Related Experiment Videos
Identification of molecular defects in a subject with type I CD36 deficiency
H Kashiwagi1, Y Tomiyama, S Kosugi
1Second Department of Internal Medicine, Osaka University Medical School, Japan.
Insights
Type I CD36 deficiency in a subject resulted from genetic mutations causing CD36 mRNA instability and reduced expression. These molecular findings explain the absence of cell surface CD36.
Area of Science:
- Molecular Biology
- Genetics
- Immunology
Background:
- CD36 is a cell surface receptor expressed on platelets and monocytes.
- CD36 deficiency can be classified into different types based on molecular mechanisms.
- Understanding the genetic basis of CD36 deficiency is crucial for diagnosing and managing related conditions.
Purpose of the Study:
- To investigate the molecular basis of a rare type I CD36 deficiency.
- To identify the genetic mutations responsible for the lack of CD36 expression on platelets and monocytes.
Main Methods:
- Molecular analysis of CD36 cDNA and genomic DNA.
- RNA blot analysis and reverse transcription-polymerase chain reaction (RT-PCR).
- Cloning and nucleotide sequence analysis of CD36 cDNA.
Main Results:
- Two different-sized CD36 cDNAs were identified: one normal and one ~150 bp smaller.
- The smaller cDNA contained a 161-bp deletion (exon 4 loss) and a dinucleotide deletion (exon 5).
- Both deletions caused frameshifts and premature stop codons, leading to greatly reduced CD36 transcripts and cell surface expression.
Conclusions:
- The identified deletions in CD36 mRNA likely cause transcript instability.
- A dinucleotide deletion in exon 5 may impact the splicing of exon 4.
- These molecular defects explain the type I CD36 deficiency observed in the subject.
Abstract:
We performed a molecular analysis of a subject whose platelets and monocytes did not express any cell surface CD36 (designated as a type I CD36 deficiency). Amplification of the 5' half of platelet and monocyte CD36cDNA (corresponding to nucleotide [nt] 191-1009 of the published CD36 cDNA sequence [Oquendo et al, Cell, 58:95, 1989]) showed that two different-sized CD36 cDNAs existed. One cDNA was of predicted normal size, whereas the other was about 150 bp smaller than that predicted for normal CD36 cDNA. Amplification of the 3' region of CD36 cDNA (nt 962-1714) in this subject showed only normal-sized CD36 cDNA. Cloning and nt sequence analysis of the cDNAs showed that the smaller sized CD36 cDNA had 161-bp deletion (from nt 331 to 491), and a dinucleotide deletion starting at nt position 539. The same dinucleotide deletion was also detected in the normal sized CD36 cDNA. Both deletions caused a frameshift leading to the appearance of a translation stop codon. RNA blot analysis and quantitative assay using the reverse transcription-polymerase chain reaction (RT-PCR) showed that the CD36 transcripts in both platelets and monocytes were greatly reduced. Comparison of the determined cDNA sequences with the genomic DNA sequence for the human CD36 gene showed that the dinucleotide deletion was located in exon 5, and that the 161-bp deletion corresponded to a loss of exon 4. PCR-based analysis using genomic DNA showed that this subject was homozygous for the dinucleotide deletion in exon 5. Except for the dinucleotide deletion, we could not find any abnormalities around exon 3, 4, and 5 including the splice junctions. These results suggested that the deletions in CD36 mRNA were likely to be responsible for instability of the transcripts, and the dinucleotide deletion in exon 5 might affect the splicing of exon 4.