V37I connexin 26 allele in patients with sensorineural hearing loss: evidence of its pathogenicity.

Sensorineural hearing loss (SNHL) is the most common inherited sensory disorder, reported in 1-3 of every 1,000 births. It has been estimated that 50% of all cases of prelingual SNHL are genetically determined. There is tremendous genetic heterogeneity, with multiple dominant and recessive loci. Mutations of the gap junction beta-2 gene (GJB2) emerge as a leading cause of autosomal recessive non-syndromic SNHL. Over 90 sequence alterations have been reported, the pathogenicity of some of them being unknown or unclear. The status of the V37I allele of connexin 26 (GJB2 amino acid product) with regards to its association with SNHL has been controversial. This study examines the pathogenicity of V37I by comparing the frequency of this allele in 40 patients with SNHL of Chinese and Caucasian descent with the frequency of the allele in 100 anonymized, ethnically matched controls. The V37I allele was identified in 43.75 and 11.5% of the patient and control alleles of Chinese ethnicity, respectively, but was not found in either Caucasian cohort. We also compiled the audiograms of 15 individuals with SNHL homozygous for the V37I allele, and showed that these individuals present with a mild to moderate SNHL. These results indicate that (1) the V37I allele is common in individuals of Chinese descent but rarely present in individuals of Caucasian decent; and (2) the V37I allele is pathogenic, but produces milder hearing loss compared to nonsense mutations of connexin 26 such as the 35delG mutation.

Leukocyte count as a predictor of death during remission induction in acute myeloid leukemia.

Acute myeloid leukemia (AML) presenting with a high leukocyte count has been associated with an increase in induction mortality and poor results in a number of other survival measures. However, the level at which an elevated leukocyte count has prognostic significance in AML remains unclear. In this report on a series of 375 adult (non-M3) AML patients undergoing induction chemotherapy at a single institution, leukocyte count analyzed as a continuous variable is shown to be a better predictor of induction death (ID) and overall survival (OS) than a leukocyte count of > or = 100 x 10(9)/L, a value characteristically associated with "hyperleukocytosis" (HL). In this patient cohort, a presenting leukocyte count of > or = 30 x 10(9)/L had high sensitivity and specificity for predicting ID, and both performance status (PS) and leukocyte count more accurately predicted for ID than age. Considering these parameters in newly-diagnosed AML patients may facilitate the development of strategies for reducing induction mortality.

Isodicentric Yp: prenatal diagnosis and outcome in 12 cases.

OBJECTIVES: 1. To present the prenatal cytogenetic findings and postnatal outcome of 12 cases with an isodicentric chromosome composed of the short arm of the Y chromosome.2. To review the literature and provide recommendations for cytogenetic analysis and counseling. METHODS: Prenatal and postnatal cytogenetic data and clinical findings of isodicentric Yp ascertained in six institutions were gathered and reviewed. RESULTS: Nine of the twelve cases were referred for advanced maternal age (AMA), one of which was a twin pregnancy with one twin having an increased nuchal translucency measurement. The remaining cases were referred owing to a family history of hemophilia and an abnormal maternal serum screen, respectively. Nine of these pregnancies resulted in the birth of a normal-appearing male infant with subsequent normal growth and psychomotor development. Follow-up ranged from birth to 7 years. In two cases, the pregnancy was terminated and the fetuses showed male external genitalia. In the case ascertained because of an increased nuchal translucency measurement, the prenatal diagnosis of 45,X was made. At birth, there were ambiguous genitalia, and postnatal cytogenetic studies found an isodicentric Yp. In 11 of the 12 cases, mosaicism was present. CONCLUSION: Our cases show that the prenatal finding of an isodicentric Yp, with or without 45,X mosaicism, is compatible with normal male phenotype in most cases, particularly in the absence of other anomalies. To ensure accuracy in cytogenetic reporting and prenatal counseling, the identification of a structurally abnormal or small Y chromosome, either alone or in the presence of 45,X colonies, should be followed immediately by confirmatory molecular cytogenetic investigations as well as by ultrasound determination of the phenotypic sex of the fetus.

Recurrent trisomy 21: four cases in three generations.

