Carole A. Sargent

Conservation of PCDHX in mammals; expression of human X/Y genes predominantly in brain

Abstract. Protocadherins are members of the cadherin superfamily involved in cell-cell interactions critical in the development of the central nervous system. This paper describes the isolation, sequence, and expression analysis of two novel protocadherin genes from the hominid specific Yp11.2/Xq21.3 block of homology between the sex chromosomes. The X-(PCDHX) and Y-linked (PCDHY) genes share 98.1% nucleotide and 98.3% amino acid identity and have an identical gene structure of six exons. The open reading frames of PCDHX and PCDHY encode proteins of 1025 and 1037 amino acids respectively and specify seven extracellular cadherin domains. Small differences in amino acid sequence affect regions that potentially have a large impact on function: thus, the X and Y genes may be differentiated in this respect. Sequence analysis of cDNA clones shows that both the X and Y loci are transcribed. RT-PCR expression analysis of mRNA from a variety of tissues and cell lines has demonstrated that both transcripts are expressed predominantly in the brain, with differential regional expression. From studies in the NTERA pluripotential cell line (which differentiates along neuronal and spermatogenic pathways in response to retinoic acid), it emerges that the X and Y-linked genes are regulated differently. This indicates that PCDHX and PCDHY possess different promoter regions. These findings suggest a role for PCDHX and PCDHY in the brain, consistent with the involvement of protocadherins in segmental brain morphogenesis and function. The implications of Y-linked genes expressed predominantly in tissues and organs other than the testis are considered within the context of the concept of sexual selection.

The critical region of overlap defining the AZFa male infertility interval of proximal Yq contains three transcribed sequences

The position of deletion breakpoints in a series of fourAZFa male infertility patients has been refined using new markers derived from BAC clone DNA sequence covering the AZFa male infertility interval. The proximal half of the AZFa interval is occupied by pseudogene sequences with homology to Xp22. The distal half contains an anonymous expressed sequence tag (named AZFaT1) found transcribed in brain, testis, and skeletal muscle and theDFFRY and DBYgenes. All the patients have AZFaT1 andDFFRY deleted in their entirety and three patients additionally have DBY deleted. The three patients with AZFaT1, DFFRY, andDBY deleted show a severe Sertoli cell only syndrome type I phenotype, whereas the patient that has retainedDBY shows a milder oligozoospermic phenotype. The expression of DBY in a cell line from this latter patient is unaltered; this shows that it is the loss of genes lying within the deletion that is responsible for the observed oligozoospermia. RT-PCR analysis of mouse testis RNA from normal and XXSxra mice (devoid of germ cells) has shown that Dby is expressed primarily in somatic cells and that the level of expression is unaltered during germ cell differentiation. This contrasts withDffry where no transcripts are detectable in XXSxra mouse testis and expression occurs specifically in testis mRNA in a germ cell dependent fashion.

A mapping and evolutionary study of porcine sex chromosome genes

A combination of FISH and RH mapping was used to study the evolution of sex chromosome genes in the pig. In total, 19 genes were identified, including 3 PAR genes (STS, KAL, PRK). The gene order of the porcine X Chromosome (Chr) closely resembled the human X Chr (PRK/STS/KAL–AMELX–EIF2s3X/ZFX–USP9X–DBX–SMCX), suggesting that the porcine X has undergone very little rearrangement during evolution. For the porcine Y Chr, two linkage groups of 10 NRY genes were found, and the following order was established: Ypter–(AMELY–EIF2S3Y/ZFY–USP9Y–DBY/UTY)–(TSPY–SMCY–UBE1Y–SRY)–CEN. This gene order showed greater conservation with the murine Y than with the human Y Chr. In addition, all porcine Y Chr genes mapped to Yp, which is similar to the mouse and included EIF2s3Y and UBE1Y, which are not present in humans. Interestingly, complete conservation of X/Y homologous gene order was found between the pig X and Y Chrs, indicating that the porcine Y Chr has not undergone extensive reorganisation with respect to the X. This suggests that the order of the X/Y homologous genes of the porcine X and Y Chrs may closely resemble the ancestral gene order of the eutherian sex chromosomes.

Breakpoint analysis of Turner patients with partial Xp deletions: implications for the lymphoedema gene location

BACKGROUND Turner syndrome is characterised by a 45,X karyotype and a variety of skeletal, lymphoedemic, and gonadal anomalies. Genes involved in the Turner phenotype are thought to be X/Y homologous with the X genes escaping X inactivation. Haploinsufficiency of the SHOXgene has been reported to cause the short stature seen in Turner syndrome patients. More recently, mutations of this gene have been shown to be associated with other skeletal abnormalities, suggesting that haploinsufficiency of SHOX causes all the Turner skeletal anomalies. No such gene has yet been identified for the lymphoedemic features. METHODS Fluorescence in situ hybridisation (FISH) analysis with PAC clones on nine patients with partially deleted X chromosomes was performed. RESULTS/DISCUSSION The Turner syndrome stigmata for each patient are described and correlation between the breakpoint and the phenotype discussed. A lymphoedema critical region in Xp11.4 is proposed and its gene content discussed with respect to that in the previously reported Yp11.2 lymphoedema critical region.

