Generation of two isogenic iPSC lines from a healthy male donor of European ancestry.

We have created two isogenic iPSC lines from fibroblasts of a healthy male donor of European ancestry. The cell lines express common pluripotency markers, are free of chromosomal aberrations and are able to differentiate into cells of all three germ layers. These iPSC are now a resource for genome editing with the aim of creating models of genetic disorders without having to depend on patient cells.

Generation of two iPSC lines (MHHi001-A-12 and MHHi001-A-13) carrying biallelic truncating mutations at the 3'-end of SRCAP using CRISPR/Cas9.

Non-Floating Harbour Syndrome (FLHS) neurodevelopmental disorder (NDD) is a recently described disorder caused by mutations in certain regions of the SRCAP gene. We generated two iPSC lines that contain truncating mutation on both alleles at the 3'-end of SRCAP using CRISPR/Cas9 technology. Both cell lines are pluripotent, differentiate into the 3 germ layers and contain no genomic aberrations or off-target modifications. The cell lines form part of a human disease model to investigate the effects of truncating mutations in different regions of SRCAP.

Generation of an iPSC line (UMGWi001-B) from a patient with Floating-Harbor Syndrome (FLHS) carrying a heterozygous SRCAP mutation (p.Arg2444).

Floating-Harbor syndrome (FLHS) is a rare genetic disease caused by mutations in the SRCAP gene. Here, we generated an induced pluripotent stem cell line from gingival fibroblasts of a male patient with a heterozygous mutation in exon 34 of the SRCAP gene (c.7330C > T, p.Arg2444*). The iPSC colonies have an atypical morphology with diffuse borders and disintegrate quickly upon touch. Still, the cell line expresses pluripotency markers and differentiates into three germ layers. The cell line can be used as patient-specific disease model and help elucidate the molecular mechanisms involving SRCAP in the context of FLHS.

X-linked congenital ptosis and associated intellectual disability, short stature, microcephaly, cleft palate, digital and genital abnormalities define novel Xq25q26 duplication syndrome.

Submicroscopic duplications along the long arm of the X-chromosome with known phenotypic consequences are relatively rare events. The clinical features resulting from such duplications are various, though they often include intellectual disability, microcephaly, short stature, hypotonia, hypogonadism and feeding difficulties. Female carriers are often phenotypically normal or show a similar but milder phenotype, as in most cases the X-chromosome harbouring the duplication is subject to inactivation. Xq28, which includes MECP2 is the major locus for submicroscopic X-chromosome duplications, whereas duplications in Xq25 and Xq26 have been reported in only a few cases. Using genome-wide array platforms we identified overlapping interstitial Xq25q26 duplications ranging from 0.2 to 4.76 Mb in eight unrelated families with in total five affected males and seven affected females. All affected males shared a common phenotype with intrauterine- and postnatal growth retardation and feeding difficulties in childhood. Three had microcephaly and two out of five suffered from epilepsy. In addition, three males had a distinct facial appearance with congenital bilateral ptosis and large protruding ears and two of them showed a cleft palate. The affected females had various clinical symptoms similar to that of the males with congenital bilateral ptosis in three families as most remarkable feature. Comparison of the gene content of the individual duplications with the respective phenotypes suggested three critical regions with candidate genes (AIFM1, RAB33A, GPC3 and IGSF1) for the common phenotypes, including candidate loci for congenital bilateral ptosis, small head circumference, short stature, genital and digital defects.

Methylation and expression analyses of the 7q autism susceptibility locus genes MEST , COPG2, and TSGA14 in human and anthropoid primate cortices.

