ORE identifies extreme expression effects enriched for rare variants.

MOTIVATION: Non-coding rare variants (RVs) may contribute to Mendelian disorders but have been challenging to study due to small sample sizes, genetic heterogeneity and uncertainty about relevant non-coding features. Previous studies identified RVs associated with expression outliers, but varying outlier definitions were employed and no comprehensive open-source software was developed. RESULTS: We developed Outlier-RV Enrichment (ORE) to identify biologically-meaningful non-coding RVs. We implemented ORE combining whole-genome sequencing and cardiac RNAseq from congenital heart defect patients from the Pediatric Cardiac Genomics Consortium and deceased adults from Genotype-Tissue Expression. Use of rank-based outliers maximized sensitivity while a most extreme outlier approach maximized specificity. Rarer variants had stronger associations, suggesting they are under negative selective pressure and providing a basis for investigating their contribution to Mendelian disorders. AVAILABILITY AND IMPLEMENTATION: ORE, source code, and documentation are available at https://pypi.python.org/pypi/ore under the MIT license. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

TRGT-denovo: accurate detection of de novo tandem repeat mutations.

Motivation: Identifying de novo tandem repeat (TR) mutations on a genome-wide scale is essential for understanding genetic variability and its implications in rare diseases. While PacBio HiFi sequencing data enhances the accessibility of the genome's TR regions for genotyping, simple de novo calling strategies often generate an excess of likely false positives, which can obscure true positive findings, particularly as the number of surveyed genomic regions increases. Results: We developed TRGT-denovo, a computational method designed to accurately identify all types of de novo TR mutations-including expansions, contractions, and compositional changes-within family trios. TRGT-denovo directly interrogates read evidence, allowing for the detection of subtle variations often overlooked in variant call format (VCF) files. TRGT-denovo improves the precision and specificity of de novo mutation (DNM) identification, reducing the number of de novo candidates by an order of magnitude compared to genotype-based approaches. In our experiments involving eight rare disease trios previously studiedTRGT-denovo correctly reclassified all false positive DNM candidates as true negatives. Using an expanded repeat catalog, it identified new candidates, of which 95% (19/20) were experimentally validated, demonstrating its effectiveness in minimizing likely false positives while maintaining high sensitivity for true discoveries. Availability and implementation: Built in Rust, TRGT-denovo is available as source code and a pre-compiled Linux binary along with a user guide at: https://github.com/PacificBiosciences/trgt-denovo.

Parent of origin effects.

A major weakness of most genome-wide association studies has been their inability to fully explain the heritable component of complex disease. Nearly all such studies consider the two parental alleles to be functionally equivalent. However, the existence of imprinted genes demonstrates that this assumption can be wrong. In this review, we describe a wide variety of different mechanisms that underlie many other parent of origin and trans-generational effects that are known to operate in both humans and model organisms, suggesting that these phenomena are perhaps not uncommon in the genome. We propose that the consideration of alternative models of inheritance will improve our understanding of the heritability and causes of human traits and could have significant impacts on the study of complex disorders.

Recurrent reciprocal deletions and duplications of 16p13.11: the deletion is a risk factor for MR/MCA while the duplication may be a rare benign variant.

BACKGROUND: Genomic disorders are often caused by non-allelic homologous recombination between segmental duplications. Chromosome 16 is especially rich in a chromosome-specific low copy repeat, termed LCR16. METHODS AND RESULTS: A bacterial artificial chromosome (BAC) array comparative genome hybridisation (CGH) screen of 1027 patients with mental retardation and/or multiple congenital anomalies (MR/MCA) was performed. The BAC array CGH screen identified five patients with deletions and five with apparently reciprocal duplications of 16p13 covering 1.65 Mb, including 15 RefSeq genes. In addition, three atypical rearrangements overlapping or flanking this region were found. Fine mapping by high-resolution oligonucleotide arrays suggests that these deletions and duplications result from non-allelic homologous recombination (NAHR) between distinct LCR16 subunits with >99% sequence identity. Deletions and duplications were either de novo or inherited from unaffected parents. To determine whether these imbalances are associated with the MR/MCA phenotype or whether they might be benign variants, a population of 2014 normal controls was screened. The absence of deletions in the control population showed that 16p13.11 deletions are significantly associated with MR/MCA (p = 0.0048). Despite phenotypic variability, common features were identified: three patients with deletions presented with MR, microcephaly and epilepsy (two of these had also short stature), and two other deletion carriers ascertained prenatally presented with cleft lip and midline defects. In contrast to its previous association with autism, the duplication seems to be a common variant in the population (5/1682, 0.29%). CONCLUSION: These findings indicate that deletions inherited from clinically normal parents are likely to be causal for the patients' phenotype whereas the role of duplications (de novo or inherited) in the phenotype remains uncertain. This difference in knowledge regarding the clinical relevance of the deletion and the duplication causes a paradigm shift in (cyto)genetic counselling.

