Androgen receptor YAC transgenic mice carrying CAG 45 alleles show trinucleotide repeat instability

X-linked spinal and bulbar muscular atrophy (SBMA) is caused by a CAG repeat expansion in the first exon of the androgen receptor (AR) gene. Disease-associated alleles (37-66 CAGs) change in length when transmitted from parents to offspring, with a significantly greater tendency to shift size when inherited paternally. As transgenic mice carrying human AR cDNAs with 45 and 66 CAG repeats do not display repeat instability, we attempted to model trinucleotide repeat instability by generating transgenic mice with yeast artificial chromosomes (YACs) carrying AR CAG repeat expansions in their genomic context. Studies of independent lines of AR YAC transgenic mice with CAG 45 alleles reveal intergenerational instability at an overall rate of approximately 10%. We also find that the 45 CAG repeat tracts are significantly more unstable with maternal transmission and as the transmitting mother ages. Of all the CAG/CTG repeat transgenic mice produced to date the AR YAC CAG 45 mice are unstable with the smallest trinucleotide repeat mutations, suggesting that the length threshold for repeat instability in the mouse may be lowered by including the appropriate flanking human DNA sequences. By sequence-tagged site content analysis and long range mapping we determined that one unstable transgenic line has integrated an approximately 70 kb segment of the AR locus due to fragmentation of the AR YAC. Identification of the cis - acting elements that permit CAG tract instability and the trans -acting factors that modulate repeat instability in the AR YAC CAG 45 mice may provide insights into the molecular basis of trinucleotide repeat instability in humans.      

Androgen receptor CAG polymorphism (Xq11-12) status and human spermatogenesis: a prospective analysis of infertile males and their offspring conceived by intracytoplasmic sperm injection

We determined the association of androgen receptor (AR) (CAG)n lengths among fertile and infertile males and offspring conceived by intracytoplasmic sperm injection (ICSI). Assessment of (CAG)n repeats in the AR was performed in a Caucasian population by gene sequencing in fertile men (n=13), infertile men (n=64), boys conceived after ICSI (n=21), and boys conceived naturally (n=11). In the AZF region of the Y chromosome, a total of 22 STSs were analyzed by multiplex PCR; selected spermatozoa were also analyzed by fluorescent in situ hybridization (FISH) for chromosomes 18, 21, X, and Y. The average age was 43.7+/-7 yr for infertile, 44.8+/-7 yr for fertile men, and 5.0+/-0.5 yr for the children. The mean (CAG)n was 22.2+/-3 for the infertile men and 19.3+/-5 for fertile controls. There was a significant difference in CAG repeat length in the severely oligo-/azoo-spermic men vs. controls (p=0.02). An inverse correlation was evident between CAG length and semen parameters. For ICSI male children, the AR (CAG)n lengths were 21.4+/-3.2 vs. 20.8+/-3.4 for boys conceived naturally. While all peripheral karyotypes of fertile and infertile men were normal, de novo gonosomal abnormalities were observed in the ICSI offspring. The incidence of Y microdeletions was 1.6% in infertile men; all the ICSI sons had an intact Y chromosome. In conclusion, severely oligo- and azoospermic men had longer CAG repeat length than fertile controls, suggesting that certain AR gene mutations may have a negative effect on spermatogenesis. An increased incidence of de novo gonosomal abnormalities was found in the ICSI offspring when compared to children conceived naturally. Our assessment of the polymorphic region of the AR gene, in the absence of other specific genomic abnormalities, suggests that the fertility of children conceived by ICSI may be conserved.     

The size of the CAG repeat in exon 1 of the androgen receptor gene shows no significant relationship to impaired spermatogenesis in an infertile Caucasoid sample of German origin

The androgen receptor (AR) gene, located on the X-chromosome at Xq11-12, contains in exon 1 a polymorphic CAG repeat which codes for a polyglutamine tract. Contractions of the CAG repeat are said to be related to prostate cancer. In contrast, sizeable expansion of the CAG repeat can cause spinal and bulbar muscular atrophy (SBMA). In infertile patients of Chinese origin and in a Melbourne multinational population impaired sperm production has been postulated to be related to moderate expansions of the polyglutamine tract. In a study of a Swedish population of infertile patients these findings could not be corroborated. The aim of our investigation was to examine the correlation between the length of the CAG repeat and impaired sperm production in an infertile Caucasoid patient sample of German ethnic origin. We found no statistically significant relationship between the size of the CAG repeat or polyglutamine tract and idiopathic impaired sperm production in the population studied. The variability of the results by various investigators may be attributed to different ethnic origins and hence different genetic modifiers of the populations studied and/or to the high probability that these infertile males may represent a heterogeneous group with respect to the causes of defective spermatogenesis.     

