due to a gynecological infection during pregnancy.

2.1.2 Early Infancy (1 Month to 7 Months)

The signs began at the age of one month, initially presented with horizontal nystagmus, which later became associated with strabismus. Initial neonatal examinations were normal, and the patient had a normal appearance. At seven months of age, she was observed to have persistent symptoms of horizontal nystagmus and strabismus, raising concerns about potential vision loss.

2.1.3 Childhood (Up to 5 Years)

Throughout her early childhood, the patient neither exhibited any motor or learning difficulties nor had any speech disorders. However, she experienced joint laxity until the age of 5. There was no history of renal or skin disorders, and the patient had no history of seizures. Ocular signs showed a brief improvement over time.

2.1.4 Age 6 Years

At the age of 6, the patient began taking L-carnitine and vitamin B1. The reason for administering these supplements is not explicitly linked to the diagnosis of mitochondrial disorders in the context provided.

2.1.5 Current Status (Age 10 Years)

At the age of 10, the patient presented with eye deviation and persistent horizontal nystagmus, accompanied by a high degree of nearsightedness (high myopia). The patient’s clinical features included horizontal nystagmus, high myopia, visual loss, hypermobile joints, and strabismus. There was no significant family history of similar symptoms.

2.2 Compliance with Ethical Standards

All procedures performed in this study involving human participants were in accordance with the ethical standards of the Arak University of Medical Sciences and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The study was approved by the Medical Ethics Committee of Arak University of Medical Sciences (Approval no: IR.ARAKMU.REC.1401.116). Written informed consent was obtained from the patient’s parents to publish this report in accordance with the journal's patient consent policy.

2.3 DNA Extraction

The salting-out procedure [14] was used to extract genomic DNA from 10 mL of peripheral blood samples obtained from the proband and her mother. A NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Inc., Wilmington, DE, USA) was used to assess the concentration and quality of genomic DNA.

2.4 Whole Exome Sequencing (WES)

The Illumina HiSeq4000 platform produced 101-bp paired-end reads for WES on the genomic DNA of the proband. The SureSelectXT2 V6 kit was used to enrich the exonic and surrounding exon-intron border areas. Raw sequencing data (approximately 100 gigabases) were aligned to the human reference genome (GRCh37/hg19) using Burrows-Wheeler Aligner (BWA) [10]. The Picard tool (v. 1.118) eliminated the polymerase chain reaction (PCR) duplicates. Sequencing covered more than 95% of the targeted regions with a sensitivity of over 99% and a mean coverage of 100×. The Genome Analysis Toolkit (GATK v 3.7) called variants, and the ANNOVAR program annotated them. Variants were identified using a minor allele frequency of 0.01 in databases such as the 1000 Genomes Project, dbSNP 138, NHBL Exome Variant Server (EVS), ESP6500, and Iranome. Additional filtering based on pathogenic variant pathogenicity and impact, prevalence in the public population, clinically relevant variant databases, and known associations with the phenotype was performed according to the literature. The variants were prioritized based on their phenotypic plausibility.

2.5 Sanger Sequencing

Sanger sequencing verified the pathogenic variants in the proband. The c.2416C>T was also assessed in the mother of the proband for segregation analysis. The ABI Sequencer 3500XL PE and BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies; Thermo Fisher Scientific, Shanghai, China) were used for Sanger sequencing (Applied Biosystems, CA, USA). Standard procedures were followed for PCR amplification, product purification, and Sanger sequencing. Specific primers targeting exons 7 and 16 of the COL18A1 locus and its flanking intronic regions were designed using the Primer3 Web-based server. The quality and specificity of the designed primers were checked using the OligoAnalyzer Tool and NCBI primer BLAST, respectively (Table 1).

Table 1 The sequence of designed primers that were used in this study.

The acquired sequences were aligned with the reference sequence in GenBank using Chromas Lite software (Technelysium Pty Ltd., Australia) and CodonCode Aligner v.8.0.2. In silico tools, including BayesDel_addAF, MutationTaster2, FATHMM software, and DANN, were utilized to evaluate the pathogenicity of the identified variants.

