Comprehensive Overview of Mutations in Neurofibromatosis Type 1 Among Iranian Patients

Neurofibromatosis type 1 (NF1) is a hereditary disease caused by mutations in the NF1 gene, characterized by almost complete penetrance and high phenotypic variability. This study aims to identify NF1 mutations in Iranian patients using whole-genome exome sequencing technology and to expand the mutation spectrum of this gene, thereby facilitating genetic counseling for affected families.

Research Background

Neurofibromatosis type 1 (NF1) is a neurocutaneous disorder with a global prevalence ranging from ¹⁄₂₁₃₂ to ¹⁄₄₇₁₂. The main manifestations include multiple café-au-lait spots, neurofibromas, inguinal freckling, optic gliomas, and Lisch nodules. Additionally, some patients develop tumors in the skin and central nervous system, increasing the potential risk of malignancy. Many patients also present atypical symptoms such as breathing difficulties, abdominal pain, and headaches.

NF1 is an autosomal dominant disorder with high penetrance and variability. Recent studies have shown that other modifier genes also play a significant role in the diverse clinical presentations of NF1 patients, including tumor protein 53 (TP53), cyclin-dependent kinase inhibitor 2A (CDKN2A), and the tumor suppressor gene PTEN. The NF1 gene has a spontaneous mutation rate of about 50% and is located on chromosome 17 at position q11.2. The encoded neurofibromin protein is involved in regulating multiple signaling pathways in the body.

Research Source

This article was jointly completed by Shahram Savad et al. from multiple Iranian research institutions such as Nilou Laboratory, Alborz University of Medical Sciences, Shiraz University of Medical Sciences, etc., and published in the journal “Neuromolecular Medicine” Volume 26, Page 28, 2024.

Research Process

Case Selection and Screening

The study included 47 NF1 cases from 33 Iranian families, all of Persian descent. Diagnosis was based on the revised International Neurofibromatosis Diagnostic Criteria. Data sampling and genetic testing were conducted between 2017 and 2023 at Genome-Nilou Laboratory in Tehran and Pars-Genome Laboratory in Karaj, Iran.

Genomic DNA Extraction and Sequencing

Genomic DNA was extracted from EDTA-anticoagulated whole blood using the salt precipitation method. The required DNA amount was 4-6μg per reaction, with a concentration of 50-200ng/μl. DNA quantity was verified using a Nanodrop device, and its integrity was confirmed by gel electrophoresis. Agilent SureSelect V7 was used for target region enrichment, and an Illumina NovaSeq sequencer achieved an average coverage depth of 100x for each target base position. Subsequently, 150bp paired-end sequences were aligned to the human reference genome (GRCh37/hg19), with sensitivity for single nucleotide variants and small insertions/deletions analysis exceeding 97%.

Bioinformatics Analysis

Variant screening underwent three stages of prioritization: 1. First stage: Low-frequency or unrecorded variants were screened based on frequency in the GnomAD (v3.1) database. 2. Second stage: Variants were screened based on clinical significance in the ClinVar database. 3. Third stage: The pathogenicity of variants was scored using tools such as Varsome, MutationTaster, SIFT, and the final variants were manually verified.

Identification of Novel Mutations

Through whole-genome exome sequencing analysis, the study identified a total of 31 variants, including 30 point mutations and 1 large deletion. In eight cases, the variants were hereditary, while the rest were sporadic. Seven of the variants were newly discovered, including c.5576 T > G, c.6658_6659insC, c.2322dupT, c.92_93insAA, c.4360C > T, c.3814C > T, and c.4565_4566delinsC, which had not been previously reported in the ClinVar and Leiden Open Variation databases.

Research Results

Mutation Characteristics

Some variants were concentrated in functional domains of the protein, especially in the GAP-related domain (GRD) and C-terminal domain (CTD), which play important regulatory roles. Most were heterozygous mutations, with some cases showing special phenotypic variations. For example, in family F14, only one monozygotic twin had Lisch nodules and skeletal developmental abnormalities. The father and daughter in family F4 had different clinical presentations despite having the same variant.

Clinical Manifestations

The study also reported some common clinical manifestations such as café-au-lait spots (97.87%), axillary and inguinal freckling (65.9%), and neurofibromas (50%). High phenotypic variability was observed in families with the same genetic variants. This may be attributed to the effects of modifier genes or environmental factors.

Conclusion

This study represents the largest sample size of Iranian NF1 cases to date. The mutations identified through whole-genome exome sequencing technology have expanded the mutation spectrum of the NF1 gene, demonstrating its high complexity in genetic variation. The results will help improve genetic counseling and personalized treatment strategies for NF1 patients and lay the foundation for future gene therapy approaches.

Significance of the Research

The main contribution of this research lies in the identification of many novel mutations in NF1 patients through whole-genome exome sequencing. These findings will promote the development of personalized medicine and early intervention strategies. Further functional assessments and genotype-phenotype correlation studies will deepen the understanding of the specific mechanisms of NF1 variants in disease manifestation.