Recurrent trisomy 21: four cases in three generations. While gonadal mosaicism can lead to recurrence of trisomy 21 (T21) for a single couple, the recurrence of free T21 in multiple members of a single pedigree has rarely been reported. We present an unusual pedigree with four cases of Down syndrome (DS) with free T21 were born to four separate women related through three generations of one family. The mothers were aged 18, 21, 29, and approximately 30 years at the time of the births. Using microsatellite markers, we excluded most of chromosome 21, excepting two small regions within 21q11.1 and 21q22.3, as being shared among the mothers of the DS children. However, two members of the pedigree, including one DS mother with a normal G-banded karyotype, carried supernumerary alleles at markers 2503J9TG, D21S369, and D21S215, which span the region from 21pter to 21q11.1. Fluorescence in situ hybridization using a centromeric probe hybridizing to chromosomes 13 and 21 did not reveal a novel location, ruling out a cryptic centromeric translocation between chromosome 21 and any chromosome other than chromosome 13. The level of meiotic recombination on chromosome 21 was unusually high in this family as well. We hypothesize that a cryptic rearrangement within the highly repetitive region of 21q11.1 is present in this family, disrupting pairing and leading to an increased risk of non-disjunction of chromosome 21 in this family.

Risk of mosaicism and uniparental disomy associated with the prenatal diagnosis of a non-homologous Robertsonian translocation carrier.

OBJECTIVE: To estimate the fetal risk of uniparental disomy (UPD) associated with the presence of a Robertsonian translocation (RT) in a parent or in the fetus, to determine whether it is clinically indicated to test these pregnancies for UPD. METHODS: Retrospective analysis of our Centre's experience in testing prenatal specimens for UPD in cases of known familial RTs or fortuitous RT finding. In addition, all reports dealing with prenatal UPD testing in similar populations obtained from PUBMED and the 1995-2001 American Society of Human Genetics Meeting's abstracts were assessed. RESULTS: No case of UPD 14 or 15 was found among the 51 tests performed at our Centre. Meta-analysis identified one case of UPD13 out of 687 UPD studies, conducted in 400 prenatal diagnoses. The 95% confidence interval of the risk of UPD in the population studied (1 in 738) is 0.02-0.76%. In one report, trisomy mosaicism for one of the chromosomes involved in the translocation was found in 3 cases out of 169 (95% confidence interval: 0.1-3 %). CONCLUSIONS: Fetuses carrying a Robertsonian translocation have a risk of UPD of 0.02-0.76% (95% CI). In this population, trisomy mosaicism is more frequent than UPD. This finding justifies the study of additional colonies in all cases of prenatally diagnosed RT.

Cytogenetic and molecular study of a premature male infant with 46,XX derived from ICSI: case report.

We investigated the aetiology of the male phenotype in a premature infant derived from ICSI with a 46,XX karyotype. A karyotypically normal couple underwent ICSI because of obstructive azoospermia in the male partner. Sperm were retrieved by testicular sperm extraction (TESE), cryopreserved, and later used for ICSI. The pregnancy after ICSI ended at 20 weeks. A normal-appearing male was delivered but he did not survive. Umbilical cord blood and placenta were sampled and used for molecular and cytogenetic investigation. The 46,XX karyotype from G-banding in this male infant correlated to a balanced female comparative genomic hybridization (CGH) profile in placental tissue. No PCR amplification of SRY on the p arm of the Y chromosome was observed while fluorescence in-situ hybridization (FISH) with the SRY probe also could not detect the gene in cord blood or placental tissues. CGH and FISH, with X and Y centromeric probes, failed to detect mosaicism in the trophoblast, stroma and amnion. Skewed X-chromosome inactivation (81%) was found in the chorionic villi. The molecular and cytogenetic studies indicated a 46,XX male infant without the SRY gene or 46,XX/XY mosaicism. The possible mechanism in this SRY-negative XX male by ICSI is discussed.

Fulminant tumour lysis syndrome in acute myelogenous leukaemia with inv(16)(p13;q22).

