Shifts in uterine bacterial communities associated with endogenous progesterone and 17ß-estradiol concentrations in beef cattle.

The relation between circulating concentrations of progesterone and 17ß-estradiol prior to insemination play a key role in optimizing fertility in cattle. This study aimed to determine the impact of endogenous progesterone (P4) and estradiol (E2) concentrations on uterine bacterial community abundance and diversity in beef cattle. Angus-influenced heifers were subjected to an industry standard estrous synchronization protocol. Uterine flushes were collected on d -2 (endogenous P4) and d 0 (endogenous E2) and used for targeting the V4 hypervariable region of 16S rRNA bacterial gene. Plasma was collected on d -2 and 0 for quantification of P4 and E2 concentrations by radioimmunoassay, respectively. Heifers were allotted to one of the following groups: High P4 + High E2 (H-H; n = 11), High P4 + Low E2 (H-L; n = 9), Low P4 + High E2 (L-H; n = 9), Low P4 + Low E2 (L-L; n = 11). Results indicated that Shannon's diversity index tended to be greater for H-L heifers compared to L-H heifers on d 0 (P = 0.10). For H-L heifers from d -2 to d 0, the relative abundance of Actinobacteria decreased and Tenericutes increased (P < 0.01). Within phylum Actinobacteria, the relative abundance of Corynebacterium decreased from d -2 to d 0 in treatment groups H-H, H-L, and L-L (P < 0.05); however, did not differ by d for L-H heifers. Within phylum Tenericutes, the relative abundance of Ureaplasma increased from d -2 to d 0 for H-L heifers (P = 0.01). Additionally for H-L heifers, the relative abundance of Bacteroidetes tended to increase from day -2 to on d 0 (P = 0.07). For H-L heifers, uterine pH increased from day -2 to d 0 (P = 0.05). These results suggest that differing endogenous concentrations of P4 and E2 may be associated with shifts in uterine microbiota and pH, and this could ultimately impact fertility outcomes in beef cattle.

Towards an in situ non-lethal rapid test to accurately detect the presence of the nematode parasite, Anguillicoloides crassus, in European eel, Anguilla anguilla.

Anguillicoloides crassus is an invasive nematode parasite of the critically endangered European eel, Anguilla anguilla, and possibly one of the primary drivers of eel population collapse, impacting many features of eel physiology and life history. Early detection of the parasite is vital to limit the spread of A. crassus, to assess its potential impact on spawning biomass. However accurate diagnosis of infection could only be achieved via necropsy. To support eel fisheries management we developed a rapid, non-lethal, minimally invasive and in situ DNA-based method to infer the presence of the parasite in the swim bladder. Screening of 131 wild eels was undertaken between 2017 and 2019 in Ireland and UK to validate the procedure. DNA extractions and PCR were conducted using both a Qiagen Stool kit and in situ using Whatman qualitative filter paper No1 and a miniPCR DNA Discovery-System™. Primers were specifically designed to target the cytochrome oxidase mtDNA gene region and in situ extraction and amplification takes approximately 3 h for up to 16 individuals. Our in-situ diagnostic procedure demonstrated positive predictive values at 96% and negative predictive values at 87% by comparison to necropsy data. Our method could be a valuable tool in the hands of fisheries managers to enable infection control and help protect this iconic but critically endangered species.

Progesterone dose during synchronization treatment alters luteinizing hormone receptor and steroidogenic enzyme mRNA abundances in granulosa cells of Nellore heifers.

The objective was to investigate effects of progesterone (P4) dose on abundance of luteinizing hormone receptor (LHCGR), aromatase (CYP19A1), 3ß-hydroxysteroid dehydrogenase (HSD3B1), and other steroidogenic mRNA transcripts in granulosa cells from dominant follicles. Nellore heifers were assigned to one of six groups: new, first-use controlled internal drug release device (CIDR1) inserted for 5 days (Large-P4-dose-D5; n = 7) or 6 days (Large-P4-dose-D6; n = 8), prostaglandin (PG)F2α administered on D0 and 1 previously-used CIDR (CIDR3) inserted for 5 days (Small- P4-dose-D5; n = 8) or 6 days (Small-P4-dose-D6; n = 8), CIDR1 inserted on D0 and removed plus PGF2α on D5 (Large-P4-dose-proestrus (PE); n = 7), and CIDR3 and PGF2α on D0 and 1, CIDR3 removed plus PGF2α on D5 (Small-P4-dose-PE; n = 7). Duration of P4 treatment (D5 compared to D6) affected abundances of CYP19A1 mRNA transcripts, with there being greater abundances on D6 than D5 (P ≤ 0.05). Heifers treated with the large dose of P4 had a smaller dominant follicle, less serum and intra-follicular estradiol (E2) concentrations (P ≤ 0.05) and lesser LHCGR, CYP19A1, and HSD3B1 transcript abundances (P ≤ 0.05). Heifers treated to induce PE had a larger follicle diameter (P = 0.09), greater intra-follicular E2 concentrations and larger abundances of CYP19A1 mRNA transcript (P ≤ 0.05) than heifers of the D6 group. Overall, treatment with larger doses of P4 resulted in lesser abundances of LHCGR, HSD3B1, and CYP19A1 mRNA transcripts; thus, potentially leading to development of smaller dominant follicles and lesser E2 concentrations.

