The protective variant rs7173049 at LOXL1 locus impacts on retinoic acid signaling pathway in pseudoexfoliation syndrome.

LOXL1 (lysyl oxidase-like 1) has been identified as the major effect locus in pseudoexfoliation (PEX) syndrome, a fibrotic disorder of the extracellular matrix and frequent cause of chronic open-angle glaucoma. However, all known PEX-associated common variants show allele effect reversal in populations of different ancestry, casting doubt on their biological significance. Based on extensive LOXL1 deep sequencing, we report here the identification of a common non-coding sequence variant, rs7173049A>G, located downstream of LOXL1, consistently associated with a decrease in PEX risk (odds ratio, OR = 0.63; P = 6.33 × 10-31) in nine different ethnic populations. We provide experimental evidence for a functional enhancer-like regulatory activity of the genomic region surrounding rs7173049 influencing expression levels of ISLR2 (immunoglobulin superfamily containing leucine-rich repeat protein 2) and STRA6 [stimulated by retinoic acid (RA) receptor 6], apparently mediated by allele-specific binding of the transcription factor thyroid hormone receptor beta. We further show that the protective rs7173049-G allele correlates with increased tissue expression levels of ISLR2 and STRA6 and that both genes are significantly downregulated in tissues of PEX patients together with other key components of the STRA6 receptor-driven RA signaling pathway. siRNA-mediated downregulation of RA signaling induces upregulation of LOXL1 and PEX-associated matrix genes in PEX-relevant cell types. These data indicate that dysregulation of STRA6 and impaired retinoid metabolism are involved in the pathophysiology of PEX syndrome and that the variant rs7173049-G, which represents the first common variant at the broad LOXL1 locus without allele effect reversal, mediates a protective effect through upregulation of STRA6 in ocular tissues.

R831X mutation of the androgen receptor gene in an adolescent with complete androgen insensitivity syndrome and bilateral testicular hamartomata.

An 18-year old, phenotypically female individual was examined for primary amenorrhea. Three months before her referral, the patient underwent surgery and a pelvic mass was removed. The physical examination revealed normal female external genitalia, normal breast development, sparse pubic hair and absence of axillary hair. The gynecological examination revealed a short blind vagina pouch and absence of cervix and uterus. Serum testosterone and dihydrotestosterone levels were very high. Karyotype was that of a normal male (46,XY). The transabdominal ultrasound, computed tomography (CT) and Magnetic resonance imaging (MRI) showed absence of uterus and fallopian tubes and revealed testis-like gonads located at the internal opening of the inguinal canal bilaterally. Bilateral gonadectomy was subsequently performed. The pathology report was that of "hamartomatous testes" and associated paratesticular leiomyoma. The clinical, laboratory, imaging, genetic and histological findings confirmed the diagnosis of complete androgen insensitivity syndrome. DNA analysis revealed a R831X mutation in exon 7 of the androgen receptor gene. A Sertoli-cell dynamic test showed elevated basal serum inhibin-B and anti-Müllerian hormone levels without further rise following FSH stimulation. The patient was started on hormone replacement therapy with conjugated estrogens. Complete androgen insensitivity syndrome must be considered in any case of primary amenorrhea. Gonadectomy must be planned to eliminate the risk of gonadal malignancy.

Molecular and Clinical Investigation of Cystinuria in the Greek-Cypriot Population.

BACKGROUND AND AIMS: Cystinuria represents 3% of nephrolithiasis in humans. Two genes have been identified as the main genetic causes of cystinuria, SLC3A1 and SLC7A9, with an autosomal recessive mode of inheritance. In the present study, we studied for the first time, genetically and clinically, all the cystinuric families identified so far in the Greek-Cypriot population. METHODS: Discovery of mutations was performed through polymerase chain reaction (PCR)-single analysis and DNA resequencing. New families were investigated through PCR-RFLPs. Clinical data were collected through the hospital patients' records and analytical follow-up of the families. RESULTS AND DISCUSSION: We found a total of five mutations in 28 Greek-Cypriot cystinuric patients belonging in 12 families. The most frequent mutation among the 28 Greek-Cypriot patients is the SLC3A1-p.T216M, which is also the second most frequent mutation in Europe, representing a genetic founder effect. Sixteen of the 28 patients are homozygous for this mutation. Even though a consanguinity loop was obvious in only one family, other patients were from families in small villages where endogamy was practiced for many centuries. Timely clinical and genetic diagnosis, accompanied by early treatment, is significant for the good health of most of our patients. Only ∼14% of them developed chronic renal failure, and only one reached end-stage renal disease (ESRD). CONCLUSION: Five SLC3A1 and SLC7A9 mutations appear to be responsible for the genetic basis of cystinuria in the Greek-Cypriot patients; having such a limited number of causative mutations will simplify diagnostics for this population.