Tumour lysis syndrome (TLS) is caused by rapid breakdown of malignant cells resulting in electrolyte disturbances and acute renal failure. TLS has rarely been described in patients with acute myelogenous leukaemia (AML). Between November 1997 and July 2001, 114 consecutive adult AML patients aged <60 yr received induction chemotherapy consisting of cytosine arabinoside 1.5 g m(-2) q 12 h x 12 doses and daunorubicin 45 mg m(-2) d(-1) x 3 doses. During induction chemotherapy (CT), seven patients (6.1%, 95% CI 2.5-12.2) developed fulminant TLS, resulting in acute renal failure; five of these seven patients had inversion of chromosome 16 [inv(16)(p13;q22)], and one patient had a biological equivalent [t(16,16)(p13;q22)]. Four of the TLS patients underwent leukapheresis for a presenting white blood cell (WBC) count > 100 x 10(9) L(-1) prior to commencing chemotherapy, and six patients subsequently required haemodialysis for a median of 2 (range 1-8) wk. One TLS patient died of intracerebral hemorrhage on day 10 and another patient of multiorgan failure on day 17. Of the other five patients, all entered a complete remission (CR) and recovered normal renal function. Four patients remain in continuous CR [median follow-up 20 (range 12-25) months]. One patient relapsed at 12 months and again developed TLS on re-induction. In univariate analysis, TLS patients were more likely to have an elevated presentation and pre-chemotherapy WBC counts, elevated serum creatinine, and uric acid levels at presentation, as well as an inv(16). In multivariate analysis, only serum creatinine and inv(16) remained statistically significant (P < 0.001 for each). Patients with an inv(16) are a unique AML subgroup at high risk for fulminant TLS.

Recurrent trisomy 21 in a couple with a child presenting trisomy 21 mosaicism and maternal uniparental disomy for chromosome 21 in the euploid cell line.

Recurrence of trisomy 21 was observed in a family in which both parents had a normal chromosome complement. Mosaic trisomy 21 was found in a blood karyotype of the first child, a second pregnancy ended in spontaneous abortion, and a full trisomy 21 was found at prenatal diagnosis of the third pregnancy of this same couple. Although recurrent trisomy 21 may be due to chance, the possibility of germline mosaicism for trisomy 21 in one of the parents has important implications for recurrence risk. Molecular analysis was therefore undertaken in this family to determine the parental origin and the stage of nondisjunction of the extra chromosome 21 in both cases. Although a maternal origin of both instances of trisomy 21 was observed, the mosaic case showed homozygosity for all markers along the duplicated maternal chromosome. Such a finding would normally suggest a postzygotic origin of the trisomy 21. However, the diploid cell line in this same case showed maternal uniparental disomy 21, implying that it was the result of a trisomic conception. We suggest that a somatic nondisjunction in the maternal germ cells is the most likely explanation for these findings. The apparent meiotic II stage of nondisjunction of the nonmosaic trisomy 21 fetus was consistent with maternal mosaicism. A review of the literature for recurrent trisomy 21 cases studied by molecular means, suggests that mosaicism in germ cells may account for more cases than is detected cytogenetically. These results also show that DNA marker analysis does not provide a valuable tool for patient counseling in case of recurrent trisomy 21.

An association between skewed X-chromosome inactivation and abnormal outcome in mosaic trisomy 16 confined predominantly to the placenta.

Skewed X-chromosome inactivation (XCI) is frequently found in the diploid fetal tissues of individuals with mosaic trisomy that originated from a 'trisomic zygote rescue' event. This may result from a high number of trisomic cells in the embryonic cell pool at the time of XCI, which are subsequently eliminated by selection. We hypothesize that extremely skewed XCI in these mosaic cases will be associated with a poor fetal outcome due to failure to completely eliminate the trisomy from all fetal tissues. To test this hypothesis, XCI status was evaluated in 17 cases of prenatally detected trisomy 16 mosaicism. Ten of the 15 informative cases showed extreme XCI skewing ( > or = 90% inactivation of one allele) in blood or other diploid fetal tissues compared to six of the 111 controls (p < 0.001). Among these 10 'skewed' cases, 6 showed an abnormal outcome, defined as developmental abnormalities and/or intrauterine or neonatal death. In contrast, of the 5 cases without extreme skewing, none showed abnormal outcome, although outcome information was incomplete in 1 case. An additional 6 cases analyzed, involving trisomy mosaicism for other chromosomes, showed similar results. Further studies are warranted to determine if XCI status adds useful information to the prediction of pregnancy outcome in prenatally detected mosaic trisomy.

