Molecular characterization of breakpoints in patients with holoprosencephaly and definition of the HPE2 critical region 2p21

Holoprosencephaly (HPE) is a common developmental defect involving the brain and face in humans. Cytogenetic deletions in patients with HPE have localized one of the HPE genes (HPE2) to the chromosomal region 2p21. Here we report the molecular genetic characterization of nine HPE patients with cytogenetic deletions or translocations involving 2p21. We have determined the parental origin of the deleted chromosomes and defined the HPE2 critical region between D2S119 and D2S88/D2S391. As a first step towards cloning the HPE2 gene which is crucial for normal brain development we have constructed a YAC contig which spans the smallest region of deletion overlap. Several of these YACs could be identified which span three different 2p21 breakpoints in HPE patients. These YACs narrow the HPE2 critical region to less than 1 Mb and are now being further analyzed to identify the gene causing holoprosencephaly on chromosome 2.      

Birth from cryopreserved embryos following in-vitro maturation of oocytes and intracytoplasmic sperm injection

This case report describes the birth of a baby following the transfer of cryopreserved embryos generated from intracytoplasmic sperm injection (ICSI) carried out on the second day after oocyte pick-up of in-vitro-matured metaphase I and germinal vesicle stage oocytes. The couple had a history of three failed intrauterine insemination attempts and reduced fertilization rates in two previous in-vitro fertilization (IVF) cycles. In the IVF-ICSI treatment cycle, 6/11 mature oocytes became fertilized following ICSI on the first day. However, the patient failed to conceive following the transfer of three embryos. Five oocytes were immature (two at metaphase I stage and three with a germinal vesicle) and these were cultured overnight. All had extruded a polar body by the following day and ICSI was therefore performed; four oocytes became fertilized, and were cryopreserved at the pronulear stage in propanediol. In the next treatment cycle, transfer of frozen embryos was planned. The pronuclear zygotes were thawed and cultured for 24 h prior to the transfer of two embryos in a cycle stimulated with low doses of follicle stimulating hormone. This resulted in a pregnancy and the delivery of a healthy baby boy. In-vitro maturation of metaphase I and germinal vesicle oocytes which are routinely collected in IVF-ICSI cycles, followed by second day ICSI fertilization, may provide a valuable source of embryos for infertile couples.      

Analysis of CAG repeat of the Machado-Joseph gene in human, chimpanzee and monkey populations: a variant nucleotide is associated with the number of CAG repeats

Machado-Joseph disease (MJD) is an autosomal dominant neurodegenerative disorder associated with an unstable and expanded CAG repeat. We analyzed this locus from various sources including MJD families, Acadian, African American, Caucasian, Greenland Inuit and Thai populations. The range of the CAG repeat size was 14-40 in the normal alleles while the MJD alleles contained 73-78 repeats in our studies. We found 25 different alleles on normal chromosomes with a heterozygosity of 0.86 in combined populations. The most common alleles were 23 (22.9%) and 14 (25.5%) repeats. We also examined 16 chimpanzees and various Old World monkeys: a pigtail macaque, a mangabey and 12 rhesus macaques. The DNA sequences surrounding the CAG repeat did not vary among species. The range of the number of the CAG repeats is 13-14 in macaques, 16 in mangabey and 14-20 in chimpanzees. Variant CAA or AAG triplets in the CAG repeat tracts were found in all 268 human, 28 monkey and 32 chimpanzee chromosomes. As reported in a previous study [Kawaguchi et al. (1994) Nature Genet. 8, 221-228] the common variant positions were the third (CAA), fourth (AAG) and sixth (CAA) positions. However, we found three human chromosomes containing CAG at the sixth position and the mangabey had AAG at the ninth position. In addition, we found CAG at the fourth position and AAG at the sixth position in all macaque chromosomes. The nucleotide following the CAG repeat tract was usually G in all species studied. However, we sometimes found C at this position in human and chimpanzee chromosomes. Interestingly, this variant C was found in all expanded chromosomes and in 54.5% of chromosomes with 27-40 CAG repeats but it was not found in any chromosomes with less than 20 CAG repeats. We hypothesize that the variant C may be associated with CAG repeat instability.      