2.6 Prediction of Protein Structure Using I-TASSER

The entire COL18A1 protein sequence was introduced into Iterative Threading ASSEmbly Refinement (I-TASSER) to predict the protein structure and structure-based functions. Similarly, the sequence of the frameshifted protein was introduced into I-TASSER to compare the wild type and its mutated counterpart. Pymol 2.5.4 visualized the predicted structures.

2.7 Compiling Reported Cases of KS Molecularly and Clinically

An extensive literature search compiled all reported cases of KS, both molecular and clinical. Multiple electronic databases, including PubMed, ClinVar, and OMIM, were systematically searched for relevant keywords and MeSH terms. The search included studies published in English up to July 2024 and studies reporting on patients with a confirmed diagnosis of KS, focusing on pathogenic variants in the COL18A1 gene and their corresponding phenotypes. The exclusion criteria were studies unrelated to KS or insufficient molecular or clinical information. Two independent reviewers screened titles, abstracts, and full texts to select relevant studies. Discrepancies were resolved through consensus or consultation with a third reviewer. Information obtained from the selected studies included demographic information, clinical manifestations, identified pathogenic variants in the COL18A1 gene, and outcomes. These standard literature reviews create an overview of KS-reported cases in terms of molecular findings and clinical characteristics, as presented in Table 2.

Table 2 Overview of molecular findings and clinical characteristics of the reported cases of KS.

3. Results

The latest literature review, which introduces a new case of a unique variant in the COL18A1 gene associated with KS, was included in this study. Seventy-one patients from previous studies, including our research, were identified (Table 2), with 47 reported variants in COL18A1. Among these variants, the truncating pathogenic variant c.4759_4760delCT was more prevalent in seven families. The data indicated that there were 31 female individuals, including our case, and 40 male individuals.

The analysis revealed a diverse distribution of COL18A1 variants across different nationalities, with the highest representation from Arabic (18), Chinese (13), Turkish (5), and British (5) populations. Other nationalities represented include Indian (4), Belgian (3), Northern European (3) Pakistani (2), Palestinian (2), Slovak (2), El Salvadorean (2), African (1), Afghan (1), Spanish (1), Jewish (1), Chilean (1), Persian (1), and Algerian (1) Additionally, there are 5 cases with unspecified or missing nationality information (NA). Zygosity analysis indicated that the majority of patients exhibited a homozygous state for the identified gene variants, with 57 cases being mutant homozygous. Compound Heterozygous variants were detected in 14 patients.

The variants were classified into different pathogenic variant types. Nonsense pathogenic variants were the most prevalent, followed by missense and non-coding pathogenic variants. The classification of variants into different pathogenicity categories highlighted a significant number of pathogenic and likely pathogenic variants (28 pathogenic and 11 likely pathogenic), emphasizing their deleterious effects on KS.

KS is caused by homozygous or compound heterozygous pathogenic variants in the COL18A1 and is characterized by both ophthalmic and non-ophthalmic clinical presentations. The presence of family history and consanguinity in affected families is consistent with this pattern of inheritance.

Genetic counseling is vital in light of these findings. Furthermore, the coexistence of comorbidities such as infection, proteinuria, hypoalbuminemia, and various other manifestations underscores the intricate multisystem involvement and complexity associated with KS.

In the present study, a total read base of 6.8 million bp was gathered for one case using WES. The proband had approximately 86000 variations. Variants with a lower frequency of 1% regarding gnomAD, 1K genome projects, ExAC, and ESP were selected for further study; consequently, 700 variants remained for further consideration. The causative variants, identified by their presence in the HGMD and ClinVar databases, were further assessed for conservation and pathogenicity using tools such as PhastCons100way, PhyloP100way, SiPhy29way, fitCons-gm, statistics, and various BayesDel and MutationTaster models (Table 3).

Table 3 The pathogenicity and conservation score according to online databases.

