Molecular diagnostics for galactosemia

Details Molecular diagnostics for galactosemia Molecular diagnostics for galactosemia

1999-10-06

2001-03-27

Fredman, Jeffrey (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C536S023500, C536S024310, C536S024330

Reexamination Certificate

active

06207387

ABSTRACT:

FIELD OF THE INVENTION
The field of this invention is the diagnosis of galactosemia using nucleic acid-based techniques.
BACKGROUND OF THE INVENTION
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
, Internat. Pediatr
. 8:97-100). Although neonatal infants were screened and treated within days of life with a galactose-restricted diet, older children had unexpectedly poor outcomes. Dysfunctions included ovarian failure, verbal dyspraxia, growth and developmental delays, and neurological signs of cortical and extrapyramidal tract impairment. Since the GALT enzyme structure is highly conserved throughout evolution, the human GALT cDNA was cloned using probes homologous to known sequences in the bacterial enzyme. When the human cDNA and gene were sequenced (see Flach, Reichardt, and Elsas, 1990, Mol. Biol. Med. 7:365-369; and Leslie et al. 1992, Genomics 14:474-480), the first mutations in the GALT gene were associated with human galactosemia (Reichardt et al. 1991, Am. J. Hum. Genet. 49:860-867; Reichardt et al, 1992, Genomics 12: 596-600) and relationships between the mutations and the resulting phenotype were being reported (Elsas et al. 1993, Internat Pediatr. 8:101). The Q188R mutation, first reported in 1991, is the most common mutation among galactosemic patients of Caucasian ethnicity (Reichert et al. 1991, Ibid.) and causes a complete loss in GALT activity. The S135L mutation is common among African-Americans (Lai et al., J. Pediatr 1996, 128: 89-95), and results in severe reduction in GALT activity. The N314D mutation, also known as the Duarte allele (Elsas et al., 1994, Am J. Hum Genet 54: 1030-1036), causes an instability in the GALT protein that leads to a 50% reduction in activity in homozygous patients. The K285N mutation is the second most common mutation among white galactosemic patients, and is the most prevalent among patients in southern Germany and Austria (Leslie et al., 1992, Ibid.). Other common mutations identified in the white population are R148W and L195P (Reichart et al. 1992, Ibid.).
These reports led to the realizations that different GALT mutations can result in different phenotypes and that therapy recommendations may vary depending on the specific mutation.
Methods currently used in newborn screening determine only the level of enzymatic activity or the accumulation of precursors. Results from current techniques are altered by the sampling parameters, such as ambient temperature and time of feeding relative to collection of sample. Also, they do not reveal the specific mutations that cause the change in enzyme activity. Certain mutations cause a mild disease, while others cause severe effects. Identifying the mutation is necessary to make a diagnosis, initiate appropriate therapy, estimate prognosis, and provide appropriate genetic counseling for the family.
There remains a need for a test with increased sensitivity and specificity for screening and diagnosis of galactosemia and asymptomatic carriers of galactosemia. There is a need for a test that can be run simultaneously on a large number of samples, such as in a micro titer plate format now commonly in use in clinical diagnostic laboratories. There is a need for a testing protocol that can quickly and specifically identify the majority of the alleles that can contribute to galactosemia in the human populations.
The claimed invention describes a nucleic acid-based method that can detect and specify the mutation involved in over 85% of individuals with some form of galactosemia rapidly and economically. Such a screen would be applicable to universal newborn screening and should supplement or completely replace current screening strategies. The screen can also be used to detect heterozygous mutations and the presence of multiple mutations in the GALT gene, which are necessary for understanding and counseling heritability and recurrence risks to family members.
SUMMARY OF INVENTION
The invention as claimed is a nucleic acid-based method that can detect and specify the mutation involved in the great majority of individuals with some form of galactosemia rapidly and economically. The method involves the detection of the mutation in amplified DNA from a patient utilizing probes that span the mutation. Six mutations are identified as being responsible for over 85% of the mutations in the United States population, and a detection kit is described for detecting these six mutations. The methods of this invention can also be used to determine the zygosity of the mutations and to detect the presence of multiple mutations in the GALT gene, both of which are necessary for understanding and counseling heritability and recurrence risks to family members.
DETAILED DESCRIPTION OF THE INVENTION
Definitions:
“GALT” means the enzyme galactose-1-phosphate uridyltransferase.
“Galactosemia” is a deficiency in the level or expression of GALT which results in detectable deviations from normalcy in animals.
As used in the claims, “a” can mean one or more, depending on the context of the claim.
Detailed Description of Invention
The invention described herein involves the use of a group or set of DNA probes to detect mutations in the GALT gene. Prior to this invention, only enzyme activity analyses have been used in clinical screening applications. With the practice of this invention, a rapid and economical screening system for detecting galactosemia in human populations, particularly in newborns, is provided. Based on the discovery of the frequency of the K285N mutation and the existence and frequency of the Y209C mutation, a process for detecting specific mutations in the GALT gene that cause over 85% of the mutations leading to galactosemia currently known in the U.S. population is provided. A preferred embodiment of this invention comprises assaying for the presence of six mutations, listed in Table 1A. In a survey of newborns the prevalence of these mutations is shown in Table 1B. If desired, this process can be followed by additional screening for less common mutations, e.g. listed in Table 2. The process comprises the collection of an RNA- or DNA-containing sample from a patient or a newborn infant, amplification of the DNA (following reverse transcription of the RNA, if needed) that encodes either a portion of or the entire GALT gene, hybridization of the amplified DNA, either serially or in parallel, with each member of a set of detectable probes, each probe being at least 10 nucleotides in length, the set comprising probes having sequences complementary to the sequences set forth in Table 1A, under hybridization conditions that allow only exactly complementary strands to remain as hybrids, and detecting the presence or absence of the hybrids.
TABLE 1A
Approx % found in
galactosemic
Designation#
Sequence*
population
S135L
TGGT
T
GGAT
62% of African-
American population

Q188R
TGCC
G
GGTA
70% of
white population

L195P
TTCC
C
GCCA
8% of all ethnic
groups

K285N
CCAA
T
TATG
17% of
white populations

Y209C
GCCT
G
TAAG
6-20% of all
ethnic groups

N314D
CTGG
G
ACCA
8% of all ethnic
groups

[deletion]
[absence
in Ashkenazi
of signal
Jewish population
with any probe]
#The number in the designation refers to the amino acid position in the human GALT protein (see, e.g. Leslie et al., ibid., or SEQ ID NO:15). The single lettersor on either side of the number identify the amino acid in the wild-type protein (preceding the amino acid number) and the amino acid present in the mutant protein.
*underlined nucleotide is the one mutated

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