Further delineation of the 15q13 microdeletion and duplication syndromes: a clinical spectrum varying from non-pathogenic to a severe outcome.

BACKGROUND: Recurrent 15q13.3 microdeletions were recently identified with identical proximal (BP4) and distal (BP5) breakpoints and associated with mild to moderate mental retardation and epilepsy. METHODS: To assess further the clinical implications of this novel 15q13.3 microdeletion syndrome, 18 new probands with a deletion were molecularly and clinically characterised. In addition, we evaluated the characteristics of a family with a more proximal deletion between BP3 and BP4. Finally, four patients with a duplication in the BP3-BP4-BP5 region were included in this study to ascertain the clinical significance of duplications in this region. RESULTS: The 15q13.3 microdeletion in our series was associated with a highly variable intra- and inter-familial phenotype. At least 11 of the 18 deletions identified were inherited. Moreover, 7 of 10 siblings from four different families also had this deletion: one had a mild developmental delay, four had only learning problems during childhood, but functioned well in daily life as adults, whereas the other two had no learning problems at all. In contrast to previous findings, seizures were not a common feature in our series (only 2 of 17 living probands). Three patients with deletions had cardiac defects and deletion of the KLF13 gene, located in the critical region, may contribute to these abnormalities. The limited data from the single family with the more proximal BP3-BP4 deletion suggest this deletion may have little clinical significance. Patients with duplications of the BP3-BP4-BP5 region did not share a recognisable phenotype, but psychiatric disease was noted in 2 of 4 patients. CONCLUSIONS: Overall, our findings broaden the phenotypic spectrum associated with 15q13.3 deletions and suggest that, in some individuals, deletion of 15q13.3 is not sufficient to cause disease. The existence of microdeletion syndromes, associated with an unpredictable and variable phenotypic outcome, will pose the clinician with diagnostic difficulties and challenge the commonly used paradigm in the diagnostic setting that aberrations inherited from a phenotypically normal parent are usually without clinical consequences.

Clinical and molecular delineation of the 17q21.31 microdeletion syndrome.

BACKGROUND: The chromosome 17q21.31 microdeletion syndrome is a novel genomic disorder that has originally been identified using high resolution genome analyses in patients with unexplained mental retardation. AIM: We report the molecular and/or clinical characterisation of 22 individuals with the 17q21.31 microdeletion syndrome. RESULTS: We estimate the prevalence of the syndrome to be 1 in 16,000 and show that it is highly underdiagnosed. Extensive clinical examination reveals that developmental delay, hypotonia, facial dysmorphisms including a long face, a tubular or pear-shaped nose and a bulbous nasal tip, and a friendly/amiable behaviour are the most characteristic features. Other clinically important features include epilepsy, heart defects and kidney/urologic anomalies. Using high resolution oligonucleotide arrays we narrow the 17q21.31 critical region to a 424 kb genomic segment (chr17: 41046729-41470954, hg17) encompassing at least six genes, among which is the gene encoding microtubule associated protein tau (MAPT). Mutation screening of MAPT in 122 individuals with a phenotype suggestive of 17q21.31 deletion carriers, but who do not carry the recurrent deletion, failed to identify any disease associated variants. In five deletion carriers we identify a <500 bp rearrangement hotspot at the proximal breakpoint contained within an L2 LINE motif and show that in every case examined the parent originating the deletion carries a common 900 kb 17q21.31 inversion polymorphism, indicating that this inversion is a necessary factor for deletion to occur (p<10(-5)). CONCLUSION: Our data establish the 17q21.31 microdeletion syndrome as a clinically and molecularly well recognisable genomic disorder.

CNVs and genetic medicine (excitement and consequences of a rediscovery).

