Identifying mutations for MYH-associated polyposis.

Polyposis associated with mutations in the gene MYH is an autosomal recessive syndrome characterized by the development of colorectal adenomas and cancer. Two common mutations, p.Tyr165Cys (exon 7) and p.Glu382Asp (exon 13), have been shown to account for the majority of the mutations occurring in individuals of Caucasian ancestry. Other mutations have been found throughout the gene and many have been shown to have very low frequencies. Ethnic differences in the mutation spectrum have also been observed. Thus, in order to achieve the highest clinical sensitivity, it is necessary to perform whole-gene sequencing of the MYH gene. The sequencing protocol described allows one to identify mutations throughout the MYH gene.

A positive modifier of spinal muscular atrophy in the SMN2 gene.

Spinal muscular atrophy (SMA) is a common autosomal-recessive motor neuron disease caused by the homozygous loss of the SMN1 gene. A nearly identical gene, SMN2, has been shown to decrease the severity of SMA in a dose-dependent manner. However SMN2 is not the sole phenotypic modifier, because there are discrepant SMA cases in which the SMN2 copy number does not explain the clinical phenotype. This report describes three unrelated SMA patients who possessed SMN2 copy numbers that did not correlate with the observed mild clinical phenotypes. A single base substitution in SMN2, c.859G>C,, was identified in exon 7 in the patients' DNA. We now show that the change creates a new exonic splicing enhancer element and increases the amount of full-length transcripts, thus resulting in the less severe phenotypes. This demonstrates that the c.859G>C substitution is a positive modifier of the SMA phenotype and that not all SMN2 genes are equivalent. We have shown not only that the SMA phenotype is modified by the number of SMN2 genes but that SMN2 sequence variations can also affect the disease severity.

Consensus characterization of 16 FMR1 reference materials: a consortium study.

Fragile X syndrome, which is caused by expansion of a (CGG)(n) repeat in the FMR1 gene, occurs in approximately 1:3500 males and causes mental retardation/behavioral problems. Smaller (CGG)(n) repeat expansions in FMR1, premutations, are associated with premature ovarian failure and fragile X-associated tremor/ataxia syndrome. An FMR1-sizing assay is technically challenging because of high GC content of the (CGG)(n) repeat, the size limitations of conventional PCR, and a lack of reference materials available for test development/validation and routine quality control. The Centers for Disease Control and Prevention and the Association for Molecular Pathology, together with the genetic testing community, have addressed the need for characterized fragile X mutation reference materials by developing characterized DNA samples from 16 cell lines with repeat lengths representing important phenotypic classes and diagnostic cutoffs. The alleles in these materials were characterized by consensus analysis in nine clinical laboratories. The information generated from this study is available on the Centers for Disease Control and Prevention and Coriell Cell Repositories websites. DNA purified from these cell lines is available to the genetics community through the Coriell Cell Repositories. The public availability of these reference materials should help support accurate clinical fragile X syndrome testing.

Molecular classification of patients with unexplained hamartomatous and hyperplastic polyposis.

CONTEXT: Significant proportions of patients with hamartomatous polyposis or with hyperplastic/mixed polyposis remain without specific clinical and molecular diagnosis or present atypically. Assigning a syndromic diagnosis is important because it guides management, especially surveillance and prophylactic surgery. OBJECTIVE: To systematically classify patients with unexplained hamartomatous or hyperplastic/mixed polyposis by extensive molecular analysis in the context of central rereview of histopathology results. DESIGN, SETTING, AND PATIENTS: Prospective, referral-based study of 49 unrelated patients from outside institutions (n = 28) and at a comprehensive cancer center (n = 21), conducted from May 2, 2002, until December 15, 2004. Germline analysis of PTEN, BMPR1A, STK11 (sequence, deletion), SMAD4, and ENG (sequence), specific exon screening of BRAF, MYH, and BHD, and rereview of polyp histology results were performed. MAIN OUTCOME MEASURES: Molecular, clinical, and histopathological findings in patients with unexplained polyposis. RESULTS: Of the 49 patients, 11 (22%) had germline mutations. Of 14 patients with juvenile polyposis, 2 with early-onset disease had mutations in ENG, encoding endoglin, previously only associated with hereditary hemorrhagic telangiectasia; 1 had hemizygous deletion encompassing PTEN and BMPR1A; and 1 had an SMAD4 mutation. One individual previously classified with Peutz-Jeghers syndrome had a PTEN deletion. Among 9 individuals with an unknown hamartomatous polyposis, 4 had mutations in STK11 (1), BMPR1A (2), and SMAD4 (1). Of the 23 patients with hyperplastic/mixed polyposis, 2 had PTEN mutations. Substantial discrepancies in histopathology results were seen. CONCLUSIONS: Systematic molecular classification of 49 patients with unexplained hamartomatous or hyperplastic polyposis uncovered a potential novel susceptibility gene, ENG, for juvenile polyposis. Importantly, given the substantial proportion of patients found to have germline mutations, more extensive analysis of the known susceptibility genes is indicated. Rereview of histology results by a dedicated gastrointestinal pathologist should be considered routinely, as organ-specific surveillance rests on defining syndromic diagnosis.

Experience and strategy for the molecular testing of Duchenne muscular dystrophy.

Mutations in the dystrophin gene result in both Duchenne and Becker muscular dystrophies (DMD and BMD). Approximately two-thirds of the affected patients have large deletions or duplications. Using the multiplex polymerase chain reaction and Southern blotting techniques, the detection of these larger mutations is relatively straightforward. Detection of the point mutations in the remaining one-third of the patients has been challenging, mainly due to the large gene size and lack of hotspots or prevalent mutations. However, with the addition of some of the newer molecular screening methods, it is becoming more feasible for clinical laboratories to test for point mutations in the larger genes like dystrophin. Here we review the clinical features, describe the mutation distributions, evaluate current molecular strategies, and illustrate how the genetic findings have impacted the current clinical diagnostics of Duchenne and Becker muscular dystrophies.