The Downrange Acoustic Toolbox: An Active Solution for Combat-Related Acute Acoustic Trauma.

Frontline military personnel are at high risk of acute acoustic trauma (AAT) caused by impulse noise, such as weapon firing or blast. This can result in anatomic disruption of the tympanic membrane and damage to the middle and inner ear, leading to conductive, sensorineural, or mixed hearing loss that may be temporary or permanent. AAT reduces warfighters' operational effectiveness and has implications for future quality of life. Hearing protection devices can mitigate AAT but are not completely protective. Novel therapeutic options now exist; therefore, identification of AAT as soon as possible from point of injury is vital to ensure optimal treatment and fulfillment of the duty of care. Early recognition and treatment of frontline AAT can maintain the deployed team's capabilities, avoid unnecessary case evacuation (CASEVAC), and raise awareness of military occupational AAT. This will help prioritize hearing preservation, maintain the fighting force, and ultimately retain personnel in service. The UK Defence hearWELL research collaboration has developed a frontline protocol for the assessment of AAT utilizing future-facing technology developed by the US Department of Defense: the Downrange Acoustic Toolbox (DAT). The DAT has been operationally deployed since 2019 and has successfully identified AAT requiring treatment, thereby improving casualties' hearing and reducing unnecessary repatriation.

Size and position of the reproductive tract impacts fertility outcomes and pregnancy losses in lactating dairy cows.

There are multiple factors that contribute to reduced fertility in lactating dairy cows. Recently, a reproductive tract size and position score (SPS) system was developed as a management tool to identify dairy cows with decreased fertility. The aim of this study was to evaluate the association between the SPS on fertility outcomes such as ovulation failure, pregnancy per artificial insemination (P/AI), concentration of pregnancy-associated glycoproteins (PAGs), and pregnancy loss in lactating dairy cows. Primiparous and multiparous lactating Holstein cows (n = 869) were enrolled at two locations. Location 1 (Loc. 1) in Minas Gerais, Brazil (n = 613) and location 2 (Loc. 2) in Agassiz, British Columbia, Canada (n = 256). At the time of AI (d 0), cows were classified as SPS (small [SPS1], medium [SPS2], or large [SPS3] sized reproductive tract) and ovulation failure was determined at 48 h and 7 d post-AI via ultrasonography (Loc. 2 only). Blood samples were collected on d 24 and 31 of gestation for quantification of PAGs and pregnancy diagnosis was performed via ultrasonography at d 31 and 60 post-AI (Loc. 1) and at d 31 ± 3 and 60 ± 3 post-AI (Loc. 2). Cows diagnosed pregnant at d 31 post-AI but not pregnant at d 60 were defined to have undergone late embryonic pregnancy loss. Parity was found to impact SPS (P < 0.01), as primiparous cows had a higher frequency of SPS1 and lower frequency of SPS3 when compared with multiparous cows (SPS1: 42.6 vs. 15.0%; SPS3: 7.0 vs. 22.0%, respectively). Cows classified as SPS3 had greater ovulation failure at 48 h (P = 0.04) and 7 d post-AI (P = 0.05). Cows classified as SPS1 had greater P/AI when compared to SPS2 and SPS3 (45.9 ± 3.3 vs. 37.4 ± 2.6 and 29.1 ± 3.5%, respectively; P = 0.004). There was no interaction between parity and SPS on P/AI. Pregnancy loss between 31 and 60 d post-AI was increased in cows classified as SPS3 compared to SPS2 and SPS1 (24.3 ± 0.05 vs. 11.6 ± 0.02 and 9.4 ± 0.02%, respectively; P = 0.04). Cows classified as SPS1 and SPS2 had greater concentrations of PAGs at 31 d post-AI when compared to SPS3 at both Loc.1 (P < 0.01) and Loc. 2 (P < 0.01). There was no interaction between SPS and pregnancy loss on PAGs at 24 and 31 d post- AI for either Loc. 1 (P = 0.75 and P = 0.76, respectively) or Loc. 2 (P = 0.61 and P = 0.81, respectively). In conclusion, cows that were classified as SPS3 had greater ovulation failure, reduced P/AI, similar concentrations of PAG on d 24, but decreased on d 31, and a greater incidence of pregnancy loss. Thus, size and position of the reproductive tract is associated with fertility and this scoring system could be used to make reproductive management decisions on dairy operations.