Association of the (TAAAA)n repeat polymorphism in the sex hormone-binding globulin (SHBG) gene with polycystic ovary syndrome and relation to SHBG serum levels.

SHBG levels are frequently low in women with polycystic ovary syndrome (PCOS) and may contribute to increased tissue exposure to free androgens. A (TAAAA)n repeat polymorphism in the promoter of the SHBG gene has been described recently, and its transcriptional activity has been shown to be related to the number of tandem repeats. Recent evidence also suggests that prenatal exposure to androgen excess may program for the development of the PCOS phenotype during adulthood. Our aim was to investigate the possible association of the functional (TAAAA)n polymorphism in the promoter of the SHBG gene with PCOS and its relation to SHBG levels. We studied 185 women with PCOS and 324 normal controls. Genotype analysis revealed six (TAAAA)n alleles containing 6-11 repeats. The distribution of these alleles was different in the two groups. Women with PCOS had a significantly greater frequency of longer (TAAAA)n alleles (more than eight repeats) than normal women who had shorter alleles (less than eight repeats) in higher frequency (P = 0.001). Furthermore, in the PCOS group, carriers of the longer allele genotypes had lower SHBG levels [1.17 +/- 0.68 micro g/dl (35.1 +/- 20.5 nmol/liter)] than those with shorter alleles [1.51 +/- 0.93 microg/dl (45.3 +/- 28 nmol/liter P = 0.02). A novel (TAAAA)n allele, which has not been previously reported, was found in low frequency, mainly in the control population. From these results, there is evidence that there may be a genetic contribution to decreased SHBG levels frequently seen in women with PCOS. The SHBG gene may act as a susceptibility gene for PCOS and may provide the genetic link for the developmental origin hypothesis for PCOS that was recently proposed on the basis of experimental observation in prenatally androgenized sheep and primates.

Polymerase chain reaction (PCR) and sequence specific oligonucleotide probes (SSOP) genotyping assay for detection of genes associated with rheumatoid arthritis and multiple sclerosis.

In this paper an assay for the detection of genes associated with rheumatoid arthritis (RA) and multiple sclerosis, using polymerase chain reaction (PCR) and sequence specific oligonucleotide probes (SSOP) is presented, in order to be further applied in a portable Lab-On-Chip (LOC) device. A substantial part of these reagents were based on the literature (11th International Histocompatibility Workshop, IHW), bearing the advantage of proven successful implementation in genotyping, while others were designed for this study. More precisely, our methodology discriminates HLA-DRB1 as DRB1*01, *04 and *10, which include shared epitope (SE) alleles associated with RA and additionally DRB1*15 allele, including DRB1*1501 associated with MS (broad genotyping method). To further present the basic elements of the assay for high resolution genotyping of SE DRB1 alleles, we provide as an example the case of HLA-DRB1*10 alleles (HLADRB1* 100101, *100102, *100103, *1002 and *1003). Regarding the methodology for developing a detection assay, for SNPs associated with RA or MS the basic steps are presented. DNA sequence data are obtained from IMGT/HLA and SNP database. Online software tools are used to define hybridization specificity of primers and probes towards human DNA, leading to hybridization patterns that uniquely designate a target allele and evaluate parameters influencing PCR efficiency. Respecting current technological limitations of autonomous molecular-based LOC systems the approach of broad genotyping of HLA-DRB1*01/*04/*10/*15 genes, is intended to be initially used, leaving, high resolution genotyping of SE alleles for future implementations. This method is easy to be updated and extended to detect additional associated loci with RA or MS.