Application of Multiplex Ligation-Dependent Probe Amplification in the Genetic Testing of Oculocutaneous Albinism
Oculocutaneous albinism (OCA) is an autosomal recessive disorder characterized by mutations that affect melanin synthesis and melanosome biogenesis. With a global prevalence of approximately 1 in 17,000, OCA presents significant challenges in diagnosis and management due to its variable clinical phenotypes. While symptomatic treatment is available, there is no definitive cure for albinism. The heterogeneity of clinical manifestations makes it difficult to classify OCA subtypes based solely on physical features. Therefore, molecular and genetic analyses have become essential tools for accurate diagnosis, carrier screening, and prenatal testing.
Traditional genetic testing methods, such as Sanger sequencing and next-generation sequencing (NGS), have been widely used to identify mutations associated with OCA. However, these methods often fail to detect the second mutation in some patients, particularly those involving copy number variations (CNVs). CNVs, which include large deletions or duplications of genomic segments, account for a significant proportion of disease-causing mutations. Multiplex ligation-dependent probe amplification (MLPA), a technique first reported in 2002, has emerged as a powerful tool for detecting CNVs, including small intragenic rearrangements.
In this study, 12 unrelated patients with OCA were selected for genetic analysis. These patients had previously been identified with only one allelic point mutation in either the tyrosinase (TYR) gene or the oculocutaneous albinism II (OCA2) gene using Sanger sequencing or NGS. Some NGS data suggested the presence of large heterozygous deletions. Clinically, all patients exhibited varying degrees of hypopigmentation in the skin, hair, and iris, accompanied by nystagmus, photophobia, and visual impairment. Their unaffected family members displayed normal pigmentation.
MLPA was employed to detect potential large heterozygous deletions in these patients. The results revealed that eight out of the 12 cases (66.7%) contained large heterozygous deletions of one or more exons. Seven distinct types of large heterozygous deletions were identified: two in the TYR gene and five in the OCA2 gene. Among these, the Ex1–24 deletion in OCA2 found in Patient 2 and the Ex1–5 deletion in TYR found in Patient 6 were previously unreported in the Human Gene Mutation Database or the 1000 Genomes Database. All identified mutations were classified as pathogenic based on the standards and guidelines for the interpretation of sequence variants published by the American College of Medical Genetics and Genomics. Notably, two of these mutations were de novo, meaning they were not inherited from either parent.
OCA1, caused by mutations in the TYR gene, results in the complete or partial loss of tyrosinase activity. OCA1A, the most severe form, is characterized by white skin and hair from birth and a lifelong absence of melanin. In contrast, OCA1B patients may exhibit some pigmentation over time due to residual tyrosinase activity. OCA2, caused by mutations in the OCA2 gene, is associated with the regulation of melanosome pH and tyrosinase activity. Patients with OCA2 typically have white skin, yellow hair, and light-colored irises, along with nystagmus, photophobia, and impaired visual acuity. However, their vision may improve with age.
The variability in OCA phenotypes underscores the importance of molecular analyses for definitive diagnosis and genetic counseling. While Sanger sequencing and NGS are effective for detecting point mutations, they often miss CNVs, which are responsible for approximately 5.5% of disease-causing mutations. MLPA, with its high sensitivity and specificity, serves as a complementary tool for identifying CNVs. Unlike NGS, which can detect CNVs across the entire genome, MLPA is limited to specific target regions. However, its ease of use and ability to confirm suspected CNVs make it invaluable in the genetic diagnosis of OCA.
In summary, this study highlights the critical role of MLPA in the genetic testing of OCA, particularly in cases where traditional methods fail to identify the second mutation. By detecting large heterozygous deletions, MLPA provides a more comprehensive understanding of the genetic basis of OCA, aiding in accurate diagnosis and informed genetic counseling for affected families.
doi.org/10.1097/CM9.0000000000000356
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