Pregnancy loss in beef cattle: A meta-analysis.

Pregnancy loss in beef cattle causes both management and economic challenges to a producer. A meta-analysis was conducted to quantify reproductive failures that occur during fertilization, early embryonic development, and late embryonic/early fetal development periods of gestation in beef cattle. The meta-analysis included more than 56,000 diagnostic records in 159 studies from 48 papers with 12 studies included in fertilization and pre- blastocyst loss analysis (FERT; days 1-7 of gestation), 107 in early embryo (EEM; days 7-32), and 40 in late embryo/early fetal period (LEF; days 32-100) analysis. Although fertilization rates are reportedly high in beef cattle, significant developmental failure occurs within the first 7 days of gestation. Approximately 28.4 % of embryos will not develop past day 7 of gestation with most embryonic losses occurring before day 4. By the conclusion of the first month of gestation, 47.9 % of cows submitted to a single insemination at day 0 will not be pregnant. Overall, LEF between days 32-60 and 100 was 5.8 %. Bos indicus animals had greater (P = 0.001) EEM compared to Bos taurus, but there was no difference (P = 0.39) for the LEF period between subspecies. Primiparous cows had greater EEM (P = 0.002) compared to nulliparous heifers and multiparous cows; and nulliparous heifers had a greater LEF compared to primiparous and multiparous cows (P = 0.048). Collectively, these cumulative findings provide a baseline assessment of pregnancy loss specific to beef cattle.

Viscoelastic drops moving on hydrophilic and superhydrophobic surfaces.

So-called "superhydrophobic" surfaces are strongly non-wetting such that fluid droplets very easily roll off when the surface is tilted. Our interest here is in understanding if this is also true, all else held equal, for viscoelastic fluid drops. We study the movement of Newtonian and well-characterised constant-viscosity elastic liquids when various surfaces, including hydrophilic (smooth glass), weakly hydrophobic (embossed polycarbonate) and superhydrophobic surfaces (embossed PTFE), are impulsively tilted. Digital imaging is used to record the motion and extract drop velocity. Optical and SEM imaging is used to probe the surfaces. In comparison with "equivalent" Newtonian fluids (same viscosity, density surface tension and contact angles), profound differences for the elastic fluids are only observed on the superhydrophobic surfaces: the elastic drops slide at a significantly reduced rate and complex branch-like patterns are left on the surface by the drop's wake including, on various scales, beads-on-a-string-like phenomena. The strong viscoelastic effect is caused by stretching filaments of fluid from isolated islands, residing at pinning sites on the surface pillars, of order ∼30 µm in size. On this scale, the local strain rates are sufficient to extend the polymer chains, locally increasing the extensional viscosity of the solution, retarding the drop.

Clinical relevance of findings in trials of CBT for depression.

Cognitive behavioural therapy (CBT) is beneficial in depression. Symptom scores can be translated into Clinical Global Impression (CGI) scale scores to indicate clinical relevance. We aimed to assess the clinical relevance of findings of randomised controlled trials (RCTs) of CBT in depression. We identified RCTs of CBT that used the Hamilton Rating Scale for Depression (HAMD). HAMD scores were translated into Clinical Global Impression - Change scale (CGI-I) scores to measure clinical relevance. One hundred and seventy datasets from 82 studies were included. The mean percentage HAMD change for treatment arms was 53.66%, and 29.81% for control arms, a statistically significant difference. Combined active therapies showed the biggest improvement on CGI-I score, followed by CBT alone. All active treatments had better than expected HAMD percentage reduction and CGI-I scores. CBT has a clinically relevant effect in depression, with a notional CGI-I score of 2.2, indicating a significant clinical response. The non-specific or placebo effect of being in a psychotherapy trial was a 29% reduction of HAMD.

