Rapid molecular diagnosis of mutations associated with generalized thyroid hormone resistance by PCR-coupled automated direct sequencing of genomic DNA: Detection of two novel mutations

Seto, Donald; Weintraub, Bruce D.

doi:10.1002/(sici)1098-1004(1996)8:3<247::aid-humu8>3.0.co;2-6

Cited by 10 publications

(2 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Furthermore, human error becomes a problem when large numbers of chromatograms must be evaluated. Despite this, a number of smaller studies rely on this straightforward method for detecting mutations (Pereira et al, 1995;Aronovich et al, 1996;Seto and Weintraub, 1996). Although most researchers who perform fluorescencebased sequencing have access to printouts of chromatograms, free software (Editview) from Perkin-Elmer that can display and print individual chromatograms can be downloaded off the Internet from the Perkin-Elmer Web site (http://www2.perkin-elmer.com/ga/index.htm.…”

Section: Candidate Genes For Mutationsmentioning

confidence: 99%

Heterozygote Detection Using Automated Fluorescence‐Based Sequencing

et al. 2002

View full text Add to dashboard Cite

Identifying mutations at the sequence level is the critical final step in identifying pathologic mutations in autosomal dominant disorders as well as in identifying carriers of autosomal recessive and X-linked disorders. In addition, the identification of the rapidly expanding collection of single-nucleotide polymorphisms (SNPs), which are now being used in the study of complex genetic traits in large populations, also requires a method for identifying heterozygote sequence variation. Although there are many types of mutations that can occur, point mutations frequently cause missense, nonsense, or splicing mutations in disease genes. Point mutations are also the most common type of polymorphism. These mutations or polymorphisms can occur in one allele, while the other allele retains the normal sequence; detecting such sequence variants in a heterozygous individual's DNA, carrying both the normal allele and the mutant allele, can be difficult. In this unit, a strategy is presented for detecting such sequence variation using automated fluorescence-based sequencing traces, and analysis with software tools that assist with the identification of sequence variation. A protocol is given that has been used to identify heterozygote mutations in TSC1 and TSC2, the causative genes for the autosomal dominant disorder tuberous sclerosis complex (TSC), as well as for a number of other inherited disorders in humans.Common genetic diseases like hypertrophic cardiomyopathy (HCM) are caused by autosomal dominant mutations in a subset of sarcomere proteins. Again, sequencing is by far the most sensitive method to define a heterozygous mutation in one of these genes. DNAs from a large cohort of individuals with HCM are often screened by direct sequencing of the major candidate genes comprising of >100 exons. Projects involving large-scale sequencing were facilitated by the improvement of PCR techniques, simplification of the purification steps, and most-importantly, advances in speed and quality of fluorescent dye sequencing with the introduction of the automated capillary sequencer. The Alternate Protocol describes an approach for large-scale sequencing adapted for serial screening for HCM mutations (http://cardiogenomics.med.harvard.edu). BASIC PROTOCOL IDENTIFYING HETEROZYGOTE MUTATIONS FROM SEQUENCE TRACESThe steps for identifying heterozygote mutations from sequence traces are outlined below and include: (1) amplification of exons from genomic DNA using PCR, (2) purification of PCR amplicons, (3) dye terminator cycle sequencing, and (4) sequence analysis. Protocols for PCR amplification and cycle sequencing are included here. Protocols for preparing genomic DNA and a variety of mutation screening methods that can be used to screen exons (or noncoding regions) for sequence variation are covered elsewhere in this chapter (see . The commentary includes a discussion of the features of a variety of sequence analysis software tools that are useful for identifying sequence variations in chromatograms generated by automated sequencers....

show abstract

Section: Candidate Genes For Mutationsmentioning

confidence: 99%

Heterozygote Detection Using Automated Fluorescence‐Based Sequencing

et al. 2002

View full text Add to dashboard Cite

show abstract

“…As a result, the mutant THR interferes with the function of normal THRs leading to a dominant negative action, which explains the dominant inheritance for this syndrome [18]. Both p.Ala317Ser or p.Arg438Pro are reported here for the first time, however mutations in residues 317 and 438 have already been described in patients with RTH worldwide including Brazilian patients [14,[20][21][22][23][24]. Those mutations are located in the ligand-binding domain, within 2 regions involving residues 310-353 and 429-461, respectively, known as "hot-spots" for mutations [1].…”

mentioning

confidence: 96%

Two Novel Mutations in the Thyroid Hormone Receptor β in Patients with Resistance to Thyroid Hormone (RTH β): Clinical, Biochemical, and Molecular Data

et al. 2015

View full text Add to dashboard Cite

The syndrome of resistance to thyroid hormone (RTH β) is an inherited disorder characterized by variable tissue hyposensitivity to 3,5,30-L-triiodothyronine (T(3)), with persistent elevation of free-circulating T(3) (FT(3)) and free thyroxine (FT(4)) levels in association with nonsuppressed serum thyrotropin (TSH). Clinical presentation is variable and the molecular analysis of THRB gene provides a short cut diagnosis. Here, we describe 2 cases in which RTH β was suspected on the basis of laboratory findings. The diagnosis was confirmed by direct THRB sequencing that revealed 2 novel mutations: the heterozygous p.Ala317Ser in subject 1 and the heterozygous p.Arg438Pro in subject 2. Both mutations were shown to be deleterious by SIFT, PolyPhen, and Align GV-GD predictive methods.

show abstract