Claire R. Quilter

Rapid detection of chromosomes X and Y aneuploidies by quantitative fluorescent PCR

Quantitative fluorescent polymerase chain reaction (QF‐PCR) assays and small tandem repeat (STR) markers have been successfully employed for the rapid detection of major numerical aneuploidies affecting human autosomes. So far, the analysis of chromosomes X and Y disorders has been hampered by the rarity of highly polymorphic markers which could distinguish normal female homozygous PCR patterns from those seen in patients with Turners syndrome. A new marker (X22) of the X/Y chromosomes has been identified which maps in the Xq/Yq pseudoautosomal region PAR2; used together with the HPRT it allows the rapid diagnosis of numerical aneuploidies of the sex chromosomes. Blood samples from normal male and female subjects and from patients with X and Y chromosome disorders (45,X and 47,XXY) have been tested by QF‐PCR with the X22 polymorphic pentanucleotide (12 alleles) together with the HPRT and P39 markers. The samples were also tested by multiplex QF‐PCR with STRs specific for chromosomes 21,18,13 and amelogenin (AMXY). Tested by QF‐PCR, all samples from normal females were heterozygous for either the X22 or the HPRT marker with fluorescent peak ratios near 1:1, thus allowing a correct, rapid diagnosis of their chromosome complement. Turners patients (45,X) showed only one X22 and one HPRT fluorescent peak, thus documenting the presence of a single X chromosome. Turners patients with mosaicism showed a major fluorescent peak for the X22 and HPRT markers and a minor peak revealing the presence of a second minor population of cells. Two 47,XXY cases could also be diagnosed. Multiplex analyses can be performed using simultaneously STR markers for chromosomes 21,18,13 X and Y. The diagnostic value of a third X‐linked marker (P39) was also investigated. These results suggest that rapid diagnosis of major numerical anomalies of the X and Y chromosomes can be performed using QF‐PCR with a new highly polymorphic X‐linked marker, X22, which maps in the Xq/Yq pseudoautosomal region PAR 2. Multiplex QF‐PCR tests—using the X22 STR in association with HPRT and, in rare cases, a third P39 marker—allow the rapid diagnosis of major aneuploidies affecting chromosomes 21, 18, 13, X and Y. The X22 marker can also be employed for the detection of fetal cells present in maternal peripheral blood or the endocervical canal. Copyright

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.

Impact on offspring methylation patterns of maternal gestational diabetes mellitus and intrauterine growth restraint suggest common genes and pathways linked to subsequent type 2 diabetes risk

Size at birth, postnatal weight gain, and adult risk for type 2 diabetes may reflect environmental exposures during developmental plasticity and may be mediated by epigenetics. Both low birth weight (BW), as a marker of fetal growth restraint, and high birth weight (BW), especially after gestational diabetes mellitus (GDM), have been linked to increased risk of adult type 2 diabetes. We assessed DNA methylation patterns using a bead chip in cord blood samples from infants of mothers with GDM (group 1) and infants with prenatal growth restraint indicated by rapid postnatal catch‐up growth (group 2), compared with infants with normal postnatal growth (group 3). Seventy‐five CpG loci were differentially methylated in groups 1 and 2 compared with the controls (group 3), representing 72 genes, many relevant to growth and diabetes. In replication studies using similar methodology, many of these differentially methylated regions were associated with levels of maternal glucose exposure below that defined by GDM [the Hyperglycemia and Adverse Pregnancy Outcome (HAPO) study] or were identified as changes observed after randomized periconceptional nutritional supplementation in a Gambian cohort characterized by maternal deprivation. These studies provide support for the concept that similar epigenetic modifications may underpin different prenatal exposures and potentially increase long‐term risk for diseases such as type 2 diabetes.—Quilter, C. R., Cooper, W. N., Cliffe, K. M., Skinner, B. M., Prentice, P. M., Nelson, L., Bauer, J., Ong, K. K., Constância, M., Lowe, W. L., Affara, N. A., Dunger, D. B., Impact on offspring methylation patterns of maternal gestational diabetes mellitus and intrauterine growth restraint suggest common genes and pathways linked to subsequent type 2 diabetes risk. FASEB J. 28, 4868–4879 (2014). www.fasebj.org

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.

Cytogenetic and molecular investigations of Y chromosome sequences and their role in Turner syndrome

It has been proposed that all live born females with Turner syndrome carry a cell line containing two sex chromosomes, which may be present at a low level of mosaicism (Hook & Warburton, 1983; Hassold et al. 1985; 1988; Connor & Loughlin, 1989). If the second sex chromosome is a Y, these patients are at risk of developing gonadoblastoma. In this study, 50 patients found to have a 45,X karyotype by conventional cytogenetic analysis, were screened by the polymerase chain reaction (PCR), for the presence of Y chromosome sequences. Two patients were positive for six of the eight Y chromosome loci tested and additional cytogenetic analysis confirmed the presence of a marker chromosome, in 8% and 3% of cells respectively. Fluorescence in situ hybridization (FISH) was used to confirm that the markers were of Y chromosome origin and helped to elucidate their structure. In addition, four other patients were found to have a Y chromosome by initial routine cytogenetic analysis. FISH, in conjunction with PCR, elucidated the structure of the Y chromosomes. This study illustrates the value of using a combination of cytogenetic and molecular techniques, to identify Y chromosome sequences in Turner syndrome.