Study of aneuploidy in normal and abnormal germ cells from semen of fertile and infertile men

This study was undertaken with the aim of investigating the cytogenetic constitution of normal as well as abnormal spermatozoa and immature germ cells found in semen of normal men and infertile patients. A specific protocol of double in-situ hybridization for chromosomes 1 and 17 based on colorimetric detection of the hybridization signals (ISH) and brightfield microscopy analysis of cellular morphology was applied. Also the influence of paternal age on sperm aneuploidy was investigated. We found that, at least in the age range analysed (28-54 years) and for semen of good quality (total normal motile counts above 10 x 10(6)) (n = 17), paternal age has no influence on baseline rates of sperm aneuploidy. However, with decreasing semen quality (total normal motile sperm counts below 5 x 10(6)) (n = 6) significantly higher rates of sperm aneuploidy for autosomes 1 and 17 were scored (0.8 versus 1.43%) (P < 0.001). Regardless of the type of semen analysed, a number of morphologically abnormal spermatozoa were found to be hyperhaploid or diploid in a high percentage of cases (20 and 10% respectively). The same was found for immature germ cells (aneuploidy rate of 18%). We conclude that in infertile men with poor quality semen a direct relationship may exist between the impairment of the spermatogenesis process (as reflected by an increased production of morphologically and cytogenetically abnormal germ cells) and rates of baseline aneuploidy occurring in normal spermatozoa. Infertile couples undergoing assisted reproduction treatment need to be counselled about the risk of using spermatozoa which may carry higher rates of non- disjunction for different chromosomes. While sperm hyper- or hypohaploidy for some chromosomes (X,Y) implies counselling couples about the risk of abnormal phenotype in their offspring, most autosomal sperm aneuploidies probably translate only into lower rates of embryo fertilization and survival.      

Radiographic damage in large joints in early rheumatoid arthritis: relationship with radiographic damage in hands and feet, disease activity, and physical disability

An assessment of the onset of radiographic damage in the large joints (hip, knees, shoulders, elbows, ankles and tarsus) in patients with early rheumatoid arthritis, and the relationship of the progression of large joint damage with joint damage in hands and feet, with physical disability, and with cumulative disease activity, was performed in a prospective 6 yr follow-up study. Large joint damage appeared to be an early phenomenon with 20% of the patients having some damage in at least one large joint within 1 yr, and 50% of the patients within 6 yr after disease onset. Radiographic damage in large joints was significantly related to the damage in hands and feet, the physical disability index, and the cumulative disease activity. The initial disease activity at study entry was the only prognostic factor that reached significance.      

HTLV-III infection after bone marrow transplantation

We prospectively documented the development of a fatal, secondarily acquired severe immunodeficiency in a 19-year-old man who underwent uncomplicated bone marrow transplantation. He had no graft v host disease (GVHD) and had normal recovery of his immune system as determined by lymphocyte phenotyping, mitogenic responses of his peripheral blood lymphocytes, and his ability to secrete immunoglobulin. This alteration in immunity was associated with the acquisition of antibody to HTLV-III. His only risk factor for the development of HTLV-III infection was the transfusions he had received during the transplant and recovery period. Two of his 54 transfusions were from an asymptomatic individual at high risk for acquired immunodeficiency syndrome (AIDS), who was subsequently found to be seropositive for anti-HTLV-III and from whom HTLV-III was isolated. The loss of immunocompetence in patients without chronic GVHD disease is unusual, and our data support the view that this patient's immunodeficiency was due to HTLV-III. When bone marrow transplant recipients without chronic GVHD develop late opportunistic infections, consideration should be given to transfusion-associated AIDS.     

