| Summary: | 碩士 === 國立臺灣大學 === 臨床醫學研究所 === 99 === Introduction :
In recent years, with the rapid development of genetic diagnostic technologies, more and more diseases have been linked to genetic mutation. Because isolation of high quality DNA from dried blood spots on traditional filter paper blotters for subsequent DNA analysis is not easy, Taiwan''s current newborn screening is still limited to congenital metabolic and endocrine diseases. Newborn genetic screening is still not available now. So, how to create a newborn genetic screening technology, has become an important issue without delay.
Congenital central hypoventilation syndrome (CCHS) is a rare neurological disorder characterized by abnormal autonomic central nervous system control of breathing during sleep. In the past, CCHS was diagnosed on the basis of clinical criteria proposed by the American Thoracic Society in 1999. CCHS is characteristically diagnosed in the newborn period almost without family history. Some cases of sudden infant death may be related to this disease.The paired-like homeobox gene PHOX2B, which is active during neuronal development, is the disease-defining gene for CCHS. Approximately 92% of individuals with the CCHS phenotype will be heterozygous for a polyalanine repeat expansion mutation (PARM); the normal allele will have 20 alanines and the affected allele will have 24–33 alanines (genotypes 20/24–20/33). The remaining 8% of individuals with CCHS will have a non-PARM (NPARM) in the PHOX2B gene; these will be missense, nonsense, or frameshift. CCHS is inherited in an autosomal dominant manner with a stable mutation.
Early detection of CCHS is important because of the significant morbidity,
especially neurologic consequences, and the risk of death in the undiagnosed subject. We hope to use congenital central hypoventilation syndrome as a disease model to establish newborn genetic screening technology.
Materials and Methods
Part 1
First, genomic DNA was collected from peripheral whole blood of 60 clinical suspected CCHS patients; newborn babies with hypoxia even death and their 27 family members from the National Taiwan University Hospital and the referring hospitals. We used PCR, CE, and direct sequencing to find out mutation points of PHOX2B gene.
Part 2
1520 health individuals’ peripheral whole blood was obtained and analyzed for PARM, using PCR, CE, and direct sequencing techniques, to test the stability of our PARM screening techniques and to investigate polyalanine polymorphism in normal population.
Part 3
Finally, We completed 3480 PARM tests of DNA from dried blood spots on filter paper blotters including QIAcard, Whatman 903, and IDBS; these three commercially available cards. We adopted magnetic beads separation of trace DNA techniques, combined with PCR, CE, and direct sequencing to study the feasibility of newborn genetic screening.
Results
In 60 clinical suspected CCHS patients; newborn babies with hypoxia even death, 10 individuals have polyalanine expansions, and 2 individuals have frameshift mutations. The PHOX2B mutation rate is 20% (12/60). The polyalanine expansion mutation is not found in 1520 health individuals and 3480 newborn babies in our population study. There are some types of polyalanine contraction found in these 5000 individuals. With a frequency of 95.01% in the sample population, (GCN)20 is the most common allele in the 20-residue polyalanine domain while (GCN)15 is second in the order of allele prevalence with a frequency of 4.25%. Additionally, the (GCN)14, (GCN)13, (GCN)7 and (GCN)6 alleles ( 0.04%,0.55%,0.14%,and 0.01%, respectively ) are also identified in the sample.
Conclusion
We successfully establish the newborn genetic screening technology in CCHS to advance genetic diagnosis to the time point of the neonatal period. With early treatment of this disease, the prognosis will be greatly improved. We found all the PHOX2B gene mutation points are located in the area around the polyalanine repeats, which shows this area as the Taiwanese population PHOX2B gene mutation hot spot. Our newborn PHOX2B screening methods focus on the polyalanine repeat region, which can detect all patients with PARM and the NPARM patients located in this region. In addition, QIAcard, Whatman 903, IDBS; these three commercially available filter paper blotters can provide both high quality and high stability of the DNA sample source for the extraction and subsequent analysis, and can be stored for a long time for re-use. This newborn genetic screening technology will be widely used in other single gene disorders in the future, such as a higher incidence of deafness genes.
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