Metabolic and reproductive characteristics of replacement beef heifers subjected to an early-weaning regimen involving high-concentrate feeding.

In an effort to better understand the consequences of early weaning (EW) for replacement beef heifers, a two-phase experiment was conducted investigating the impact on metabolic function and documenting reproductive characteristics. In phase 1, Angus×Simmental heifers (n=35) were stratified by BW and sire, and randomly assigned to either a normal weaning (NW, n=18) or EW (n=17) treatment. EW heifers were weaned at 107±3 days of age and provided access to a concentrate-based ration ad libitum with limit-fed mixed grass hay. NW heifers remained with their dams until 232±3 days of age, at which point heifers from both treatments were comingled and grazed on mixed summer pasture. Following NW, weekly blood samples were collected from all heifers for progesterone analyses used to determine the onset of puberty. Pelvic and ovarian size was measured before breeding. All heifers were subjected to an estrous synchronization protocol with timed artificial insemination (AI) at 437±4 days of age. During phase 2 of the experiment, a subset of pregnant heifers (n=16) were divided into two replicates and subjected to a glucose tolerance test, epinephrine challenge and progesterone clearance analysis. Neither age nor BW at puberty differed between EW and NW heifers. Likewise, no differences in pelvic area or ovarian size were observed. Thus, it appears that the reproductive maturity of EW and NW heifers was similar. Heifers studied during phase 2 of the experiment were restricted to those that had become pregnant to their first AI. Within this cohort, EW heifers tended to have lower overall circulating progesterone concentrations than those that were NW (P=0.14). Aspects of glucose and insulin dynamics were also altered, as EW heifers tended to have lower baseline glucose concentrations (P=0.10) despite similar baseline insulin concentrations. Compared with NW heifers, EW heifers had lower insulin area under the curve (P<0.05), which was partly the result of a tendency for lower peak insulin concentrations (P=0.11). Results of the glucose tolerance test indicate that a lesser insulin response was necessary to properly clear the glucose in the EW heifers, suggesting enhanced insulin sensitivity. Collectively, these results indicate that EW is not detrimental for the growth or reproductive development of replacement beef heifers, although some differences in glucose and insulin dynamics persist into adulthood.

A simplified method to estimate body growth parameters of the European eel Anguilla anguilla.

A simple approach is proposed to fit a body growth model for the European eel Anguilla anguilla to data-poor case studies. The model is a modified von Bertalanffy curve allowing for delayed sex determination and sexual dimorphism. The proposed procedure provides preliminary estimates of model parameters on the basis of average age and body length of silver eels.

A systematic review of the clinical relevance of repetitive transcranial magnetic stimulation.

OBJECTIVE: Repetitive transcranial magnetic stimulation (rTMS) is an approved treatment for depression. The clinical relevance of its efficacy is unclear. The clinical relevance of findings in the rTMS literature was assessed by translating Hamilton Depression Rating Scale (HAMD) data into Clinical Global Impression-Improvement scale (CGI-I) scores. METHOD: We performed electronic searches of MEDLINE, Embase, PsycINFO, PubMed and Cochrane Central Register of Controlled Trials for RCTs and non-RCT trials on rTMS using Hamilton Depression Rating Scale (HAMD). Articles were included if published in English before January 2014. We translated HAMD scores into nominal CGI-I scores for rTMS for depression and for treatment-resistant depression (TRD). RESULTS: About 960 abstracts were retrieved. Sixty-three studies were included, yielding 130 study arms. For depression, the mean percentage change in HAMD scores in all sham-controlled rTMS treatment arms was 35.63 (SD 16.35) and for sham-rTMS 23.33 (SD 16.51). For TRD, active rTMS in sham-controlled studies showed a mean HAMD percentage reduction of 45.21 (SD 10.94) versus 25.04 (SD 17.55) for sham-rTMS. When aggregated scores were translated into notional CGI-I scores, for the treatment of depression, the notional CGI-I score difference between rTMS and sham-rTMS was 0.5 in favour of rTMS; for TRD, it was 0.75 in favour of rTMS. Differences between rTMS and sham-rTMS were bigger when all study arms were combined. CONCLUSION: Whilst rTMS appears to be efficacious for both non-refractory and treatment-resistant depression, the clinical relevance of its efficacy is doubtful.