Mitotic and meiotic stability of the CAG repeat in the X-linked spinal and bulbar muscular atrophy gene

X-linked spinal and bulbar muscular atrophy (SBMA) occurs due to an expansion of the trinucleotide repeat (CAG)_n in the androgen receptor gene. Anticipation is relatively rare in SBMA in contrast to spinocerebellar ataxia type 1 (SCA1), and dentatorubral and pallidoluysian atrophy (DRPLA) which show obvious paternal anticipation. The differences in the CAG repeat number were compared among sperm, leukocytes and skeletal muscles of SBMA patients. In SBMA, the sperm of most patients and the skeletal muscle of all patients showed the same repeat number as their leukocytes, whereas the increase in the repeat number from leukocytes to sperm was evident in SCA1 and DRPLA patients. The higher mosaicism level in sperm compared with leukocytes was common in SBMA, SCA1 and DRPLA, and the level of sperm was lower in SBMA than in SCA1 and DRPLA. Thus, spermatogenesis was suggested to be strongly associated with paternal anticipation. The mosaicism level was smaller in SBMA than in other (CAG)_n expanded disorders, and smallest in the SBMA carrier females. These findings demonstrate that the CAG repeat in SBMA is relatively stable in mitotic and meiotic processes, and there is a possibility that the lower mosaicism level of the carrier females compared with the SBMA patients is associated with X-linked recessive inheritance.     

Genotyping of Israeli infertile men with idiopathic oligozoospermia

Microdeletions of the long arm of the Y chromosome involving the azoospermia factor (AZF) region are associated with severe oligo- or azoospermia. Abnormal androgen receptor (AR) structure or function has also been implicated in male infertility. To assess the contribution of these genetic defects to male infertility, 61 Israeli men with severe oligo- (n = 15) or azoospermia (n = 46), were screened for Y chromosome microdeletions, and the AR-(CAG)n repeat length. Fifty fertile Israeli men were similarly analyzed. PCR amplification of 20-54 simple tag sequences (STSs) located at Yq was used to determine the rate and extent of Y chromosome microdeletions. PCR with primers flanking the AR-(CAG)n region and subsequent size fractionation on gradient acrylamide gels were used to determine AR-(CAG)n length. Five azoospermic individuals (5/61-8.2% and 5/46-10.8% of azoospermic patients) displayed Y chromosome microdeletions. The mean CAG repeat number in infertile men was 18.6 +/- 3.0 compared with 16.6 + 2.7 in fertile men (n = 50), a statistically significant difference (p = 0.003). Y chromosome microdeletions contribute to male infertility in our azoospermic population, and the mean length of the AR-CAG is significantly longer in our infertile population than in fertile men.     

Somatic stability of the expanded CAG trinucleotide repeat in X-linked spinal and bulbar muscular atrophy

Expansion of trinucleotide repeats has now been associated with eight inherited diseases: X-linked spinal and bulbar muscular atrophy, two fragile X syndromes, myotonic dystrophy, Huntington's disease, spinocerebellar ataxia type I, dentatorubral pallidoluysian atrophy and Machado-Joseph disease. It has been shown that these expanded DNA repeats are unstable in number when transmitted from parents to offspring (“meiotic instability”), while somatic variation in repeat number has also been found in the fragile X syndrome and myotonic dystrophy. Moderate meiotic instability has been demonstrated in X-linked spinal and bulbar muscular atrophy (SBMA, Kennedy's disease). In order to determine if the expanded CAG repeat in SBMA also shows somatic instability, we compared different tissues from two patients with SBMA. We then examined the in vitro stability of the CAG repeat expansion by analyzing fibroblast cell cultures. Length comparison of expanded CAG repeats from all these materials clearly demonstrates that the CAG trinucleotide repeat in SBMA does not exhibit somatic variation. © 1996 Wiley-Liss, Inc.     

