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Molecular diagnostics for galactosemia

a galactose and diagnostic technology, applied in the field of galactose diagnostics, can solve problems such as deviations from normalcy in animals

Inactive Publication Date: 2001-09-27
EMORY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This method provides a sensitive and specific screening tool for galactosemia, enabling accurate diagnosis and personalized therapy recommendations, as well as identifying asymptomatic carriers, by directly detecting mutations in the GALT gene, thereby improving patient outcomes and family counseling.

Problems solved by technology

"Galactosemia" is a deficiency in the level or expression of GALT which results in detectable deviations from normalcy in animals.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

[0032] A. Oligonucleotides Useful in Detecting GALT Gene Mutations

[0033] Oligonucleotides useful in detecting the GALT gene mutation can be synthesized by techniques known in the art. The oligonucleotides can be from nine to twenty-five nucleotides in length; nucleotides shorter than 9 nucleotides will not provide accurate and reproducible results. Nucleotides longer than 25 nucleotides in length for detecting a single mutation in GALT can be used, but they provide no advantages over oligonucleotides 9-25 nucleotides in length. The single nucleotide mutation can be located anywhere in the oligonucleotide, as long as it is at least 3 nucleotides from either end of the oligonucleotide. One set of oligonucleotides of this invention, wherein the mutation is located exactly in the middle of the oligonucleotide, is presented in Table 3.

[0034] B. Amplification of DNA

[0035] 1. GALT Gene PCR

[0036] Samples are collected from any tissue or organ of a patient from which RNA or DNA can be amplif...

example 2

[0054] GALT Mismatched Hybrids Assay

[0055] To use multiple probes in a single hybridization format, an assay was developed by determining an optimal length for the probes so that the same temperature changes would result in the melting of the mismatched hybrids. Oligonucleotides between 9 and 25 nucleotides could satisfy this requirement, and those of 21 nucleotides, with the mutation at position 11, (see table 3) were found to be especially well-suited to a single temperature for disassociation of the hybrid. In addition, the use of tetramethyl ammonium chloride (TMAC) to the hybridization buffer allowed the hybridization temperature to be standardized at 55 C for all probes.

[0056] Hybridization Buffers and Techniques.

[0057] Amplified DNA in 5 .mu.l aliquots are spotted on a prewetted positively charged nylon membrane, denatured (using 0.5M NaOH, 2.0M NaCl, 25 mM EDTA) and UV-crosslinked. The membranes are prehybridized at 55.degree. C. for 30 minutes in a hybridization buffer (3.0...

example 3

[0058] Screening of a Galactosemia-Positive Patient for Mutation Type(s)

[0059] It is sometimes desirable to determine the exact nature of the mutation or mutations that are contributing to a patient's galactosemia. Such a determination will become critical as gene therapy techniques are implemented to correct these deficiencies. One example for analyzing a single patient's GALT gene for the presence of any or all of the known mutations in the GALT gene is provided herein. Synthetic oligonucleotides carrying each of the mutations to be screened are immobilized individually onto a support, such as a membrane, for example, as "dots" on a nitrocellulose membrane. A sample of the patient's labeled, amplified GALT gene (see Example 1 above for methods of amplification) is hybridized to all the dots on the membrane under conditions that promote only the hybridization of exactly matched sequences. The membrane is washed to remove mismatched and nonspecific hybridizing material, and the memb...

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Abstract

A process for detecting mutations in the gene responsible for galactosemia, galactose-1-phosphate uridyl transferase (GALT), is described. In one embodiment, the process can be used to detect over 85% of the mutations known to cause galactosemia in the United States population by using six different oligonucleotide probes, which span single-nucleotide Missense or nonsense mutations in the GALT gene. Hybridization conditions which can distinguish a single nucleotide mismatch are used to detect both the presence and zygosity of mutations in the GALT gene to aid in genetic counseling. A kit for use in detecting mutations in the GALT gene is also disclosed.

Description

[0001] This application claims priority in U.S. Provisional Application No. 60 / 103,286 filed Oct. 6, 1998.[0003] The field of this invention is the diagnosis of galactosemia using nucleic acid-based techniques.[0004] Galactosemia, an inherited metabolic disorder of milk sugar metabolism, is caused by certain mutations in the gene coding for the enzyme, galactose-1-phosphate uridyl transferase (GALT), which result in defective enzyme activity. This disorder is potentially fatal, so newborns throughout the U.S. and developed countries are screened for galactosemia, which when detected and treated early, saves lives and money. Evaluation of outcome in galactosemic patients began in the 1980s, and by 1990, an enigma was revealed (for example, see Kornrower, G M, J. Inher. Metab. Dis., 1982, 5:96-104; Waggoner and Buist, 1993, Intenat. Pediatr. 8:97-100). Although neonatal infants were screened and treated within days of life with a galactose-restricted diet, older children had unexpecte...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C12Q1/6883
CPCC12Q1/6883C12Q2600/156
Inventor ELSAS, LOUIS J. IIMURALIDHARAN, KASINATHAN
Owner EMORY UNIVERSITY
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