The extensive variability of individual human genomes contributes to phenotypic variability. Structural genomic variants, and copy number variants (CNVs) in particular, have recently been rediscovered as contributors to the genomic plasticity and evolution and as pathoetiologic elements for both monogenic and complex traits. Herein we review some of the consequences of CNVs in the context of human inherited diseases.

Integrative transcriptomic analysis reveals key drivers of acute peanut allergic reactions.

Mechanisms driving acute food allergic reactions have not been fully characterized. We profile the dynamic transcriptome of acute peanut allergic reactions using serial peripheral blood samples obtained from 19 children before, during, and after randomized, double-blind, placebo-controlled oral challenges to peanut. We identify genes with changes in expression triggered by peanut, but not placebo, during acute peanut allergic reactions. Network analysis reveals that these genes comprise coexpression networks for acute-phase response and pro-inflammatory processes. Key driver analysis identifies six genes (LTB4R, PADI4, IL1R2, PPP1R3D, KLHL2, and ECHDC3) predicted to causally modulate the state of coregulated networks in response to peanut. Leukocyte deconvolution analysis identifies changes in neutrophil, naive CD4+ T cell, and macrophage populations during peanut challenge. Analyses in 21 additional peanut allergic subjects replicate major findings. These results highlight key genes, biological processes, and cell types that can be targeted for mechanistic study and therapeutic targeting of peanut allergy.

Glaucoma screening clinic in general practice: prevalence of occult disease, and resource implications.

BACKGROUND: Previous studies have shown that for every known case of glaucoma there is another case of occult disease. Most cases of glaucoma are detected by optometrists. AIM: This study set out to determine the prevalence of occult glaucoma in a practice population and assess the likely resource implications of introducing a glaucoma screening programme into a general practice setting. METHOD: The 1153 patients registered with one practice in Leicester who were aged 55-69 years on 1 January 1992 and who were not known to have glaucoma prior to screening were invited to a screening clinic. Prior to screening there were 11 known cases of glaucoma in this age group. Screening was carried out by a practice nurse. Patients who failed the screening tests were referred according to the study protocol to the ophthalmology department of the Leicester Royal Infirmary and examined by one ophthalmologist. The number of cases of occult glaucoma and other eye disease detected, the cost per case screened and case detected, and the number of referrals generated were evaluated. RESULTS: Nine hundred and fifty people (82%) accepted the invitation and attended for glaucoma screening. Of those screened 115 (12%) were referred for ophthalmic assessment. Glaucoma was confirmed in 14 of the referred patients (12%) while a further 15 (13%) were found to have ocular hypertension. All but one of those people diagnosed as having glaucoma recalled having been examined by their optician within the last five years; for 50% the period was less than two years. Nineteen of the patients referred (17%) had other ocular pathology detected by the ophthalmologist and no abnormality was detected in 65 patients referred (57%). The estimated cost to the practice (excluding hospital outpatient costs) per case screened using the study protocol was 6 pounds and the cost per case detected was 408 pounds. CONCLUSION: Glaucoma screening may be successfully undertaken in a general practice setting by non-ophthalmically trained staff who have received tuition in the use of the equipment. It is well received by the population served but the capital cost of equipment is likely to be too high for most practices to afford. The reaffirmation of at least one occult case of glaucoma for every known case is particularly alarming in the absence of a national screening programme and the asymptomatic course of this treatable, blinding disease. Closer cooperation between general practitioners and optometrists will be the practical way ahead for most practices.

Concentrations of progesterone in milk as a monitor of early pregnancy diagnosis in dairy cows.

