Comprehensive screening of CREB-binding protein gene mutations among patients with Rubinstein-Taybi syndrome using
Toru Udaka1, Hazuki Samejima, Rika Kosaki
1Division of Medical Genetics, Department of Pediatrics, Keio University School of Medicine, Shinanomachi, Tokyo, Japan.
Insights
Genetic testing for Rubinstein-Taybi syndrome (RSTS) is now more accessible. A new denaturing high-performance liquid chromatography (DHPLC) method efficiently detects CREBBP gene mutations in RSTS patients.
Area of Science:
- Genetics
- Molecular Biology
- Clinical Diagnostics
Background:
- Rubinstein-Taybi syndrome (RSTS) is caused by mutations in the CREBBP gene.
- Current genetic testing for CREBBP is complex and costly, limiting clinical application.
- There is a need for a more efficient and accessible diagnostic method for RSTS.
Purpose of the Study:
- To develop and validate a sensitive and specific automated denaturing high-performance liquid chromatography (DHPLC) assay for analyzing the CREBBP gene.
- To assess the utility of the DHPLC method for detecting CREBBP mutations in patients with RSTS.
Main Methods:
- Developed a DHPLC-based protocol to analyze the entire coding region of the CREBBP gene.
- Utilized a 96-well format PCR plate for simultaneous amplification of all CREBBP exons using 41 primer pairs.
- Employed a custom computer script for serial analysis of PCR amplicons under optimized conditions.
Main Results:
- Identified heterozygous CREBBP mutations in 12 out of 21 RSTS patients (57% detection rate).
- Detected various mutation types including frameshift, nonsense, splice-site, and missense mutations.
- The DHPLC method demonstrated high sensitivity and specificity comparable to existing techniques.
Conclusions:
- The DHPLC-based mutation analysis offers a sensitive, specific, and potentially more accessible method for detecting CREBBP mutations in RSTS.
- This approach can aid medical geneticists in confirming RSTS diagnoses and providing genetic counseling.
- The methodology can be adapted for genetic analysis of other genes associated with dysmorphic syndromes.
Abstract:
Mutations in the CREBBP (CREB-binding protein gene) cause Rubinstein-Taybi syndrome (RSTS). At present, however, genetic testing of CREBBP is not commonly applied in clinical settings because the currently available assays are technically and financially demanding, mainly because of the size of the gene. In the present study, we took advantage of a highly sensitive and specific, automated denaturing high-performance liquid chromatography (DHPLC) technique. First, we developed a DHPLC-based protocol to analyze the entire coding region of CREBBP. Second, we analyzed genetic samples from 21 RSTS patients using DHPLC. The coding region was amplified by 41 primer pairs, all of which have the same cycling conditions, aliquoted on a 96-well format PCR plate. In this manner, all the exons were simultaneously amplified using a single block in a PCR machine. We then wrote a computer script to analyze all the PCR amplicons generated from various portions of the CREBBP gene in a serial manner at optimized conditions determined individually for each amplicon. Heterozygous CREBBP mutations were identified in 12 of the 21 patients: five frameshift mutations, three nonsense mutations, two splice-site mutations, and two missense mutations. The resulting detection rate of 57% was comparable to the outcome of previous studies. The relatively high detection rate in the present study demonstrates the enhanced sensitivity of the DHPLC-based mutation analysis, as exemplified by mutation analyses of other genes. The implementation of similar methodologies for other dysmorphic syndromes will help medical geneticists to confirm their clinical impressions and to provide accurate genetic counseling for patients and their families.
