Methodology for direct sequencing of DYSF and CAPN3

Rapid direct sequence analysis of the dystrophin gene.

Flanigan KM, von Niederhausern A, Dunn DM, Alder J, Mendell JR, Weiss RB.

Mutations in the dystrophin gene result in both Duchenne and Becker muscular dystrophy (DMD and BMD), as well as X-linked dilated cardiomyopathy. Mutational analysis is complicated by the large size of the gene, which consists of 79 exons and 8 promoters spread over 2.2 million base pairs of genomic DNA. Deletions of one or more exons account for 55%-65% of cases of DMD and BMD, and a multiplex polymerase chain reaction method-currently the most widely available method of mutational analysis-detects approximately 98% of deletions. Detection of point mutations and small subexonic rearrangements has remained challenging. We report the development of a method that allows direct sequence analysis of the dystrophin gene in a rapid, accurate, and economical fashion. This same method, termed “SCAIP” (single condition amplification/internal primer) sequencing, is applicable to other genes and should allow the development of widely available assays for any number of large, multiexon genes.

Am J Hum Genet. 2003 Apr;72(4):931-9 (PDF)

Evidence-based path to newborn screening for Duchenne muscular dystrophy.

JR Mendell, C Shilling, ND Leslie, KM Flanigan, R Al-Dahhak, J Gastier-Foster, K Kneile, DM Dunn, B Duval, A Aoyagi, C Hamil, M Mahmoud, K Roush, L Bird, C Rankin, H Lilly, N Street, R Chandrasekar, RB Weiss

Objective: Creatine kinase (CK) levels are increased on dried blood spots in newborns related to the birthing process. As a marker for newborn screening, CK in Duchenne muscular dystrophy (DMD) results in false‐positive testing. In this report, we introduce a 2‐tier system using the dried blood spot to first assess CK with follow‐up DMD gene testing.

Methods: A fluorometric assay based upon the enzymatic transphosphorylation of adenosine diphosphate to adenosine triphosphate was used to measure CK activity. Preliminary studies established a population‐based range of CK in newborns using 30,547 deidentified anonymous dried blood spot samples. Mutation analysis used genomic DNA extracted from the dried blood spot followed by whole genome amplification with assessment of single‐/multiexon deletions/duplications in the DMD gene using multiplex ligation‐dependent probe amplification.

Results: DMD gene mutations (all exonic deletions) were found in 6 of 37,649 newborn male subjects, all of whom had CK levels >2,000U/l. In 3 newborns with CK >2,000U/l in whom DMD gene abnormalities were not found, we identified limb‐girdle muscular dystrophy gene mutations affecting DYSF, SGCB, and FKRP.

Interpretation: A 2‐tier system of analysis for newborn screening for DMD has been established. This path for newborn screening fits our health care system, minimizes false‐positive testing, and uses predetermined levels of CK on dried blood spots to predict DMD gene mutations.

Annals of Neurology, Jan 201271 (3), pp. 304–313.(PDF)

Automated genomic sequence analysis of the three collagen VI genes: applications to Ullrich congenital muscular dystrophy and Bethlem myopathy.

A K Lampe, D M Dunn, A C von Niederhausern, C Hamil, A Aoyagi, S H Laval, S K Marie, M-L Chu, K Swoboda, F Muntoni, C G Bonnemann, K M Flanigan, K M D Bushby and R B Weiss

Introduction: Mutations in the genes encoding collagen VI (COL6A1, COL6A2, and COL6A3) cause Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD). BM is a relatively mild dominantly inherited disorder with proximal weakness and distal joint contractures. UCMD is an autosomal recessive condition causing severe muscle weakness with proximal joint contractures and distal hyperlaxity.

Methods: We developed a method for rapid direct sequence analysis of all 107 coding exons of the COL6 genes using single condition amplification/internal primer (SCAIP) sequencing. We have sequenced all three COL6 genes from genomic DNA in 79 patients with UCMD or BM.

Results: We found putative mutations in one of the COL6 genes in 62% of patients. This more than doubles the number of identified COL6 mutations. Most of these changes are consistent with straightforward autosomal dominant or recessive inheritance. However, some patients showed changes in more than one of the COL6 genes, and our results suggest that some UCMD patients may have dominantly acting mutations rather than recessive disease.

Discussion: Our findings may explain some or all of the cases of UCMD that are unlinked to the COL6 loci under a recessive model. The large number of single nucleotide polymorphisms which we generated in the course of this work may be of importance in determining the major phenotypic variability seen in this group of disorders.

J Med Genet. 2005 Feb; 42(2): 108–120.(PDF)