Autism is a neurodevelopmental disorder with an estimated prevalence of 1 in 150 children which makes it more common than childhood cancer and juvenile diabetes. It is estimated that there are more than 100,000 individuals affected by autism in Canada and tens of millions worldwide. It is well established that genetic factors play important role in the pathophysiology of autism; still, our current understanding of these genetic factors is limited and cause of autism remains an important question. During the past decade, after completion of human genome, several new high throughput genome scan technologies have been developed such as microarrays. In the present study, we undertook the challenge of identifying X-chromosomal genes involved in autism by performing genome-wide copy number variation analysis of more than 400 probands with autism using Affymetrix 500K single nucleotide polymorphism (SNP) microarrays. We identified copy number variants implicating several genes on the chromosome X such as PTCHD1, IL1RAPL1, IL1RAPL2 and TSPAN7 as autism candidate genes. We also demonstrated that autism and intellectual disability may share some of these genes as etiologic factors. We performed a comprehensive analysis of PTCHD1 locus and showed that mutations at this locus are associated with autism in ~1 % of the cases. This study also demonstrated that PTCHD1 mutations can cause intellectually disability with or without autism, and that the PTCHD1 protein may act as a receptor in Hedgehog signaling pathway. We have also carried out a detailed analysis of TSPAN7 and IL1RAPL1 to explore the contributions of these genes in autism. We identified one family with intronic deletion of IL1RAPL1 and another case with a missense mutation in this gene, thus implicating this known intellectual disability gene in autism. Our findings highlight the importance of the X chromosome in the etiology of autism, and demonstrate the power of copy number variation analysis coupled with other technologies in identification of disease genes, in particular for complex genetic disorders such as autism.

Autism is a neurodevelopmental disorder with an estimated prevalence of 1 in 150 children which makes it more common than childhood cancer and juvenile diabetes. It is estimated that there are more than 100,000 individuals affected by autism in Canada and tens of millions worldwide. It is well established that genetic factors play important role in the pathophysiology of autism; still, our current understanding of these genetic factors is limited and cause of autism remains an important question. During the past decade, after completion of human genome, several new high throughput genome scan technologies have been developed such as microarrays. In the present study, we undertook the challenge of identifying X-chromosomal genes involved in autism by performing genome-wide copy number variation analysis of more than 400 probands with autism using Affymetrix 500K single nucleotide polymorphism (SNP) microarrays. We identified copy number variants implicating several genes on the chromosome X such as PTCHD1, IL1RAPL1, IL1RAPL2 and TSPAN7 as autism candidate genes. We also demonstrated that autism and intellectual disability may share some of these genes as etiologic factors. We performed a comprehensive analysis of PTCHD1 locus and showed that mutations at this locus are associated with autism in ~1 % of the cases. This study also demonstrated that PTCHD1 mutations can cause intellectually disability with or without autism, and that the PTCHD1 protein may act as a receptor in Hedgehog signaling pathway. We have also carried out a detailed analysis of TSPAN7 and IL1RAPL1 to explore the contributions of these genes in autism. We identified one family with intronic deletion of IL1RAPL1 and another case with a missense mutation in this gene, thus implicating this known intellectual disability gene in autism. Our findings highlight the importance of the X chromosome in the etiology of autism, and demonstrate the power of copy number variation analysis coupled with other technologies in identification of disease genes, in particular for complex genetic disorders such as autism.

No description available.

Three point mutations have been found in the hormone-binding domain (HBD) of the human androgen receptor (hAR): one in the N-terminal end (Ile663Asn in a family with partial androgen insensitivity syndrome (PAIS)); one in the middle, (Leu820Val in a family with PAIS); and one in the C-terminal end (Pro903Ser, in a family with complete AIS). The positions 663 and 903 were the most terminal mutation sites in the HBD found to date. The three mutant hARs have been previously characterized biochemically in genital skin fibroblasts. In the family with the Leu820Val substitution, the mother and the grandmother were found to be carriers for the same mutation. To prove their pathogenicity, each of the three mutations has been reproduced in an hAR expression vector that was transfected into COS-1 cells. In COS-1 cells, the complexes from Pro903Ser and Leu820Val had: increased thermolability; increased dissociation rates; decreased affinity; and abnormal transactivation. There was a hierarchy in the severity of the mutations expressed in kinetic and transactivation assays that correlated with the severity of the clinical phenotype. The pathogenicity of the Pro903Ser and the Leu820Val mutations was thereby confirmed. In COS-1 cells, the AR with Ile663Asn had normal thermolability, normal dissociation rates, and normal transactivation, but a decreased affinity. Although this sequence alteration has only been found in a PAIS patient, its pathogenicity is not considered to be proven. More sensitive assays are needed for this purpose.