The autism susceptibility locus on human chromosome 7q32 contains the maternally imprinted MEST and the non-imprinted COPG2 and TSGA14 genes. Autism is a disorder of the 'social brain' that has been proposed to be due to an overbalance of paternally expressed genes. To study regulation of the 7q32 locus during anthropoid primate evolution, we analyzed the methylation and expression patterns of MEST, COPG2, and TSGA14 in human, chimpanzee, Old World monkey (baboon and rhesus macaque), and New World monkey (marmoset) cortices. In all human and anthropoid primate cortices, the MEST promoter was hemimethylated, as expected for a differentially methylated imprinting control region, whereas the COPG2 and TSGA14 promoters were completely demethylated, typical for transcriptionally active non-imprinted genes. The MEST gene also showed comparable mRNA expression levels in all analyzed species. In contrast, COPG2 expression was downregulated in the human cortex compared to chimpanzee, Old and New World monkeys. TSGA14 either showed no differential regulation in the human brain compared to chimpanzee and marmoset or a slight upregulation compared to baboon. The human-specific downregulation supports a role for COPG2 in the development of a 'social brain'. Promoter methylation patterns appear to be more stable during evolution than gene expression patterns, suggesting that other mechanisms may be more important for inter-primate differences in gene expression.

A high density of human communication-associated genes in chromosome 7q31-q36: differential expression in human and non-human primate cortices.

The human brain is distinguished by its remarkable size, high energy consumption, and cognitive abilities compared to all other mammals and non-human primates. However, little is known about what has accelerated brain evolution in the human lineage. One possible explanation is that the appearance of advanced communication skills and language has been a driving force of human brain development. The phenotypic adaptations in brain structure and function which occurred on the way to modern humans may be associated with specific molecular signatures in today's human genome and/or transcriptome. Genes that have been linked to language, reading, and/or autism spectrum disorders are prime candidates when searching for genes for human-specific communication abilities. The database and genome-wide expression analyses we present here revealed a clustering of such communication-associated genes (COAG) on human chromosomes X and 7, in particular chromosome 7q31-q36. Compared to the rest of the genome, we found a high number of COAG to be differentially expressed in the cortices of humans and non-human primates (chimpanzee, baboon, and/or marmoset). The role of X-linked genes for the development of human-specific cognitive abilities is well known. We now propose that chromosome 7q31-q36 also represents a hot spot for the evolution of human-specific communication abilities. Selective pressure on the T cell receptor beta locus on chromosome 7q34, which plays a pivotal role in the immune system, could have led to rapid dissemination of positive gene variants in hitchhiking COAG.

Chromosomal copy number changes in patients with non-syndromic X linked mental retardation detected by array CGH.

Several studies have shown that array based comparative genomic hybridisation (CGH) is a powerful tool for the detection of copy number changes in the genome of individuals with a congenital disorder. In this study, 40 patients with non-specific X linked mental retardation were analysed with full coverage, X chromosomal, bacterial artificial chromosome arrays. Copy number changes were validated by multiplex ligation dependent probe amplification as a fast method to detect duplications and deletions in patient and control DNA. This approach has the capacity to detect copy number changes as small as 100 kb. We identified three causative duplications: one family with a 7 Mb duplication in Xp22.2 and two families with a 500 kb duplication in Xq28 encompassing the MECP2 gene. In addition, we detected four regions with copy number changes that were frequently identified in our group of patients and therefore most likely represent genomic polymorphisms. These results confirm the power of array CGH as a diagnostic tool, but also emphasise the necessity to perform proper validation experiments by an independent technique.

Cloning, characterization and chromosomal localization of the Sus scrofa SLC31A1 gene.

Copper is an essential element necessary for normal function of numerous enzymes in all living organisms. Uptake of copper into the cell is thought to occur through the membrane protein, SLC31A1 (CTR1), which has been described in a variety of species including yeast, human and mouse. In this study, we present cloning, gene structure, chromosomal localization and expression pattern of the Sus scrofa SLC31A1 gene, which encodes a 189 amino acid protein. The (SSC) SLC31A1 gene is organized in four exons and spans an approximately 2.3 kb genomic region. We have localized the gene to chromosome 1q28-q2.13 using a somatic cell hybrid panel. This region shows conservation of synteny with human chromosome 9, where the human SLC31A1 (CTR1) gene has been localized. Expression studies suggest that SLC31A1 mRNA is transcribed in all tissues examined.