Distribution, prevalence and intensity of Anguillicola crassus (Nematoda) infection in Anguilla anguilla in the Republic of Ireland.

This study is the first comprehensive documentation of the geographical range of Anguillicola crassus in its host, the European eel Anguilla anguilla, in the Republic of Ireland. The prevalence and intensity of infections across 234 sites and 93 river basins in Ireland comprising rivers, lakes and transitional waters (estuaries) were analysed. While only 32% of the river basins were affected by this nematode, they correspond to 74% of the total wetted area. Significant differences in infection levels among water body types were found with lakes and transitional waters yielding the highest values, which can be attributed to the proportions of juvenile (total length, L(T) < 300 mm) A. anguilla caught. There were no significant differences in infection levels between water body types for adult A. anguilla or between sexes for any water body type. Prevalence was significantly lower in juvenile compared with adult A. anguilla captured in rivers and a positive correlation between infection levels and host size-classes was found. Future efforts should focus on monitoring the spread of A. crassus infections and assessing the swimbladder health of A. anguilla in Ireland.

Application of congener based multi-matrix profiling techniques to identify potential PCDD/F sources in environmental samples from the Burrishoole Catchment in the West of Ireland.

Homologue and congener profiles of PCDD/Fs in eels, passive sampler and sediment extracts from the Burrishoole, a rural upland catchment on the western Irish seaboard were compared with potential PCDD sources. ΣPCDD/F levels in eels ranged from 2.9 to 25.9 pg g(-1) wet weight, which are elevated compared to other Irish locations. The OCDD congener dominated the pattern of ΣPCDD/Fs in all matrices from Burrishoole. Passive samplers were successfully deployed to identify for the first time the presence in the water column of PCDD/Fs and dimethoxylated octachlorodiphenyl ether (diMeOoctaCDE), impurities found in pentachlorophenol (PCP) production. Principal component analysis (PCA) identified similarities between PCDD/F profiles in technical PCP mixtures and environmental samples from the Burrishoole region. Results strongly suggest residual PCDD contamination associated with historic local use of a dioxin contaminated product in the catchment area, with pentachlorophenol a strong candidate.

3-M syndrome: a growth disorder associated with IGF2 silencing.

3-M syndrome is an autosomal recessive disorder characterised by pre- and post-natal growth restriction, facial dysmorphism, normal intelligence and radiological features (slender long bones and tall vertebral bodies). It is known to be caused by mutations in the genes encoding cullin 7, obscurin-like 1 and coiled-coil domain containing 8. The mechanisms through which mutations in these genes impair growth are unclear. The aim of this study was to identify novel pathways involved in the growth impairment in 3-M syndrome. RNA was extracted from fibroblast cell lines derived from four 3-M syndrome patients and three control subjects, hybridised to Affymetrix HU 133 plus 2.0 arrays with quantitative real-time PCR used to confirm changes found on microarray. IGF-II protein levels in conditioned cell culture media were measured by ELISA. Of the top 10 downregulated probesets, three represented IGF2 while H19 was identified as the 23rd most upregulated probeset. QRT-PCR confirmed upregulation of H19 (P<0.001) and downregulation of IGF2 (P<0.001). Levels of IGF-II secreted into conditioned cell culture medium were higher for control fibroblasts than those for 3-M fibroblasts (10.2±2.9 vs 0.6±0.9 ng/ml, P<0.01). 3-M syndrome is associated with a gene expression profile of reduced IGF2 expression and increased H19 expression similar to that found in Silver-Russell syndrome. Loss of autocrine IGF-II in the growth plate may be associated with the short stature seen in children with 3-M syndrome.

Beaufort trout MicroPlex: a high-throughput multiplex platform comprising 38 informative microsatellite loci for use in resident and anadromous (sea trout) brown trout Salmo trutta genetic studies.

A flexible panel consisting of 38 informative microsatellite markers for Salmo trutta is described. These markers were selected from a pool of over 150 candidate loci that can be readily amplified in four multiplex PCR groups but other permutations are also possible. The basic properties of each markers were assessed in six population samples from both the Burrishoole catchment, in the west of Ireland, and Lough Neagh, in Northern Ireland. A method to assess the relative utility of individual markers for the detection of population genetic structuring is also described. Given its flexibility, technical reliability and high degree of informativeness, the use of this panel of markers is advocated as a standard for S. trutta genetic studies.

Pseudohypoparathyroidism type 1b due to paternal uniparental disomy of chromosome 20q.

