Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-12-04
2004-11-02
Myers, Carla J. (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023500, C536S024310, C536S024330
Reexamination Certificate
active
06811976
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a novel diagnostic method for the detection of infertility in males. In particular, the present invention relates to a diagnostic method for detecting the presence or absence of a mutation or mutations in the POLG gene in a biological sample. The invention relates also to the use of a mutant POLG gene in the detection of infertility in males and in the screening of human populations for the presence of such mutation or mutations as a predictive test for male infertility. The present invention further relates to the use of the POLG gene as an indicator of other pathological conditions associated with or related to male infertility, including those manifesting in women.
BACKGROUND OF THE INVENTION
Fertility problems have manifested increasingly in western countries due to a variety of social causes. For example, young couples delay their decision to establish a family for reasons of education and career, and tend to plan for a smaller family size due to the increasing priority placed upon quality of life. Environmental and lifestyle factors may also play a part in revealing an underlying subfertility due primarily to other causes. For many couples, prolonged attempts to become pregnant end in failure, resulting in their seeking assistance from an infertility clinic. This decision is often taken long past the time when they initially would have hoped to start a family. People are understandably reluctant to take a step that may seem humiliating. Such delays, together with the attendant stress, anguish and shame, can have a profoundly destructive effect on relationships and on quality of life generally.
Infertility can result from a great variety of causes, including anatomical, developmental, infectious and toxicological factors. The majority of cases can be attributed wholly or predominantly to one or other partner, with roughly equal numbers of cases of male and female factor infertility. In both instances, however, the primary cause is almost certainly genetic. Estimates of the true population prevalence of infertility vary, but are generally accepted to be in the range of 2 to 5%. This is, therefore, one of the commonest human genetic disorders.
Male infertility can manifest as reduced quantity (oligozoospermia) or total lack (azoospermia) of sperm, reduced quantity of motile sperm (asthenozoospermia) or morphologically abnormal sperm (teratozoospermia). These categories are not exclusive, since many infertile males appear to fall into more than one such class. Microscopic, cytological and biochemical methods are presently available for the determination of these defects. However, actual measurements can be highly variable, even for a single individual between samplings. Frequently, moreover, no actual cause for male infertility can be found. This can cause additional costs arising from the need for further clinical examinations. Selecting an appropriate method of assisted reproduction is often a haphazard and costly matter, with no guarantee of eventual success. All these difficulties and delays can place severe, additional strains on relationships. Additional means for the early detection or prediction of infertility, in particular male infertility are thus needed. Early detection of male infertility would save both money and human stress and would allow appropriate counselling or assisted reproduction to be offered to a significant population of individuals who would otherwise only discover their problem after it has caused severe damage to their quality of life.
Though it has been long suspected that male infertility in most cases is a genetic disorder, and furthermore that it is genetically heterogeneous (i.e. that many different genes are the underlying cause of the disorder in different individuals), little information has emerged relating specific genetic defects and male infertility. The two known genetic causes of male infertility are the ‘cystic fibrosis’ mutations in the CFTR gene, part of a clearly recognizable clinical syndrome affecting approximately one person in 1500 [see, e.g. Lissens, W. and Liebaers, I. Baillieres Clinical Obstetrics and Gynaecology 11 (1997) 797-817; Schnedl, W. et al., Wiener Klinische Wochenschrift 103 (1991) 29-33] and a deletion on the Y chromosome, that leads to complete azoospermia, and is found in approximately 1 to 2% of infertile males in this category [Elliott, D. J. and Cooke, H. J. BioEssays 19 (1997) 801-809]. The supposed genetic cause of male infertility in the vast majority of cases has thus far remained unknown.
Since spermatozoa are heavily dependent on respiratory energy for motility, impaired energy metabolism, whether in mature spermatozoa or at earlier stages of male germ cell differentiation, is a long hypothesized mechanism contributing to infertility. Defects in mitochondrial function [Johns, J. C. S. et al., Nat Med 3 (1997) 124-125], possibly associated with mtDNA lesions [Kao, S. H. et al., Mol Hum Reproduction 4 (1998) 657-666; Lestienne P. et al., Mol Hum Reproduction 3 (1997) 811-814], have been reported in sperm samples from infertile males, and sperm motility appears to be correlated with mitochondrial respiratory activity [Ruiz Pesini, E. et al., Clin Chem 44 (1998]. 1616-1620] and membrane potential [Troiano. L. et al., Exp Cell Res 241 (1998) 384-393]. However, no relationship between a specific mitochondrial gene and male infertility has been disclosed or even suggested.
SHORT DESCRIPTION OF THE INVENTION
It has now, surprisingly, been discovered that the frequency of a mutant genotype in the POLG gene encoding the catalytic subunit of mito-chondrial DNA polymerase (DNA polymerase &ggr;) is significantly increased in groups of infertile males. Specifically, the mutant genotype has been located in the area of the trinucleotide (CAG) microsatellite repeat of the POLG gene within the N-terminal region of the coding sequence. While the normal POLG gene contains 10 consecutive, glutamine encoding CAG codons, followed by a single CAA and two further CAGs, at least one allele with an altered CAG repeat-length in the POLG gene is found in a large population of infertile men.
The POLG mutations described in the present application represent the first significant step in efforts of elucidating the genetic nature of male infertility and provide novel means of diagnosis thereof.
An object of the invention is thus to provide a non-invasive, non-intrusive diagnostic method that is useful in identifying, detecting and characterizing male infertility in the large fraction of cases where its causes remain unsolved.
A second object is to provide a simple screening test of strong predictive value, in order to identify in advance those individuals who will require reproductive counselling and assistance. Such information will greatly enhance the expected quality of life of those who suffer from this disorder.
The present invention relates to a new method for the diagnosis of male infertility by detecting the presence or absence of a mutation or mutations in the POLG gene encoding the catalytic subunit of mitochondrial DNA polymerase in a biological sample.
The present invention also relates to the use of a mutant form of the POLG gene encoding the catalytic subunit of mitochondrial DNA polymerase for the diagnosis of male infertility.
The present invention also relates to the use of a mutant form of the POLG gene encoding the catalytic subunit of mitochondrial DNA polymerase as a diagnostic agent.
The present invention further relates to diagnostic kits containing suitable reagents to detect a mutant form of the POLG gene or the normal POLG gene.
The present invention further relates to the use of the POLG gene as an indicator of other pathological conditions associated with or related to male infertility, including those manifesting in women.
REFERENCES:
Rovio et al. Euro. Jrnl. of HUman Genetics (1999) 7, 140-146.*
Ropp and Copeland Genomics (1996) 36, 449-458.*
Rovia A et al: “Analysis of the trin
Jacobs Howard
Rovio Anja
Licentia Oy
Myers Carla J.
Sakelaris Sally
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