The 5S rRNA genes in Macaca fascicularis are organized in two large tandem repeats.

The 5S rRNA genes in Macaca fascicularis are organized in tandem repeats which are unusually large and complex. The tandem repeats consist of a 7.3 kb DNA fragment with two 5S rRNA genes linked to a 4.3 kb fragment with one gene. The total number of genes in the repeats is 50-100 per haploid genome. The 5S rDNA has an external promoter, the D box, in the same position relative to transcription start as the human gene but is transcribed less efficiently than a human 5S rRNA gene in a HeLa cell extract.

Similar splice-site mutations of the ATP7A gene lead to different phenotypes: classical Menkes disease or occipital horn syndrome.

More than 150 point mutations have now been identified in the ATP7A gene. Most of these mutations lead to the classic form of Menkes disease (MD), and a few lead to the milder occipital horn syndrome (OHS). To get a better understanding of molecular changes leading to classic MD and OHS, we took advantage of the unique finding of three patients with similar mutations but different phenotypes. Although all three patients had mutations located in the splice-donor site of intron 6, only two of the patients had the MD phenotype; the third had the OHS phenotype. Fibroblast cultures from the three patients were analyzed by reverse transcriptase (RT)-PCR to try to find an explanation of the different phenotypes. In all three patients, exon 6 was deleted in the majority of the ATP7A transcripts. However, by RT-PCR amplification with an exon 6-specific primer, we were able to amplify exon 6-containing mRNA products from all three patients, even though they were in low abundance. Sequencing of these products indicated that only the patient with OHS had correctly spliced exon 6-containing transcripts. We used two different methods of quantitative RT-PCR analysis and found that the level of correctly spliced mRNA in this patient was 2%-5% of the level found in unaffected individuals. These findings indicate that the presence of barely detectable amounts of correctly spliced ATP7A transcript is sufficient to permit the development of the milder OHS phenotype, as opposed to classic MD.

Influence of feeding intensity, grazing and finishing feeding on meat and eating quality of young bulls and the relationship between muscle fibre characteristics, fibre fragmentation and meat tenderness.

Forty-one autumn-born Friesian bull calves were allocated to two production systems (Extensive='E'and Intensive='I'). In the E-system, animals were loose-housed and fed a roughage-based diet from October to May, followed by a grazing period from May to October. Ten animals were slaughtered directly from pasture in October [360 kg body weight (BW)] and 11 after a 10-week finishing period in tie-stalls (460 kg). The E-bulls were compared with intensively-fed tie-stall-housed young bulls (I) slaughtered at comparable weights (360 kg, n=11 and 460 kg, n=9). The myofibril fragmentation index (MFI) was measured 24 h post mortem in semitendinosus (ST), longissimus dorsi (LD), and supraspinatus (SU) muscles, and meat quality characteristics and sensory evaluation of LD were performed on aged meat. Intramuscular fat content was lower (P<0.001) in all three muscles of E- compared with I-bulls. MFI of ST and LD was lower in E-bulls compared with I-bulls, but only at 360 kg. In contrast, MFI of SU was higher in E- compared with I-bulls at 360 kg. In E- compared with I-bulls, shear force value of ST was higher (P<0.003) at 360 kg, but not at 460 kg. Panel scores for tenderness, taste and juiciness were all lower (P<0.006 to 0.001) and remarks for off-flavour higher in E- compared with I-bulls, the effects being most pronounced at 360 kg. A 10-week finishing period improved all meat and eating quality characteristics of E-bulls. In LD, the correlation between MFI and tenderness was 0.79 (P<0.001), which indicates a potential of MFI as an early predictor of tenderness.

Relation between gender, etiology and survival in patients with symptomatic heart failure.