The objectives of the experiment were to evaluate the efficacy of using progesterone concentrations in milk and palpation per rectum on days 21 or 22 postbreeding to estimate pregnancy and evaluate management practices; and to investigate physiological occurrences leading to incorrect diagnosis of pregnancy when serial samples of milk were collected. Of particular interest were indications of early embyronic death and insemination of cows not in estrus. Milk samples were collected at the afternoon milking of days 0 or 1 (day 0 = day of estrus), 9 or 10, 21 or 22 and 27 or 28 following breeding in 200 lactating dairy cows. Tentative diagnosis of pregnancy was made based on concentrations of progesterone in milk on days 21 and 22 alone and on days 21 or 22 and 27 or 28. In addition all cows were palpated per rectum on days 21 or 22 postbreeding and a tentative pregnancy diagnosis was made. Pregnancy was confirmed by examination of the genital tract per rectum between 35 and 50 days after breeding. Values of 4 ng/ml or greater and/or the presence of a mature corpus luteum were considered positive signs of pregnancy. Progesterone in milk ranged from 0.1 to 18 ng/ml. On days 0 or 1, 9 or 10, 21 or 22 and 27 or 28 concentrations of progesterone in milk averaged 1.5 +/- 0.3, 11.1 +/- 0.5, 12.0 +/- 0.4 12.5 +/- 0.5 ng/ml for pregnant cows. Corresponding samples from nonpregnant cows averaged 1.2 +/- 0.2, 10.3 +/- 0.4, 3.0 +/- 0.4, 6.8 +/- 0.6 ng/ml, respectively. Ninety-six and 104 cows were classified as pregnant and nonpregnant on days 21 or 22 as compared to 78 and 118 cows diagnosed as pregnant and nonpregnant on days 21 or 22 and 27 or 28 combined. Pregnancy detection by progesterone in milk on days 21 or 22 with pregnancy determined via rectal palpation 35 to 50 days postbreeding was 77 and 100% accurate for positive and negative diagnosis, respectively. The percent agreement using progesterone in milk on days 21 or 22 and 27 or 28 combined was 95 and 100%, respectively, for positive and negative diagnosis. Diagnosis based on rectal palpation 21 or 22 days postbreeding was 63 92 (69%) and 76 88 (87%) for pregnant and nonpregnant cows, respectively. Ten of the 200 cows had progesterone concentratins in milk of > 4 ng/ml at the time of breeding. Six of these cows were pregnant from a previous insemination. The other four cows were nonpregnant and were inseminated during the luteal phase of the cycle. In conclusion, measurement of progesterone in milk is a useful tool in early detection of pregnant and nonpregnant cows and may be useful in detecting reproductive problems in a dairy herd. It will probably be most useful when used in combination with later pregnancy diagnosis per rectum .

Effect of ovarian activity and endogenous reproductive hormones on GnRH-induced ovarian cycles in postpartum dairy cows.

The objective was to determine the relationship of prior follicular development to GnRH-induced cyclic ovarian activity in dairy cows postpartum. Sixty lactating Holstein and Guernsey cows from the University of Missouri dairy herds were assigned at random to one of two groups. Group I consisted of 20 cows (control) given a single intramuscular (IM) injection of saline. Group II consisted of 40 cows given a single injection of 100 micrograms gonadotropin releasing hormone (GnRH) IM. Treatments were administered 12 to 14 days postpartum. All cows were palpated per rectum to monitor ovarian activity prior to treatment (time 0) and 2 to 3 and 7 to 9 days post-treatment. Blood was collected via tail vessel puncture at time 0 and 7 to 9 days post-treatment. The percentage of cows exhibiting a follicle 10 mm or greater in diameter prior to treatment was not different between Group I (50%) and Group II (43%). However, twenty (50%) of the GnRH-treated cows initiated cyclic ovarian activity following treatment as evidenced by palpable CL and plasma progesterone greater than or equal to 1.0 ng/ml on day 9 post-treatment in contrast to only four (20%) saline-treated cows (P less than 0.05). Seventeen of 20 cows ovulating in Group II exhibited a palpable follicle greater than or equal to 10 mm in diameter prior to treatment compared to 3 of 4 cows in Group I. Prior to treatment, plasma concentrations of LH, estradiol-17 beta and progesterone were not different (P less than .05) between cows in Groups I and II. In Group II, preinjection levels of estradiol-17 beta and LH for cows ovulating in response to GnRH were higher (P less than .01) than in cows not ovulating. Postinjection concentrations of progesterone in plasma on days 7 to 9 were higher (P less than .01) in cows ovulating than in cows not ovulating. Six (30%) cows in Group I developed ovarian cysts prior to conception as compared to 12.5% (5) of the cows in Group II (P less than .05). The present study suggests: (1) GnRH treatment as early as 12 to 14 days postpartum may initiate cyclic ovarian activity in normal dairy cows; (2) elevated preinjection concentrations of estradiol-17 beta and follicular growth are important for GnRH induced ovulations; and (3) GnRH treatment 12 to 14 days postpartum may be useful in reducing abnormal ovarian activity.

Ovarian response to gonadotropin-releasing hormone in postpartum dairy cows.