The pathogenicity ranking of homozygous/compound heterozygote variants, considering the non-consanguinity of the parents, prioritized splicing region, stop gain, and frameshift pathogenic variants, followed by the assessment of amino acid changes for nonsynonymous pathogenic variants. WES analysis identified two compound heterozygous variants in the COL18A1 gene: the c.2416C>T (p.Arg806Ter) variant, which was inherited from the mother, and the c.1698delC (p.Gly567AspfsTer45) variant, for which the inheritance status is unknown due to the unavailability of paternal testing.

Both variants were classified as pathogenic according to ACMG and ClinVar guidelines [38]. The c.2416C>T (p.Arg806Ter) variation has previously been reported as a disease-causing variant [39]. The novel frameshift pathogenic variants c.1698delC (p.Gly567AspfsTer45) has not been reported in the 1K genome project, gnomAD, ExAC, HGMD, ESP 6500, TOPMed Bravo, 4.7 KJPN, Iranome, GME Variome, Mexican DB, and GenomeAsia (Table 3). Sanger sequencing revealed that c.2416C>T (p.Arg806Ter) was heterozygous in both the proband and her mother. The proband carried the second variant, c.1698delC (p.Gly567AspfsTer45), while her mother had a normal, wild-type genotype. This suggests that the first variant was inherited from the mother, while the second variant may have originated from the father or occurred spontaneously. Because the father's information was unavailable, we could not determine whether the second variant, c.1698delC (p.Gly567AspfsTer45), was inherited from the father or occurred de novo. However, we confirmed that the first variant, c.2416C>T (p.Arg806Ter), was in a heterozygous state in both the proband and her mother, indicating segregation through the maternal lineage. This observation revealed a compound heterozygote state in the proband, causing KS (Figure 1b & c).

Furthermore, we used the I-TASSER online software to predict the potential effect of candidate pathogenic variants on the protein domain structure [40]. LOMETS identified PDB hints 2pffB (Z-score of 10.05), 1vt41 (Z-score of 4.74), and 2uur (Z-score of 3.54) as the most reliable PDB structure templates for threading alignment with the COL18A1 wild-type, c.2416C>T (p.Arg806Ter), and c.1698delC (p.Gly567AspfsTer45) variants, respectively. Based on pair-wise structure similarity, the SPICKER program predicted final protein models with a C-score = -0.19 for wild-type form, C-score = -2.88 for the stop codon, and-1.79, respectively (Figure 2).

Figure 2 The schematic view of the stimulated domain proteins using I-TASSER. a) Wild-type; b) The premature stop codon; and c) Frameshift pathogenic variant.

4. Discussion

In this study, we investigated a 10-year-old Iranian female with clinical symptoms that were suggestive of KS. WES analysis of gDNA identified two pathogenic compound heterozygous variants in the COL18A1 gene, which encodes the alpha chain of collagen type XVIII. The variants identified include a previously documented premature stop codon, c.2416C>T (p.Arg806Ter) [41], and a novel frameshift pathogenic variant, c.1698delC (p.Gly567AspfsTer45), located in exons 7 and 16 of COL18A1, respectively. The identified variants were validated through Sanger sequencing. Furthermore, the impact of the identified pathogenic variant on the COL18A1 structure was assessed using I-TASSER.

The findings of this study align with existing literature on KS, which has documented the heterogeneity of the disease's phenotypic manifestations [42]. These manifestations range from mild ocular abnormalities to severe neurodevelopmental impairment. The presence of diverse clinical presentations further underscores the complex nature of the disease and the necessity for genetic counseling [43]. Given the rarity of KS and the fact that COL18A1 is the sole causative gene, elucidating the spectrum of pathogenic variants is of paramount importance [20].

In the analysis of previously documented cases of KS, 47 distinct variants of the COL18A1 gene were identified. The most frequently observed variant was the truncating pathogenic variant c.4759_4760delCT, which was present in seven families. This finding highlights the importance of understanding the spectrum of gene variants associated with KS, as different variants may result in varied clinical outcomes. The distribution of COL18A1 variants showed variation across different nationalities, with the highest prevalence observed in populations of Arabic, Chinese, Turkish, and British descent. These findings suggest that ethnicity-specific genetic factors may contribute to the development of KS. Further studies examining the genetic landscape of KS in different populations would provide valuable insights into its pathogenesis.