OBJECTIVES: This study investigated the relation between gender, etiology and survival in patients with symptomatic heart failure. BACKGROUND: Previous work provides conflicting results concerning the relation between gender, clinical characteristics and survival in patients with heart failure. METHODS: We examined the relation of these factors in 557 patients (380 men, 177 women) who had symptomatic heart failure, predominantly nonischemic in origin (68%) and typically associated with severe left ventricular dysfunction. RESULTS: Follow-up data were available in 99% of patients (mean follow-up period 2.4 years, range 1 day to 10 years) after study entry, and 201 patients reached the primary study end point of all-cause mortality. By life-table analysis, women were significantly less likely to reach this primary end point than men (p < 0.001). A significant association was found between female gender and better survival (p < 0.001), which depended on the primary etiology of heart failure (p = 0.008 for the gender-etiology interaction) but not on baseline ventricular function. Women survived longer than men when heart failure was due to nonischemic causes (men vs. women: relative risk [RR] 2.36, 95% confidence interval [CI] 1.59 to 3.51, p < 0.001). In contrast, outcome appeared similar when heart failure was due to ischemic heart disease (men vs. women: RR 0.85, 95% CI 0.45 to 1.61, p = 0.651). CONCLUSIONS: Women with heart failure due to nonischemic causes had significantly better survival than men with or without coronary disease as their primary cause of heart failure.

Growth factor expression and effects of amrinone in monocrotaline-induced pulmonary hypertension in rats.

Platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) have been implicated in myointimal proliferative arteriopathy, a lesion seen in monocrotaline-induced pulmonary hypertension (MIPH). The purpose of this study was to examine the expression of PDGF and bFGF in the lungs of rats given monocrotaline and to examine the effects of amrinone on the hearts and lungs of these rats. Twenty-four 26-day-old rats were randomized to receive either monocrotaline (approximately 3.6 mg/kg/d) or no monocrotaline and concomitantly to receive either amrinone (100 mg/kg/d) or no amrinone for 21 days. Lungs were examined for immunohistochemical evidence of PDGF and bFGF, and hearts were examined for effects of pulmonary hypertension and amrinone. Immunohistochemical staining of lungs showed no evidence of PDGF except in bronchioles. bFGF staining was similar between groups (no monocrotaline 25%, monocrotaline 27%, monocrotaline and amrinone 22%), and the staining was confined to the arterial walls. Rats given monocrotaline showed significantly greater right ventricular (RV) weight (0.13 +/- 0.02 g versus 0.23 +/- 0.04 g [mean +/- SD], P < 0.001), right ventricular/left ventricular (RV/LV) weight ratio (0.29 +/- 0.06 versus 0.59 +/- 0.1, P < 0.001), and lung/body weight ratio (0.006 +/- 0.001 versus 0.01 +/- 0.003, P < 0.05) than controls. Rats given monocrotaline and amrinone were not significantly different from rats given only monocrotaline with regard to RV weight, RV/LV weight ratio, or lung/body weight ratio. We conclude that the vasculopathy seen in MIPH is not associated with the presence of PDGF or bFGF, suggesting that other growth factors may mediate this process. The course of MIPH is not altered by amrinone.

Effects of growth hormone and ovariectomy on performance, serum hormones, insulin-like growth factor-binding proteins, and muscle fiber properties of prepubertal Friesian heifers.