The human androgen receptor (hAR) is a ligand-activated, nuclear transcription factor. Mutations affecting the formation and/or action of the hAR cause androgen insensitivity syndrome (AIS). The majority of mutations identified to date are within the DNA- and hormone-binding domains; very few have been identified in the transactivational modulatory domain, encoded by exon 1. This work presents an analysis of exon 1 and the 5$ sp prime$-flanking region of the hAR in a set of subjects whose AIS was believed to be caused by a mutation within these regions. Six of twelve strains had a nonsense or frameshift mutation in exon 1; a seventh strain had two missense and one silent substitution; no mutations were identified in the remaining subjects. The two missense mutations were recreated, individually and together, in an hAR complementary DNA (cDNA) expression vector and expressed in heterologous COS-1 cells. Their pathogenicity could not be proven with the system and assays used. In addition, mRNA and protein levels were analyzed and correlated with the identified mutations and the subjects' phenotype.

The human androgen receptor (hAR) is a ligand-activated transcription factor, and like other nuclear receptors, consists of a N-terminal modulatory domain, a central DNA-binding domain, and a C-terminal ligand-binding domain (LBD). Several missense mutations in the LBD cause androgen insensitivity syndrome (AI), a condition in XY individuals with absent or subnormal male primary and secondary sexual characteristics. On the other hand, abnormal expansion of a polyglutamine tract in the N-terminal domain of the hAR causes spinal and bulbar muscular atrophy (SBMA) which also affects males and causes milder forms of AI, in addition to adult-onset motor neuron degeneration and gradual wasting and weakening of the muscles of the limbs, face, throat, and tongue. However, it was not clear how and to what extent these mutations contribute to the clinical phenotype of the affected individuals. In order to investigate this matter, I used PCR site-directed mutagenesis to create plasmids expressing hARs with two pairs of missense mutations in the LBD (Val865Leu and Val865Met, and Arg839His and Arg839Cys), discovered in AI individuals with varying severity of the phenotype, and two abnormal expansions of the polyglutamine repeat discovered in SBMA patients (40 and 50 glutamines). I also synthesized plasmids expressing no glutamines (0 glutamines), 12 glutamines, or 20 glutamines in the same N-terminal region of the hAR. These plasmids were transiently expressed in heterologous cells (COS-1 and PC-3), and the mutant hARs were assayed for ligand binding, stability, and transactivational capacity. / In contrast to the findings by others (McPhaul et al., 1992; Marcelli et al., 1994), in some instances involving identical mutations, I consistently observed a correlation between the biochemical phenotype of the mutant hARs and the clinical phenotype of AI individuals; that is, the more severe receptor phenotype was associated with the more severe AI. These results support the hypothesis that hAR phenotype is the dominant factor in the development of the secondary sexual characteristics in normal and affected individuals. / I also observed a tight negative correlation between polyglutamine tract length and transactivational capacity. This suggests that polyglutamine modulates the activity of the hAR, and that hAR activity might be suppressed in various androgen-sensitive tissues (including motor neurons) in SBMA individuals, thereby contributing to the age of onset and/or progression of the disease, even if it cannot be the primary pathogenic agent of the disease.

The major objective of this thesis was to determine the molecular basis of a "ligand-selective" mutant androgen receptor (AR) phenotype. Methyltrienelone (MT), a synthetic androgen, dissociates normally from this receptor but mibolerone (MB), another synthetic androgen, dissociates from it two-fold faster than normal. This mutant receptor was identified within genital skin fibroblasts (GSF) from two unrelated individuals with different degrees of androgen insensitivity (AI). Sequence analysis of the AR gene from both subjects revealed a G to A transition at nt 2969 in exon 6 that alters codon 813 from serine to asparagine (S813N). Transiently expressed hAR.S813N did not reproduce the mutant phenotype in several heterologous cells: COS-1, BHK, CHO or HeLa cells. In contrast, when AR free (R$ sp-$) GSF were used as host cells, MB-R.S813N complexes dissociated almost two fold faster than the controls (n = 4) while MT-R.S813N complexes dissociated normally. These results establish the G to A transition at nt 2969 as the cause of the ligand-selective phenotype. Such host-cell restricted expression of the mutant dissociation rate points to cell-specific factors that can suppress abnormal dissociation of A-R complexes. Host cell-restricted expression of the abnormal dissociation rates has also been observed for two other transiently expressed mutant AR, hAR.V865L and hAR.R839H (n = 3). / Expansion of the glutamine (gln) tract within the N-terminus of the AR causes spinal bulbar muscular atrophy (SBMA), a disease of motor neurons, but the mechanism of this neuropathology is unknown. To determine the effect of gln-tract expansion upon AR function, SBMA-associated mutant AR was transiently expressed and characterized in COS-1 cells. The androgen-binding parameters of the mutant receptor were normal, but it had decreased transactivation competence (50-66% of normal; n = 3). This abnormal transregulatory function may account for the expression of traits associated with minimal androgen insensitivity (MAI) that are variably expressed in the SBMA patients.