Human platelets secrete chemotactic activity for eosinophils

Thrombin-stimulated platelets liberate factors that induce chemotaxis of eosinophils and raise their cytosolic Ca2+ content ([Ca2+]i). The sources of this activity are the dense- and alpha-granules because inhibition of prostaglandin endoperoxide/thromboxane A2 formation and the platelet-activating factor receptor-antagonist WEB 2086 have no effect. Platelets from patients with Storage-Pool Deficiency show about 60% of the normal chemotactic activity with little effect on [Ca2+]i, whereas completely degranulated platelets fail to affect eosinophils. In concentrations secreted by the platelets, adenosine diphosphate (ADP), and platelet factor 4 have no effect, whereas adenosine triphosphate (ATP) induces a strong chemotactic response and increases [Ca2+]i. However, apart from ATP other modulating factors must be involved as platelet releasates induce more chemotaxis than ATP alone. Thus, platelets secrete factors that activate eosinophils and may contribute to inflammatory and allergic processes.     

The in vitro effects of Newcastle disease virus on the metabolic and antibacterial functions of human neutrophils

Live Newcastle disease virus (NDV) was used to investigate the in vitro effects of a viral infection on phagocytosis, chemiluminescence generation, superoxide production, oxygen consumption, NADPH-oxidase activity, and intracellular killing of bacteria by Ficoll-Hypaque separated human neutrophils. Phagocytosis of oil red O particles by NDV- treated PMN was inhibited by 50%. Chemiluminescence by PMN was inhibited 79% after zymosan stimulation and 86% after tetradeconyl phorbol acetate stimulation. Superoxide generation was inhibited by 68%. Oxygen consumption was inhibited in the presence of NDV by 37% after stimulation with phorbol myristate acetate, while membrane- associated NADPH-enzyme activity was decreased by 19%. The percent of surviving intracellular S. aureus was significantly elevated in NDV- treated PMN after 60 and 120 min of incubation. Purified bacterial neuraminidase markedly suppressed chemiluminescence, while neuraminic acid blocked the effects of the virus. These observations suggest that infections with myxoviruses may suppress a number of vital neutrophil functions. It appears that the effects may be partly mediated by the interaction of viral neuraminidase with the external neutrophil membrane.     

Significant aortic stenosis associated with poorer functional outcomes in patients with acute ischaemic stroke undergoing endovascular therapy

Background and aimBi-directional feedback mechanisms exist between the heart and brain, which have been implicated in heart failure. We postulate that aortic stenosis may alter cerebral haemodynamics and influence functional outcomes after endovascular thrombectomy for acute ischaemic stroke. We compared clinical characteristics, echocardiographic profile and outcomes in patients with or without aortic stenosis that underwent endovascular thrombectomy for large vessel occlusion acute ischaemic stroke.MethodsConsecutive acute ischaemic stroke patients with anterior and posterior circulation large vessel occlusion (internal carotid artery, middle cerebral artery and basilar artery) who underwent endovascular thrombectomy were studied. Patients were divided into those with significant aortic stenosis (aortic valve area <1.5 cm²) and without. Univariate and multivariate analyses were employed to compare and determine predictors of functional outcomes measured by modified Rankin scale at three months.ResultsWe identified 26 (8.5%) patients with significant aortic stenosis. These patients were older (median age 76 (interquartile range 68–84) vs. 67 (interquartile range 56–75) years, p = 0.001), but similar in terms of medical comorbidities and echocardiographic profile. Rates of successful recanalisation (73.1% vs. 78.0%), symptomatic intracranial haemorrhage (7.7% and 7.9%) and mortality (11.5% vs. 12.6%) were similar. Significant aortic stenosis was independently associated with poorer functional outcome (modified Rankin scale >2) at three months (adjusted odds ratio 2.7, 95% confidence interval 1.1–7.5, p = 0.048), after adjusting for age, door-to-puncture times, stroke severity and rates of successful recanalisation.ConclusionIn acute ischaemic stroke patients managed with endovascular thrombectomy, significant aortic stenosis is associated with poor functional outcome despite comparable recanalisation rates. Larger cohort studies are needed to explore this relationship further.