Transmission of male infertility to future generations: lessons from the Y chromosome

The introduction of ICSI and testicular sperm extraction (TESE) has allowed many infertile men to father children. The biggest concern about the wide use of these techniques is the health of the resulting offspring, in particular their fertility status. If the spermatogenic defect is genetic in origin, there is potential risk of transmitting this defect to future offspring. The most frequently documented genetic cause of male infertility is a Y chromosome deletion. The Y chromosome has acquired a large number of testis-specific genes during recent evolution, and deletions causing infertility take out a number of these genes. These deletions have been shown to be transmitted to 100% of male offspring. Also, absence of an aberration on the Y chromosome does not rule out a genetic cause of the infertility phenotype, as there are many other genes involved in spermatogenesis elsewhere in the genome, and current mapping techniques--especially on the Y chromosome--can miss many aberrations. More detailed studies of these spermatogenesis genes, which are now possible because of more precise sequence-based mapping, will lead to improved understanding of the genetic basis of male infertility and enable proper counselling of patients undergoing ICSI in the future.     

Clinical Aspects of CAG Repeat Diseases

Seven neurodegenerative disorders are known to be caused by unstable expansions of the trinucleotide CAG within human genes, and more will be discovered in the coming years. These disorders share some clinical similarities, as well as some differences, which are summarized here. These diseases have unusual clinical genetic properties related to the dynamic nature of CAG repeat expansions, including instability of the repeat expansion in meiosis, particularly male meiosis; a strong correlation between onset age and size of the repeat expansion; anticipation (earlier disease onset in succeeding generations); new mutations arising from unstable, mutable alleles with a high‐normal CAG repeat number; and reduced penetrance for alleles in the lowaffected range. Much more remains to be learned about the molecular biology and clinical pathophysiology of this new class of genetic diseases.SummaryIn the last six years, seven neurodegenerative diseases have been found to be caused by expansions of intragenic CAG repeat sequences. The diseases share a variable (usually adult) age of onset, which is highly dependent on the length of the CAG repeat, and effects on multiple systems within the central and peripheral nervous systems. The cerebral cortex is not a primary site of pathology for any of the diseases, and organs other than the nervous system are not primarily affected (except for SBMA). The diseases differ in their primary site of neuropathology, and for that reason have widely varying neurologic profiles. The distributions of normal and abnormal CAG repeat sizes vary among the diseases, and suggest that different mechanisms of mutagenesis or disease pathogenesis could exist for the different disorders.The dynamic nature of trinucleotide repeat mutations has clarified a number of clinical and genetic observations in these diseases. New mutations arising from mutable normal alleles have been reported for some of the diseases. The tendency to further expansion of an expanded allele provides a molecular correlate to the clinical observation of anticipation (171). Sex‐ and disease‐dependent meiotic instability correlates with the observation of a paternal bias among juvenile onset cases for HD, SCA1 and DRPLA. Finally, reduced penetrance for alleles at the low end of the abnormal range has been observed for some (but not all) diseases in the group. Detection of CAG repeat expansions is relatively easy and inexpensive in the clinical laboratory, and molecular diagnosis has greatly improved diagnostic accuracy for this group of disorders. However, a full understanding of the biology and pathophysiology of this new class of mutations is still to come, and is awaited eagerly by clinicians and patients alike.     

Expansions of CAG repeat tracts are frequent in a yeast mutant defective in Okazaki fragment maturation

To understand the causes of CAG repeat tract changes that occur in the passage of human disease alleles, we are studying the effect of replication and repair mutations on CAG repeat tracts embedded in a yeast chromosome. In this report, we examine the effect of a mutation in the RTH1/RAD27 gene encoding a deoxyribonuclease needed for removal of excess nucleotides at the 5'-end of Okazaki fragments. Deletion of the RTH1/RAD27 gene has two effects on CAG tracts. First, the rth1/rad27 mutation destabilizes CAG tracts. Second, although most tract length changes in wild-type yeast cells are tract contractions, approximately half of the changes that occur as a result of the rth1/rad27 mutation are expansions of one or more repeat units. These results support the hypothesis that tract expansions that occur during passage of human disease alleles bearing expanded CAG tracts result from excess DNA synthesis on the lagging strand of replication.      

