Molecular screening of genes mapped in POF loci

Abstract

Objective: Premature ovarian failure (POF) - amenorrhea with elevated gonadotropins occurring before the age of 40 - with normal karyotype may be secondary to Xq chromosome defect. Two specific regions on Xq have been defined as POF loci: POF1 Xq26-qter and POF2 Xq13-Xq22 (Am J Med Genet; 1999, 89: 186–200) The POF1 segment, the QM or RPL10 (Ribosomal protein L10) gene, which maps to Xq28, is a novel gene that belongs to a class of transcription regulatory proteins and is highly conserved throughout eukaryotic evolution. There is strong evidence in Drosophila and suggestive evidence in humans that deficiencies of ribosomal proteins may yield viable but abnormal phenotypes. The Minute phenotype from Drosophila (reduced body size and infertility) results from deficiencies at any one of the ribosomal loci spread across the genome (Genome Res; 1998, 8: 509–523). Its location and function makes the QM gene a candidate for ovarian failure mapped to that region. The only candidate gene that maps the POF2 segment is the human homologue of the Drosophila melanogaster diaphanous gene (DIA). Mutated alleles of this gene affect spermatogenesis and oogenesis in the fruit fly. A disruption, due to a breakpoint in the 3’ end of the DIA gene, mapped to Xq22, has been previously described in familial POF (Am J Hum Genet; 1998, 62: 533–541). To investigate whether QM and DIA genes are involved with oogenesis and infertility we analyze the expression of mRNA in human oocyte and screened for mutations in these genes are associated with POF. Design: In vitro experiment at Academic Medical Center. Materials/Methods: RNA was extracted from oocytes (n=14) and granulosa cells from IVF patients and blood samples from 27 POF patients and 100 controls were screened for mutations in the coding region. The RNA was extracted from samples for subsequent reverse transcriptase and polymerase chain reaction (RT-PCR). Amplified cDNA was analyzed by single-stranded conformational polymorphism (SSCP) and/or by DNA sequencing. Results: There was detectable QM mRNA in oocytes and granulosa cells. In one POF patient a missense mutation in the QM gene was disclosed. A TTG→TCG conversion at nucleotide 308 resulted in a serine at codon 103 in place of leucine, which represents a change in QM protein hydrophobicity. No mutations were found in the DIA gene, ruling out the last exon mutations in this gene as the cause of POF in these patients. Conclusions: According to these results it is tempting to speculate that QM gene might be involved on the genesis of oocyte development and infertility. Mutational analysis of this gene could be useful for diagnosis of infertility. Supported by: FAPESP 99/3366–7.