Sixteen prepubertal Friesian heifers were used to examine the effect of bovine growth hormone (GH) and ovariectomy (OVX) at 2.5 mo of age (2 x 2 factorial design) on growth, carcass quality, and fiber types, capillarization, and metabolic potentials of the longissimus muscle, and serum concentrations of estradiol-17beta (E2beta), insulin, GH, IGF-I, and IGF-binding proteins (IGFBP). Treatment with GH (15 mg/d) started at 147 +/- 3 kg BW and lasted for 15 wk. Heifers were fed a mixed roughage-based diet. Growth hormone increased ADG (P < .001), improved gain:feed (P < .007), and had a small but positive influence on lean accretion. Growth hormone reduced fat thickness (P < .009), carcass fat trim (P < .009) and i.m. fat (P < .09). Ovariectomy did not affect performance but increased dressing percentage (P < .03), full rib weight (P < .003), and fat thickness (P < .04). Ovariectomy reduced E2beta (P < .001) and insulin (P < .02), and increased the 32-kDa IGFBP (IGFBP-2) (P < .09). Growth hormone treatment increased GH, IGF-I, the 28-kDa IGFBP, and the 40- to 43-kDa IGFBP (IGFBP-3) (P < .004 or P < .001). Neither GH nor ovariectomy affected the proportion and relative area of the individual muscle fiber types, but GH tended to increase type I fiber area (P < .10). Number of capillaries per fiber increased in OVX GH-treated heifers (GH x OVX interaction, P < .02). Activities of citrate synthetase were higher in GH-treated (P < .05) and OVX (P < .02) heifers, indicating increased oxidative capacity of the longissimus muscle. The effects of GH on performance and carcass fattening were in accordance with the observed hormonal changes. When slaughter occurs before puberty, ovariectomy has no effect on performance, only few effects on carcass quality, and small effects on hormone concentrations.

Submersion injuries in children younger than 5 years in urban Utah.

Submersion injuries of children younger than 5 years in 4 urban Utah counties from 1984 through 1988 were studied retrospectively to identify associated risk factors. Infants younger than 1 year had the highest rates of both submersion injuries and deaths. The incidence of bathtub drownings was 2 to 3 times higher than reported national rates. All bathtub drownings occurred while the victim was bathing with a young sibling (10 months to 7 years of age) without adult supervision. All drownings in pools and moving bodies of water (rivers, irrigation ditches) resulted from unintentional falls into the water rather than from swimming and wading activities. Drowning prevention strategies should focus on educating parents about the risk of young children bathing with siblings in the absence of adult supervision and fencing regulations for pools and open bodies of moving water.

In vitro stability of growth hormone releasing factor (GRF) analogs in porcine plasma.

The stability of growth-hormone releasing factor (growth regulating factor; GRF) analogs in porcine plasma was examined. GRF analogs were incubated in porcine plasma at 37 degrees C, extracted and subsequently analyzed using high performance liquid chromatography (HPLC). GRF(1-29)-NH2 was rapidly broken down in the plasma with a degradation rate of t1/2 = 13 min. The primary degradation product was identified as GRF(3-29)-NH2. Substitution of Gly15 by Ala15 slightly prolonged the plasma half-life (t1/2 = 17 min) and the major degradative fragment was found to be [Ala15]GRF(3-29)-NH2. The cleavage between the 2 and 3 position of the peptide was not inhibited by trasylol at a concentration of 1,000 KIU/ml but was dramatically reduced by the combined use of diprotin A and trasylol. Absence of the free amino group at the N-terminus and/or substitution of a D-amino acid residue at the penultimate position completely prevented cleavage between the 2 and 3 position in the structural linear GRF analogs. Side-chain to side-chain cyclization between Asp8 and Lys12 amino acid residues significantly improved the stability of GRF in plasma with t1/2 greater than 2 hr. An additional stability was provided by substitution of D-Ala2 for Ala2 in the structural cyclic analog. Cyclization between Lys21 and Asp25 also improved the stability of the GRF peptide in the plasma. Stability was further enhanced by the presence of D-Ala2 or by forming a dicyclic analog through an additional linkage between Asp8 and Lys12.

Processing open and closed class-headed nonwords: left hemisphere support for separate vocabularies.

Evidence from studies of sentence production and comprehension points to a distinction between the open and closed class vocabularies. Data from the neurologically impaired suggest the left hemisphere selectively supports this distinction. This study further examines the vocabulary distinction by presenting to the different visual hemifields nonwords embedded with open or closed class portions, and asking normal subjects to make lexical decisions. Results indicate the vocabulary distinction is evident with RVF, but not LVF, presentations. This is discussed in terms of the use to which open and closed class words are put in sentence processing and also bears on the issue of the distinct cognitive styles associated with the different cerebral hemispheres.