Genetic follow-up of male offspring born by ICSI,using a multiplex fluorescent PCR-based test for Yq deletions

De-novo deletions involving AZFa, b, c and d are one of the most common chromosomal aberrations in man resulting in defective spermatogenesis and male infertility. Currently, Yq deletion screening involves either single or multiplex PCR using Y-specific sequence tagged site markers and the subsequent analysis of the amplification products on ethidium bromide-stained agarose gels. To improve the practicality of routine and high throughput Yq testing, we have developed a more sensitive multiplex fluorescent (FL)-PCR screening system using genomic DNA extracted from cheek buccal cells as a readily available PCR template. For genetic follow-up studies of ICSI-conceived children, we also developed a DNA fingerprinting system based on the co-amplification of four highly polymorphic markers to validate family samples and detect any potential extraneous DNA contamination that could cause a misdiagnosis. Multiplex FL-PCR analysis of buccal cell DNA from two infertile men who conceived three sons by ICSI demonstrated that their Yq deletions were vertically transmitted. Fine mapping with additional Yq markers revealed identical deletion endpoints involving the loss of AZFdc sequences. This firstly indicates that the extent of the Yq deletion was unchanged on ICSI transmission and secondly supports the view that AZFdc deletions may arise by a common de-novo event. Analysis of paternal, maternal and sibling DNA fingerprints showed the co-inheritance of parental alleles by each male child and confirmed the expected relationship between each family member. The application of these new FL-PCR based screening tests in genetic follow-up studies will assist in confirming transmission of specific genetic defects to male offspring conceived by ICSI and provide a basis for genetic counselling and potential treatment options as these boys approach sexual maturity.     

Is the CAG repeat of mitochondrial DNA polymerase gamma (POLG) associated with male infertility? A multi-centre French study

BACKGROUND: Recent data emphasized the implication of polymerase gamma (POLG) CAG repeats in infertility, making it a very attractive gene for study. A comparison of POLG CAG repeats in infertile and fertile men showed a clear association between the absence of the usual 10-CAG allele and male infertility, excluding azoospermia. It has also been suggested that the POLG gene polymorphism should be considered as a possible contributing factor in unexplained couple infertility where semen parameters are normal. In this study, we investigated the POLG CAG repeats, in a well-defined population of patients with severe male factor infertility. METHODS: We conducted a large study of POLG CAG repeats in 433 infertile and 91 fertile, normozoospermic and healthy males. In all subjects, phenotypic data, including semen parameters, hormonal status and clinical profiles, were available. RESULTS: Thirteen 'homozygous mutants' (3%) were found among the 433 idiopathic infertile patients. The follow-up of the 13 'homozygous mutant' resulted in pregnancy for more than half of the couples, through assisted reproductive techniques or even spontaneously. In addition, one 'homozygous mutant' was identified in 91 fertile men (1.1%) CONCLUSION: Under our conditions, our study does not confirm any relationship between the polymorphic CAG repeat in the POLG gene and male infertility.     

CAG repeat length variation in sperm from a patient with Kennedy's disease

Using a modified sperm typing protocol, the mutation frequencyof the CAG repeat region at the androgen receptor locus hasbeen measured using a rare semen sample from an individual withspinal and bulbar muscular atrophy (SBMA). Among 258 X chromo-some-containingsperm, 19% had a repeat number equal to the donor's somaticDNA (47 repeats), 66% were expansions and 15% were contractions.The average expansion was 2.7 repeats. More than half of theexpansions involved one or two repeats; the largest was 11 repeats.68% of the contractions were also one or two repeats but six(16%) were very large (12–25 repeats). One contractiongenerated an allele in an intermediate size range (33–39repeats). Such alleles have not been observed among more than900 normal and SBMA X-chromosomes that have been examined. Comparisonof the SBMA sperm typing results with mutation frequency dataon normal alleles supports the hypothesis that trinucleotiderepeat expansions may have a different molecular origin thancontractions.     