Transcriptional changes in response to X chromosome dosage in the mouse: implications for X inactivation and the molecular basis of Turner Syndrome

BackgroundX monosomic mice (39,XO) have a remarkably mild phenotype when compared to women with Turner syndrome (45,XO). The generally accepted hypothesis to explain this discrepancy is that the number of genes on the mouse X chromosome which escape X inactivation, and thus are expressed at higher levels in females, is very small. However this hypothesis has never been tested and only a small number of genes have been assayed for their X-inactivation status in the mouse. We performed a global expression analysis in four somatic tissues (brain, liver, kidney and muscle) of adult 40,XX and 39,XO mice using the Illumina Mouse WG-6 v1_1 Expression BeadChip and an extensive validation by quantitative real time PCR, in order to identify which genes are expressed from both X chromosomes.ResultsWe identified several genes on the X chromosome which are overexpressed in XX females, including those previously reported as escaping X inactivation, as well as new candidates. However, the results obtained by microarray and qPCR were not fully concordant, illustrating the difficulty in ascertaining modest fold changes, such as those expected for genes escaping X inactivation. Remarkably, considerable variation was observed between tissues, suggesting that inactivation patterns may be tissue-dependent. Our analysis also exposed several autosomal genes involved in mitochondrial metabolism and in protein translation which are differentially expressed between XX and XO mice, revealing secondary transcriptional changes to the alteration in X chromosome dosage.ConclusionsOur results support the prediction that the mouse inactive X chromosome is largely silent, while providing a list of the genes potentially escaping X inactivation in rodents. Although the lower expression of X-linked genes in XO mice may not be relevant in the particular tissues/systems which are affected in human X chromosome monosomy, genes deregulated in XO mice are good candidates for further study in an involvement in Turner Syndrome phenotype.

The human-specific Yp11.2/Xq21.3 homology block encodes a potentially functional testis-specific TGIF-like retroposon

Yp11.2/Xq21.3 is a human-specific homology block that constitutes the largest shared region among the sex chromosomes, spanning some 3.5 Mb. Only two transcribed sequences have been mapped to this segment: the protocadherin genes PCDHX/Y, and the X-linked poly(A)-binding protein PABPC5 gene, whose Y-homolog has been lost during human evolution. This paper reports the genomic structure, expression, and evolutionary conservation of a third (X-Y homologous) transcribed sequence, TGIFLX/Y (TGIF-like X/Y), mapping to this region. TGIFLX/Y has a 2666-bp mRNA encoded by two exons separated by a 96-bp intron. TGIFLX/Y are homeodomain-containing genes related to the TALE superclass gene family. Comparative DNA analysis indicates that TGIFLX originated from retrotransposition of TGIF2, located on 20q11.2-12, onto the X Chromosome. RT-PCR analysis reveals that both X- and Y-linked genes are specifically expressed in adult testis. Cloning and sequencing of TGIFLX homologs in hominoids and Old World monkeys provides evidence for an open reading frame in the eight species studied. Interestingly, a single base pair deletion in the human TGIFLY (as compared with TGIFLX) creates a different reading frame where the C-terminal residues shared by TGIFLX and other TGIF proteins are missing. The conservation, similarity to protein-encoding transcription factors, and specific expression in testis points to a transcriptional role for TGIFLX/Y in this tissue.

Analysis of X chromosome genomic DNA sequence copy number variation associated with premature ovarian failure (POF)

BACKGROUND Premature ovarian failure (POF) is a heterogeneous disease defined as amenorrhoea for >6 months before age 40, with an FSH serum level >40 mIU/ml (menopausal levels). While there is a strong genetic association with POF, familial studies have also indicated that idiopathic POF may also be genetically linked. Conventional cytogenetic analyses have identified regions of the X chromosome that are strongly associated with ovarian function, as well as several POF candidate genes. Cryptic chromosome abnormalities that have been missed might be detected by array comparative genomic hybridization. METHODS In this study, samples from 42 idiopathic POF patients were subjected to a complete end-to-end X/Y chromosome tiling path array to achieve a detailed copy number variation (CNV) analysis of X chromosome involvement in POF. The arrays also contained a 1 Mb autosomal tiling path as a reference control. Quantitative PCR for selected genes contained within the CNVs was used to confirm the majority of the changes detected. The expression pattern of some of these genes in human tissue RNA was examined by reverse transcription (RT)-PCR. RESULTS A number of CNVs were identified on both Xp and Xq, with several being shared among the POF cases. Some CNVs fall within known polymorphic CNV regions, and others span previously identified POF candidate regions and genes. CONCLUSIONS The new data reported in this study reveal further discrete X chromosome intervals not previously associated with the disease and therefore implicate new clusters of candidate genes. Further studies will be required to elucidate their involvement in POF.