Genomic clones were isolated and characterized using the human monoamine oxidase A (MAOA) cDNA to screen a phage library, constructed from a human 4X cell line (48, XXXX). The genomic contig derived from overlapping phage clones showed that the size of the MAOA gene is over 80 kb. Exon-containing fragments from these phage clones were subcloned and sequenced. The data from this showed that the MAOA gene consists of 15 exons. A YAC (yeast artificial chromosome) isolated using the MAOA cDNA was characterized. This YAC was found to contain both the MAOA and the MAOB genes. Using PFGE (pulsed-field gel electrophoresis) to investigate the YAC, it was found that the MAOA and the MAOB genes are located within 50 kb and adjacent to each other. The two genes are localized in a 3'-to-3' fashion, suggesting their expression may be regulated independently. The analysis of the homology shown by the two genes clearly demonstrated that they were derived from duplication of a common ancestral gene. A CpG island was discovered to be associated with the 5' end of both genes. A restriction map of -2.5 Mb of genomic DMA around the MAO genes was generated by PFGE. Long-range mapping defined the physical relationship between the marker L1.28 and the MAO genes as L1.28_MAOA_MAOB. A number of genetic diseases have been linked to the Xp11.3 region. Strong linkage was known to exist between the Norrie disease locus and L1.28. Studies showed that some of the Norrie patients have deletions encompassing the region which contains L1.28 as well as the MAO genes. Another YAC isolated by using L1.28 as the probe was also characterized. A phage library was constructed from the L1.28 YAC and the end clones were isolated. Studies on some of the Norrie deletion patients showed that the proximal end clone of the YAC was retained in one of the deletion patients. Previous studies had shown that the Norrie disease locus was also localized proximal to the 5' end of the MAOB gene. The combined information placed the disease locus to an interval of 240 kb within the YAC. More phage clones were characterized in order to define further the region for the Norrie locus which was finally localized within 160 kb. A YAC fragment of 160 kb was isolated and used to screen two human retinal cDNA libraries. Among the cDNAs isolated, one group was found to be deleted in some of the Norrie patients previously without any known deletion, which established their candidacy as the transcripts of the Norrie disease locus. Further characterization of the candidate gene showed that it is conserved across species. The expression of the gene was detected in various tissues. The homology shared between the NDP gene and some of the growth factor binding proteins suggests its role in neural cell proliferation and differentiation.

Two previously identified sequence alterations in the androgen receptor gene of patients with complete androgen resistance are studied to prove their pathogenicity. Family studies confirm that the mutation segregates with the phenotype and that the mothers are heterozygous carriers. In one family a sibling of the patient is identified as a heterozygous carrier. Mutant cDNAs encoding the mutant receptors are constructed and expressed in COS-1 cells. The resulting mutant receptors show a decreased apparent equilibrium constant for androgens, faster dissociation rates and impaired transactivation. Further studies reveal that both mutant receptors were either inactivated or destroyed in the presence of hormone, while the normal receptor is stabilized and up-regulated by incubation with ligand. These results prove that the sequence alterations thus identified are pathogenic and illustrate a dual mechanism of pathogenicity: an affinity defect combined with a loss of binding activity in the presence of hormone, resulting in receptors incapable of supporting normal male sexual development.

This work proves the pathogenicity of four substitution mutations in the androgen-binding domain of the human androgen receptor (hAR) gene of four subjects with varying degrees of androgen insensitivity syndrome (AIS): complete (CAIS), partial (PAIS), or mild (MAIS). Of three unrelated CAIS subjects, two have Arg830Leu, the third has Arg830Gln. Their genital skin fibroblasts (GSF) have negligible androgen binding, but in overexpressing transfectants, the mutant androgen-binding activities have increased dissociation rates and decreased affinity for androgen. Owing to the instability of AR-androgen complexes, both mutants fail to transactivate a reporter gene. Glu771Ala and Arg870Gly caused PAIS and MAIS, respectively. Their normal levels of GSF androgen-binding activity have normal androgen affinity but increased dissociation rates. In transfectants, rates of dissociation resemble those in GSF, but the androgen affinities are questionably abnormal. Instability of Glu771Ala and Arg870Gly AR-androgen complexes caused subnormal transactivation of a reporter gene.