A mouse model of spinal and bulbar muscular atrophy

Spinal and bulbar muscular atrophy (SBMA) is an adult-onset motor neuron disease, caused by the expansion of a trinucleotide repeat (TNR) in exon 1 of the androgen receptor (AR) gene. This disorder is characterized by degeneration of motor and sensory neurons, proximal muscular atrophy, and endocrine abnormalities, such as gynecomastia and reduced fertility. We describe the development of a transgenic model of SBMA expressing a full-length human AR (hAR) cDNA carrying 65 (AR(65)) or 120 CAG repeats (AR(120)), with widespread expression driven by the cytomegalovirus promoter. Mice carrying the AR(120) transgene displayed behavioral and motor dysfunction, while mice carrying 65 CAG repeats showed a mild phenotype. Progressive muscle weakness and atrophy was observed in AR(120) mice and was associated with the loss of alpha-motor neurons in the spinal cord. There was no evidence of neurodegeneration in other brain structures. Motor dysfunction was observed in both male and female animals, showing that in SBMA the polyglutamine repeat expansion causes a dominant gain-of-function mutation in the AR. The male mice displayed a progressive reduction in sperm production consistent with testis defects reported in human patients. These mice represent the first model to reproduce the key features of SBMA, making them a useful resource for characterizing disease progression, and for testing therapeutic strategies for both polyglutamine and motor neuron diseases.     

Loss of endogenous androgen receptor protein accelerates motor neuron degeneration and accentuates androgen insensitivity in a mouse model of X-linked spinal and bulbar muscular atrophy

X-linked spinal and bulbar muscular atrophy (SBMA; Kennedy's disease) is a polyglutamine (polyQ) disease in which the affected males suffer progressive motor neuron degeneration accompanied by signs of androgen insensitivity, such as gynecomastia and reduced fertility. SBMA is caused by CAG repeat expansions in the androgen receptor (AR) gene resulting in the production of AR protein with an extended glutamine tract. SBMA is one of nine polyQ diseases in which polyQ expansion is believed to impart a toxic gain-of-function effect upon the mutant protein, and initiate a cascade of events that culminate in neurodegeneration. However, whether loss of a disease protein's normal function concomitantly contributes to the neurodegeneration remains unanswered. To address this, we examined the role of normal AR function in SBMA by crossing a highly representative AR YAC transgenic mouse model with 100 glutamines (AR100) and a corresponding control (AR20) onto an AR null (testicular feminization; Tfm) background. Absence of endogenous AR protein in AR100Tfm mice had profound effects upon neuromuscular and endocrine-reproductive features of this SBMA mouse model, as AR100Tfm mice displayed accelerated neurodegeneration and severe androgen insensitivity in comparison to AR100 littermates. Reduction in size and number of androgen-sensitive motor neurons in the spinal cord of AR100Tfm mice underscored the importance of AR action for neuronal health and survival. Promoter-reporter assays confirmed that AR transactivation competence diminishes in a polyQ length-dependent fashion. Our studies indicate that SBMA disease pathogenesis, both in the nervous system and the periphery, involves two simultaneous pathways: gain-of-function misfolded protein toxicity and loss of normal protein function.     

Analysis of the dynamic mutation in the SCA7 gene shows marked parental effects on CAG repeat transmission

The gene for spinocerebellar ataxia 7 (SCA7) includes a transcribed, translated CAG tract that is expanded in SCA7 patients. We have determined expansions in 73 individuals from 17 SCA7 kindreds and compared them with repeat lengths of 180 unaffected individuals. Subjects with abnormal expansions comprise 59 clinically affected individuals and 14 at-risk currently unaffected individuals predicted to carry the mutation by haplotype analysis. For expanded alleles, CAG repeat length correlates with disease progression and severity and correlates inversely with age of onset. Increased repeat lengths are seen in generational transmission of the disease allele, consistent with the pattern of clinical anticipation seen in these kindreds. Repeat lengths in expanded alleles show somatic mosaicism in leukocyte DNA, suggesting that these alleles are unstable within individuals as well as between generations. Although dynamic repeat expansions from paternal transmissions are greater than those from maternal transmissions, maternal transmission of disease is more common, suggesting germline or embryonic effects of the repeat expansion.      