Gene expression profiling in porcine maternal infanticide: A model for puerperal psychosis†

The etiology of mental disorders remains largely unclear. Complex interactions between genetic and environmental factors are key to the development of such disorders. Puerperal psychosis is the most extreme form of postnatal mood disorder in women. Similarly, parturition in the pig can trigger extreme behavioral disturbances, including maternal infanticide. In this study, we have used a targeted cDNA microarray approach using the pig as a model to understand the genes and genetic pathways that are involved in these processes. Two subtracted cDNA libraries from porcine hypothalamus were constructed, which were enriched for genes that were over‐expressed and under‐expressed in the aberrant behavioral phenotype, compared to the matched control. In addition to this, a normalized library was constructed from hypothalamus and pituitary samples taken from pigs in a variety of reproductive states. The libraries were partially sequenced and combined represented approximately 5,159 different genes. Microarray analysis determined differences in gene expression between hypothalamus samples from nine matched pairs of infanticidal versus control animals, using a common reference design. Microarray analysis of variance (MAANOVA) identified 52 clones as being differentially expressed (P ≤ 0.002) in the infanticide phenotype, a second analysis with friendly statistics package for microarray analysis (FSPMA) identified 9 genes in common to MAANOVA, and a further 16 genes. A rapid cross‐species screen onto a human oligonucleotide array confirmed 3 genes and highlighted 61 more potential candidates. Some of these genes and the pathways in which they are involved were also implicated in a parallel QTL study on maternal infanticide.

Protocadherin X ( PCDHX) and Y ( PCDHY) genes; multiple mRNA isoforms encoding variant signal peptides and cytoplasmic domains.

The gene-poor, hominid-specific Yp11.2/Xq21.3 X–Y homology block encodes two members of the protocadherin group of cell surface molecules, PCDHX and PCDHY. These two genes, mainly expressed in brain, were known to be composed of at least six exons sharing 98.1% DNA identity. The genomic structure of PCDHX/Y has been reanalyzed in detail, uncovering the existence of at least 11 more exons spanning more than 700 kb. Many of these exons located at the 5′ and 3′ ends of PCDHX/Y undergo differential and alternative splicing. Seven of the exons have been found to use alternative splice sites. Most of these variants are expressed within the brain, although some isoforms exhibit a more ubiquitous distribution pattern. PCDHX/Y transcription appears to be driven from two alternative promoters located usptream of exon 1 and exon 4.1. Assuming that the splicing events at the 5′ and 3′ ends of these genes are independent of one another, potentially up to 360 different mRNAs could be produced. The main impact on protein function is predicted to be in the efficiency of translation, post-translational processing within the cell, and structure of the cytoplasmic domain that may influence any role the genes have in signaling.

The human RPS4 paralogue on Yq11.223 encodes a structurally conserved ribosomal protein and is preferentially expressed during spermatogenesis.

BackgroundThe Y chromosome of mammals is particularly prone to accumulate genes related to male fertility. However, the high rate of molecular evolution on this chromosome predicts reduced power to the across-species comparative approach in identifying male-specific genes that are essential for sperm production in humans. We performed a comprehensive analysis of expression of Y-linked transcripts and their X homologues in several human tissues, and in biopsies of infertile patients, in an attempt to identify new testis-specific genes involved in human spermatogenesis.ResultsWe present evidence that one of the primate-specific Y-linked ribosomal protein genes, RPS4Y2, has restricted expression in testis and prostate, in contrast with its X-linked homologue, which is ubiquitously expressed. Moreover, we have determined by highly specific quantitative real time PCR that RPS4Y2 is more highly expressed in testis biopsies containing germ cells. The in silico analysis of the promoter region of RPS4Y2 revealed several differences relative to RPS4Y1, the more widely expressed paralogue from which Y2 has originated through duplication. Finally, through comparative modelling we obtained the three dimensional models of the human S4 proteins, revealing a conserved structure. Interestingly, RPS4Y2 shows different inter-domain contacts and the potential to establish specific interactions.ConclusionsThese results suggest that one of the Y-linked copies of the ribosomal protein S4 is preferentially expressed during spermatogenesis and might be important for germ cell development. Even though RPS4Y2 has accumulated several amino acid changes following its duplication from RPS4Y1, approximately 35 million years ago, the evolution of the Y-encoded RPS4 proteins is structurally constrained. However, the exclusive expression pattern of RPS4Y2 and the novelties acquired at the C-terminus of the protein may indicate some degree of functional specialisation of this protein in spermatogenesis.