CONTEXT: Pseudohypoparathyroidism type 1b (PHP1b) is the result of end-organ resistance to PTH and other hormones such as TSH in the absence of any features of Albright's hereditary osteodystrophy. Patients with PHP1b show imprinting abnormalities at the complex GNAS locus. The molecular cause of autosomal dominant familial PHP1b has been well-defined with identification of microdeletions within the GNAS locus or the nearby STX16, but the molecular mechanism of the GNAS imprinting defects in sporadic PHP1b cases remains elusive. OBJECTIVE: We investigated the underlying molecular mechanism of GNAS imprinting defects in two patients with sporadic PHP1b. RESULTS: We identified paternal uniparental disomy of the long arm of chromosome 20 (patUPD20) in two unrelated patients with sporadic PHP1b. This provides an explanation for the patients' GNAS methylation abnormalities and hormone resistance. Our data and a review of the six published cases of patUPD20 suggest that high birth weight and/or early-onset obesity and macrocephaly may also represent features of patUPD20. CONCLUSION: We suggest that patUPD20 should be considered in the evaluation of patients with sporadic PHP1b.

Epigenotype-phenotype correlations in Silver-Russell syndrome.

BACKGROUND: Silver-Russell syndrome (SRS) is characterised by intrauterine growth restriction, poor postnatal growth, relative macrocephaly, triangular face and asymmetry. Maternal uniparental disomy (mUPD) of chromosome 7 and hypomethylation of the imprinting control region (ICR) 1 on chromosome 11p15 are found in 5-10% and up to 60% of patients with SRS, respectively. As many features are non-specific, diagnosis of SRS remains difficult. Studies of patients in whom the molecular diagnosis is confirmed therefore provide valuable clinical information on the condition. METHODS: A detailed, prospective study of 64 patients with mUPD7 (n=20) or ICR1 hypomethylation (n=44) was undertaken. RESULTS AND CONCLUSIONS: The considerable overlap in clinical phenotype makes it difficult to distinguish these two molecular subgroups reliably. ICR1 hypomethylation was more likely to be scored as 'classical' SRS. Asymmetry, fifth finger clinodactyly and congenital anomalies were more commonly seen with ICR1 hypomethylation, whereas learning difficulties and referral for speech therapy were more likely with mUPD7. Myoclonus-dystonia has been reported previously in one mUPD7 patient. The authors report mild movement disorders in three further cases. No correlation was found between clinical severity and level of ICR1 hypomethylation. Use of assisted reproductive technology in association with ICR1 hypomethylation seems increased compared with the general population. ICR1 hypomethylation was also observed in affected siblings, although recurrence risk remains low in the majority of cases. Overall, a wide range of severity was observed, particularly with ICR1 hypomethylation. A low threshold for investigation of patients with features suggestive, but not typical, of SRS is therefore recommended.

Further refinement of the critical minimal genetic region for the imprinting disorder 6q24 transient neonatal diabetes.

AIMS/HYPOTHESIS: Transient neonatal diabetes (TND) is associated with overexpression of genes within a critical region on 6q24. This study aims to refine the boundaries of this region to reduce the number of potential candidate genes for 6q24 TND. METHODS: Fifteen patients with transient neonatal diabetes and submicroscopic chromosome 6 duplications were investigated. The duplications were confirmed by microsatellite analysis and subsequently mapped using tiled chromosome 6 array Comparative Genomic Hybridisation (aCGH) and MLPA. Duplication boundaries were compared to identify the minimal shared region of duplication. These data were then used with available clinical data to identify associations between size of 6q24 duplication and severity of TND phenotype. RESULTS: Alignment of the minimal region of duplication to the human genome reduced the minimal TND critical region, formerly estimated at 440 kb, to 160-173 kb, revealing PLAGL1 (pleiomorphic adenoma gene-like 1) and HYMAI (imprinted in hydatidiform mole) to be the only genes wholly included therein. Additionally, the complete paternal duplication of a region containing the theoretical protein FAM164B was associated with the severe growth restriction observed in 6q24 duplication patients. CONCLUSIONS/INTERPRETATION: This study has significantly reduced the critical region associated with 6q24 TND. It has eliminated several previous TND candidate genes, leaving the overlapping imprinted genes PLAGL1 and HYMAI as the only remaining complete candidate genes for 6q24 TND. Moreover, these data provide the first evidence that an additional region, encompassing the theoretical protein FAM164B, may have a critical role in the growth restriction phenotype observed in many 6q24 TND patients.