Spinal and bulbar muscular atrophy: ligand-dependent pathogenesis and therapeutic perspectives

Spinal and bulbar muscular atrophy (SBMA) is a late-onset motor neuron disease characterized by proximal muscle atrophy, weakness, contraction fasciculations, and bulbar involvement. SBMA exclusively affects males, while females are usually asymptomatic. The molecular basis of SBMA is the expansion of a trinucleotide CAG repeat, which encodes the polyglutamine (polyQ) tract in the first exon of the androgen receptor (AR) gene. The histopathological hallmark is the presence of nuclear inclusions containing mutant truncated ARs with expanded polyQ tracts in the residual motor neurons in the brainstem and spinal cord, as well as in some other visceral organs. The AR ligand, testosterone, accelerates AR dissociation from heat shock proteins and thus its nuclear translocation. Ligand-dependent nuclear accumulation of mutant ARs has been implicated in the pathogenesis of SBMA. Transgenic mice carrying the full-length human AR gene with an expanded polyQ tract demonstrate neuromuscular phenotypes, which are profound in males. Their SBMA-like phenotypes are rescued by castration, and aggravated by testosterone administration. Leuprorelin, an LHRH agonist that reduces testosterone release from the testis, inhibits nuclear accumulation of mutant ARs, resulting in the rescue of motor dysfunction in the male transgenic mice. However, flutamide, an androgen antagonist promoting nuclear translocation of the AR, yielded no therapeutic effect. The degradation and cleavage of the AR protein are also influenced by the ligand, contributing to the pathogenesis. Testosterone thus appears to be the key molecule in the pathogenesis of SBMA, as well as main therapeutic target of this disease.     

The androgen receptor gene mutations database (ARDB): 2004 update

The current version of the androgen receptor (AR) gene mutations database is described. The total number of reported mutations has risen from 374 to 605, and the number of AR-interacting proteins described has increased from 23 to 70, both over the past 3 years. A 3D model of the AR ligand-binding domain (AR LBD) has been added to give a better understanding of gene structure-function relationships. In addition, silent mutations have now been reported in both androgen insensitivity syndrome (AIS) and prostate cancer (CaP) cases. The database also now incorporates information on the exon 1 CAG repeat expansion disease, spinobulbar muscular atrophy (SBMA), as well as CAG repeat length variations associated with risk for female breast, uterine endometrial, colorectal, and prostate cancer, as well as for male infertility. The possible implications of somatic mutations, as opposed to germline mutations, in the development of future locus-specific mutation databases (LSDBs) is discussed. The database is available on the Internet (http://www.mcgill.ca/androgendb/).     

Multiple founder effects in spinal and bulbar muscular atrophy (SBMA, Kennedy disease) around the world.

SBMA (spinal and bulbar muscular atrophy), also called Kennedy disease, is an X-chromosomal recessive adult-onset neurodegenerative disorder caused by death of the spinal and bulbar motor neurones and dorsal root ganglia. Patients may also show signs of partial androgen insensitivity. SBMA is caused by a CAG repeat expansion in the first exon of the androgen receptor (AR) gene on the X-chromosome. Our previous study suggested that all the Nordic patients with SBMA originated from an ancient Nordic founder mutation, but the new intragenic SNP marker ARd12 revealed that the Danish patients derive their disease chromosome from another ancestor. In search of relationships between patients from different countries, we haplotyped altogether 123 SBMA families from different parts of the world for two intragenic markers and 16 microsatellites spanning 25 cM around the AR gene. The fact that different SBMA founder haplotypes were found in patients from around the world implies that the CAG repeat expansion mutation has not been a unique event. No expansion-prone haplotype could be detected. Trinucleotide diseases often show correlation between the repeat length and the severity and earlier onset of the disease. The longer the repeat, the more severe the symptoms are and the onset of the disease is earlier. A negative correlation between the CAG repeat length and the age of onset was found in the 95 SBMA patients with defined ages at onset.