Laparotomies were performed on 17 lactating dairy cows, milked twice daily, 12 or 13 days after parturition to observe gross ovarian structures. Five cows were randomly given an IM injection of saline solution and 12 cows were given 100 micrograms of gonadotropin releasing hormone (GnRH). A 2nd laparotomy was performed 3 days later to observe changes in ovarian structures. In addition, the genital tract was palpated per rectum 5 to 8 days after the 2nd laparotomy, and blood samples for hormonal analysis were obtained prior to each laparotomy and palpation per rectum. Eight of 12 cows given GnRH and 2 of 5 cows given saline solution had developing corpora lutea with prominent ovulation papilla at the time of the 2nd laparotomy. Luteinized follicles were not observed. In GnRH-treated cows, 7 of 7 cows with follicles greater than or equal to 15 mm ovulated after GnRH treatment compared (P less than 0.01) with 1 of 5 cows with follicles less than or equal to 10 mm. Increase in plasma progesterone was not observed in GnRH-treated cows not ovulating. Thus, GnRH induced ovulation in postpartum dairy cows that had large follicles at the time of GnRH treatment.

Correlation between pregnancy diagnosis by membrane slip and embryonic mortality.

In a study involving 7,477 pregnant cows (5,426 Holstein-Friesians and 2,051 Guernseys), each pregnancy was diagnosed by rectal palpation, using the membrane slip technique. Each cow was examined twice, first by a veterinary student, then by a clinician. Of the cows diagnosed pregnant, 7,058 (94.4%) calved. The embryonic death rate in cows examined during the first 50 days of gestation was significantly (P less than 0.001) greater than that associated with examinations after 50 days of gestation. A significant difference was found between herds in different years (P less than 0.05). There was no conclusive indication that embryonic loss at the time of or shortly after early examination was iatrogenic abortion resulting from the membrane slip technique.

Cytology suggestive of glandular neoplasia: outcomes and suggested management.

Eighty-three cases having a cervical smear result showing abnormal glandular cells were identified and matched up with the diagnostic histology result. Thirty-four (41.0%) were associated with malignancy and 26 (31.3%) with a cervical intraepithelial lesion without invasion. Thirty-eight (45.8%) had conditions of the cervix as follows: 12 cases had invasive disease of the cervix; nine (10.8%) adenocarcinoma of cervix and three (3.6%) squamous carcinoma of cervix. Nineteen (22.9%) had cervical intraepithelial neoplasia (CIN/SIL) alone and seven (8.4%) had cervical glandular intraepithelial neoplasia (CGIN) +/- CIN. There were 16 (19.3%) cases with malignancies of the uterine corpus and six (7.2%) had a malignancy arising from another primary site. Twenty-three (27.7%) had no malignant or pre-malignant condition. The risk of malignancy was related to age and ranged from 18.2% in those under 35 years to 67.9% in those 55 years and over. A protocol for the management of these cases is described.

H-Y antigen in a fertile XY female horse.

The presence of significantly reduced levels of H-Y antigen in the blood of an XY mare is consistent with the view that H-Y genes comprise a system of testis determinants. Loss or suppression of a critical portion of H-Y genes and subthreshold expression of H-Y antigen could account for a failure of testicular differentiation, thereby allowing a measure of ovarian development in an XY embryo.

An assessment of the value of video recordings of receptionists.

Video recordings of receptionists at work in general practice were found to be useful for self assessment by the receptionists and enabled the doctors to see areas for improvement in the organization of the reception area.

Evidence for a cryptic 46,XX cell line in a 45,X/46,X,psu idic(Xq) patient with normal reproduction.

Gonadal dysgenesis resulting in primary infertility is one of the most common features of Turner syndrome. There have been a number of cases described of pregnancy in 45,X subjects, but whether or not the fertility is associated with a 46,XX cell line in the germ cells is not known. We describe a 45,X/46,X,psu idic(Xq) female with normal fertility, in whom a cryptic 46,XX cell line was found in the germ cells.

Xp deletions associated with autism in three females.

We report eight females with small deletions of the short arm of the X chromosome, three of whom showed features of autism. Our results suggest that there may be a critical region for autism in females with Xp deletions between the pseudoautosomal boundary and DXS7103. We hypothesise that this effect might be due either to the loss of function of a specific gene within the deleted region or to functional nullisomy resulting from X inactivation of the normal X chromosome.