Our WES analysis identified a variety of pathogenic variant types in the COL18A1 gene, with nonsense pathogenic variants being the most common, followed by missense and non-coding pathogenic variants. This classification of variants into different pathogenic variant types further emphasizes the deleterious effects of these pathogenic variants on KS. The pathogenicity of the identified variants was assessed using a range of computational tools, including PhastCons100way, PhyloP100way, SiPhy29way, fitCons-gm, and MutationTaster. These tools facilitated the prioritization of the variants based on their conservation and pathogenicity scores. Both the identified variants, c.2416C>T (p.Arg806Ter) and c.1698delC (p.Gly567AspfsTer45), were classified as pathogenic according to the ACMG and ClinVar guidelines. The c.2416C>T (p.Arg806Ter) variant has been previously identified as a pathogenic variant [41], while the c.1698delC (p.Gly567AspfsTer45) variant is characterized as a novel frameshift pathogenic variant.

To elucidate the potential structural impacts of the identified pathogenic variants on the COL18A1 protein, I-TASSER online software was employed. This software predicted alterations in the protein domain structure for both variants. The resulting models indicated a possible disruption of the protein structure, suggesting that these pathogenic variants may have significant functional implications.

Overall, this study contributes to the growing body of knowledge regarding KS and its genetic basis. The identification of pathogenic variants of COL18A1 expands our understanding of the disease and its clinical implications. The heterogeneity of the phenotypic manifestations highlights the need for personalized and comprehensive clinical management. The application of advanced genomic technologies, such as whole-exome sequencing (WES) and bioinformatics tools, provides significant insights into the genetic landscape of KS and may facilitate the development of targeted therapies. Future research should prioritize elucidating the molecular mechanisms underlying KS and identifying potential therapeutic targets. Collaborative efforts across diverse populations and countries are essential to enhance our understanding of the genetic basis of KS and improve clinical management strategies. Furthermore, long-term follow-up studies are necessary to assess the efficacy of targeted therapies and evaluate the impact of genetic counseling on individuals and families affected by KS.

In conclusion, this study advances the disciplines of genetics and ophthalmology by offering novel insights into the genetic framework of KS. The identification of novel variants of COL18A1 expands our understanding of the disease and its clinical implications. Further research and collaboration are needed to fully understand the molecular mechanisms underlying KS and develop effective therapeutic interventions.

Acknowledgments

We would like to thank all the participants of this research. The only external AI-assisted tools we utilized were commercially available grammar and clarity checkers, specifically Paperpal, to ensure the English language quality of our writing. These tools were employed solely to refine the language and improve readability, not to generate or alter the scientific content. We reaffirm that we have reviewed and edited the section generated by the AI-assisted tool for accuracy, and acknowledge full responsibility for the content of the manuscript.

Author Contributions

Mohammad-Reza Ghasemi: Conceptualization, Investigation, Methodology, Formal analysis, Writing—original draft; Maryam Mirahmadi: Investigation, Methodology, Formal analysis, Writing—original draft; Hadi Bayat: Investigation, Formal analysis, Writing—review and editing; Morteza Sheikhi Nooshabadi: Investigation, Methodology; Sanaz Jamshidi: Investigation, Methodology; Shadab Salehpour: Validation, Resources; Reza Mirfakhraie: Validation, Resources; Fatemeh Fazeli: Investigation, Methodology; Mohammad Miryounesi: Conceptualization, Validation, Supervision, Writing—review and editing; Milad Gholami: Conceptualization, Investigation, Validation, Resources, Supervision, Project administration, Funding acquisition, Writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by Arak University of Medical Sciences (Grant Number: 4130).

Competing Interests

The authors declare that they have no conflicts of interest.

Data Availability Statement

The data supporting the findings of this study are available upon request from the corresponding author. The data were not publicly available because of privacy or ethical restrictions.