A comparative study between infertile males and patients with Turner syndrome to determine the influence of sex chromosome mosaicism and the breakpoints of structurally abnormal Y chromosomes on phenotypic sex

The Y chromosome is important for male development as it contains the sex determining gene SRY 1 and many spermatogenesis genes.2 Structural abnormalities of the Y chromosome include rings, deletions, inversions, and dicentrics.3,4 These types of abnormalities are common in infertile males (1.5%), especially those with azoospermia.5,6 However, such rearrangements are unstable and an additional 45,X cell line is frequently present.3 The 45,X cell line has been shown to influence phenotypic sex so that these chromosome constitutions may also be found in patients with ambiguous genitalia and in female patients with gonadal dysgenesis and Turner syndrome.4,7 In fact, from cytogenetic studies about 4-6.2% of female Turner patients show Y chromosome mosaicism8–10 irrespective of the presence of SRY .4,11,12 Mosaicism varies widely between tissues and accurate interpretation depends on the number of cells examined and the number and types of tissues studied.13,14 It has been reported that phenotypic sex is strongly influenced by the percentage and distribution of Y chromosome containing cells in the gonads.15,16 However, studies on gonadal tissue are hindered by the fact that it is rarely available for analysis and alternative, more easily accessible tissue is usually studied. It has also been suggested that the structure of the Y chromosome may indirectly affect phenotypic sex. The repetitive sequences at the euchromatin/heterochromatin boundary of the Y chromosome long arm are thought to have an important stabilising role and loss of this region loses this effect, resulting in mosaicism with a 45,X cell line.17 In dicentrics, which are the most common abnormality of the Y chromosome,3 it has been suggested that the position of the q arm breakpoint in dicentric Yp chromosomes can influence Y chromosome stability. The more proximal the …

Porcine Maternal Infanticide as a Model for Puerperal Psychosis

Childbirth is a period of substantial rapid biological and psychological change and a wide range of psychotic disorders can occur ranging from mild ‘baby blues’ to severe episodes of psychotic illnesses. Puerperal psychosis is the most extreme form of postnatal psychosis, occurring in 1 in 1,000 births. In this study, we have used the pig as an animal model for human postnatal psychiatric illness. Our aim was to identify quantitative trait loci (QTL) associated with maternal (infanticide) sow aggression. This is defined by sows attacking and killing their own newborn offspring, within 24 hr of birth. An affected sib pair whole genome linkage analysis was carried out with 80 microsatellite markers covering the 18 porcine autosomes and the X chromosome, with the aim of identifying chromosomal regions responsible for this abnormal behavior. Analysis was carried out using the non‐parametric linkage test of Whittemore and Halpern, as implemented in the Merlin software. The results identified 4 QTL mapping on Sus scrofa chromosomes 2 (SSC2), 10 (SSC10), and X (SSCX). The peak regions of these QTL are syntenic to HSA 5q14.3‐15, 1q32, Xpter‐Xp2.1, and Xq2.4‐Xqter, respectively. Several potential candidate genes lie in these regions in addition to relevant abnormal behavioral QTL, found in humans and rodents.

Cytogenetic and Y chromosome microdeletion screening of a random group of infertile males.

OBJECTIVE To assess whether to perform routine cytogenetic and Y chromosome microdeletion screening on all infertile male patients. DESIGN A cytogenetic and Y microdeletion study of a random group of infertile men. SETTING University department. PATIENT(S) In total, 40 patients had azoospermia (21 nonidiopathic), 27 had severe oligozoospermia/oligoasthenozoospermia (<or=5 x 10(6)/mL) (5 nonidiopathic), 20 had oligozoospermia/oligoasthenozoospermia (5-20 x 10(6)/mL) (6 nonidiopathic), and 16 had asthenozoospermia (5 nonidiopathic). Many were candidates for intracytoplasmic sperm injection (ICSI). INTERVENTION(S) Collection of blood samples from all patients and buccal cells from one patient. MAIN OUTCOME MEASURE(S) Karyotype analysis, polymerase chain reaction (PCR) screening for Y chromosome microdeletions, and fluorescence in situ hybridization of abnormal chromosomes. RESULT(S) Ten (9.7%) subjects, including one nonidiopathic patient, were found to have an abnormal karyotype. Two idiopathic azoospermic patients were missing large portions of Y chromosome euchromatin, confirmed by PCR analysis and an additional idiopathic azoospermic patient had a Y chromosome microdeletion. CONCLUSION(S) Routine cytogenetic analysis of all infertile male patients is required but it may be advisable to limit routine Y chromosome microdeletion screening to patients with severe male factor infertility (<or=5 x 10(6)/mL).

Gene structure and expression of serotonin receptor HTR2C in hypothalamic samples from infanticidal and control sows

BackgroundThe serotonin pathways have been implicated in behavioural phenotypes in a number of species, including human, rat, mouse, dog and chicken. Components of the pathways, including the receptors, are major targets for drugs used to treat a variety of physiological and psychiatric conditions in humans. In our previous studies we have identified genetic loci potentially contributing to maternal infanticide in pigs, which includes a locus on the porcine X chromosome long arm. The serotonin receptor HTR2C maps to this region, and is therefore an attractive candidate for further study based on its function and its position in the genome.ResultsIn this paper we describe the structure of the major transcripts produced from the porcine HTR2C locus using cDNA prepared from porcine hypothalamic and pooled total brain samples. We have confirmed conservation of sites altered by RNA editing in other mammalian species, and identified polymorphisms in the gene sequence. Finally, we have analysed expression and editing of HTR2C in hypothalamus samples from infanticidal and control animals.ConclusionsThe results confirm that although the expression of the long transcriptional variant of HTR2C is raised in infanticidal animals, the overall patterns of editing in the hypothalamus are similar between the two states.Sequences associated with the cDNA and genomic structures of HTR2C reported in this paper are deposited in GenBank under accession numbers FR720593, FR720594 and FR744452.