Warthen DM, Moore EC, Kamath BM, Morrissette JJD, Sanchez P, Piccoli DA, et al. Jagged1 (JAG1) Mutations in Alagille Syndrome: Increasing the Mutation Detection Rate. Hum Mutat 2006;27: 436–443. (Reproduced by permission)
Alagille syndrome (AGS) is caused by heterozygous mutations in JAG1, and mutations have been previously reported in about 70% of patients who meet clinical diagnostic criteria. We studied a cohort of 247 clinically well-defined patients, and using an aggressive and sequential screening approach we identified JAG1 mutations in 94% of individuals. Mutations were found in 232 out of 247 patients studied and 83 of the mutations were novel. This increase in the mutation rate was accomplished by combining rigorous clinical phenotyping, with a combination of mutation detection techniques, including fluorescence in situ hybridization (FISH), genomic and cDNA sequencing, and quantitative PCR. This higher rate of mutation identification has implications for clinical practice, facilitating genetic counseling, prenatal diagnosis, and evaluation of living-related liver transplant donors. Our results suggest that more aggressive screening may similarly increase the rate of mutation detection in other dominant and recessive disorders.
The genetic bases of many liver diseases have been elucidated over the last decade. The most notable of these include Wilson's disease and hemochromatosis. Both of these conditions are inherited in an autosomal recessive pattern. Hemochromatosis is classically Mendelian with alterations in 1 gene (HFE gene) accounting for 95% of the cases, whereas Wilson's disease results from multiple mutant variations in the Wilson's Disease Gene (which codes for a membrane P-type ATPase, ATP7B) and variable phenotypic expression of these genotypes. It is now known that there are multiple liver diseases that have a genetic basis underlying their phenotypic expression (Table 1). Like Wilson's disease, however, many of these conditions result from multiple potential mutations, and have variable phenotypic expression. In cystic fibrosis, for instance, more than 1300 mutations have been identified in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.1 Characterization of the genetic mutations underlying progressive familial intrahepatic cholestasis and benign recurrent intrahepatic cholestasis has improved our diagnostic ability and understanding of these disorders, as well.2 In general, our modern molecular biological techniques are facilitating the identification of more polymorphisms that result in disease, allowing for more definitive individual diagnosis and family screening than has been possible in the past.
|Alpha-1 antitrypsin deficiency|
|Progressive familial intrahepatic cholestasis|
|Benign recurrent intrahepatic cholestasis|
|Cholesterol ester storage disease|
Warthen et al.3 have now accomplished methods for enhanced polymorphism detection for Alagille syndrome (AGS). Alagille syndrome, or arteriohepatic dysplasia, is an autosomal dominant multiorgan system disorder. During the neonatal period, patients with AGS develop cholestatic jaundice. Liver biopsies performed later than 6 months of age typically show the characteristic paucity of bile ducts; however, biopsy done prior to 6 months of age may show nonspecific intrahepatic cholestasis and portal inflammation. Traditionally, the diagnosis of AGS has been based on liver biopsy findings in conjunction with classic clinical criteria. Clinical diagnosis requires 3 of 5 clinical criteria which include: cholestasis with intrahepatic paucity of the bile ducts on histological evaluation, congenital cardiac disease most typically pulmonic valvular stenosis with peripheral arterial stenosis, the retinal finding of posterior embryotoxin, abnormal vertebrae (“butterfly” or hemi-vertebrae) and decrease in interpediculate distance in the lumbar spine, and characteristic facies with a broad forehead, pointed mandible and bulbous tip of the nose and in the fingers.4, 5 Despite these characteristic physical features, however, there can be great variability in the phenotypic presentation of patients with AGS, even within families, making diagnosis sometimes difficult.6
Alagille syndrome is caused by a mutation or deletions in Jagged1, a ligand in the Notch signaling pathway whose main function is in cell fate determination.7, 8 Screening for JAG1 has largely been performed by using scanning techniques such as single-strand conformation polymorphism (SSCP analysis), confirmation sensitive gel electrophoresis (CSGE), or denaturing high performance liquid chromatography. Using these techniques, mutation screening of JAG1 in patients with AGS has demonstrated mutations in 60%–70% of patients with the clinical diagnosis of AGS. The inability to detect the remaining mutations is felt to be secondary to technical difficulties in screening for this large gene.
Using a comprehensive and sequential screening approach, Warthen et al. were able to identify JAG1 mutations in 94% of individuals with clinically defined AGS (Fig. 1). Patients were initially screened using standard techniques, either by SSCP or CSGE. Of those who were screened using SSCP, JAG1 mutation was identified in 70% of individuals. Those in whom no mutation was identified underwent further investigation including CSGE, complementary DNA sequencing, genomic DNA sequencing and/or quantitative polymerase chain reaction (qPCR). Forty additional mutations were identified resulting in 94.7% of patients with AGS having a mutation identified. Of those initially screened using CSGE, mutations were identified in 67% of patients with AGS. Of those in whom no mutation was identified by this initial technique, further study by genomic DNA sequencing, complementary DNA sequencing, qPCR, and cytogenic analysis identified an additional 55 mutations for a total identification of the JAG1 mutation in 90% of patients.
This elegant study by Warthen et al. represents an important intersection between clinical medicine and molecular diagnosis. Prior to the identification of JAG1 mutation, the diagnosis of AGS was solely based on the classic clinical criteria of the patient. The large variability of presentations of AGS made the diagnosis difficult at times. With the identification of JAG1 mutations as the genetic basis of AGS, a breakthrough in understanding and diagnosing AGS was made. By now further improving mutation detection rates through their comprehensive systematic approach, Warthen et al. have further strengthened diagnostic abilities and the potential for prenatal genetic screening and genetic counseling. Ongoing improvements in molecular techniques continue to improve our understanding of the molecular basis of clinical hepatology. This study by Warthen et al. represents the continuation of that evolution.