Confirmation of linkage in X-linked infantile spasms (West syndrome) and refinement of the disease locus to Xp21.3-Xp22.1.

The syndrome of infantile spasms, hypsarrhythmia, and mental retardation (West syndrome) is a classical form of epilepsy, occurring in early infancy, which is etiologically heterogeneous. In rare families, West syndrome is an X-linked recessive condition, mapped to Xp11.4-Xpter (MIM 308350). We have identified a multi-generation family from Western Canada with this rare syndrome of infantile spasms, seen exclusively in male offspring from asymptomatic mothers, thereby confirming segregation as an X-linked recessive trait. Using highly polymorphic microsatellite CA-repeat probes evenly distributed over the entire X chromosome, linkage to markers DXS7110, DXS989, DXS1202, and DXS7106 was confirmed, with a maximum LOD score of 3.97 at a theta of 0.0. The identification of key recombinants refined the disease-containing interval between markers DXS1226 and the adrenal hypoplasia locus (AHC). This now maps the X-linked infantile spasms gene locus to chromosome Xp21.3-Xp22.1 and refines the interval containing the candidate gene to 7.0 cM. Furthermore, this interval overlaps several loci previously linked with either syndromic or non-syndromic X-linked mental retardation (XLMR), including one recognized locus implicated in neuroaxonal processing (radixin, RDXP2). Collectively, these studies lend strong support for the presence of one or more genes intrinsic to brain development and function, occurring within the critical interval defined between Xp21.3-Xp22.1.

Ethyl alcohol reduces cardiac output, stroke volume, and end diastolic volume in the embryonic chick.

It has been established that ethanol causes both human congenital cardiac malformations and structural intracardiac abnormalities in the embryonic chick. In view of a theory that reduced embryonic tissue hemodynamics are associated with the development of malformations, we attempted to determine whether or not a) ethanol altered cardiac blood flow and b) altered hemodynamics were a function of ethanol dose in the chick embryo. Cardiac function in Hamburger-Hamilton stage 19 chick embryos was recorded on videotape before and up to 10 hours after exposure to graded doses of ethanol. Parameters of cardiac function, including cardiac output, were determined from videotaped images by means of computer assistance. Cardiac output decreased in a linear fashion with dose for up to 3 hours after exposure to ethanol. The maximum relative percent decrease in cardiac output was directly related to the dose of ethanol administered. Furthermore, the time required after ethanol treatment for mean cardiac output to return to pretreatment and control values was also dose-dependent--lower doses of ethanol required less time for mean cardiac output to return to pretreatment and control values. Although relatively high doses of ethanol depress cardiac rate, we attribute the significant decrease in cardiac output primarily to parallel dose-dependent decreases in both stroke volume and end diastolic volume. Our data are consistent with the hypothesis that reduced embryonic cardiac blood flow during cardiogenesis is associated with the development of ethanol-induced intracardiac defects in chick embryos.

Cardioteratogenic dose of ethanol reduces both lactic dehydrogenase and succinic dehydrogenase activity in the bulbar ridges of the embryonic chick heart.

It has been established previously that the chick embryo is an appropriate model for studying the pathogenesis of ethanol-induced ventricular septal defect and that the growth and fusion of the proximal bulbar ridges are critical to ventricular septation within the embryonic heart. To determine whether enzyme activity was altered significantly in bulbar ridges in response to a cardioteratogenic dose of ethanol, we injected 0.32 ml of 50% ethanol in chick Ringer's saline (CRS) into the air cells of 3-day-old chick embryos (72-84 h incubation, Hamburger-Hamilton stages 18-19, Dekalb Delta strain). Embryos were harvested 48-60 h after treatment (stages 26-27). Hearts were removed, frozen and sectioned, and the tissues were processed for enzyme histochemical demonstrations of lactic and succinic dehydrogenase. Twelve ethanol-treated embryos and 12 CRS-treated controls were compared for lactic dehydrogenase (LDH) levels. Fourteen ethanol-treated embryos and 14 controls were compared for succinic dehydrogenase (SDH) levels. Formazan deposits were quantified by means of computer-assisted microdensitometry. Our results showed that ethanol decreased the mean LDH activity level by 36.3% (P = 0.005) and the mean SDH level by 40.8% (P < 0.02). Furthermore, ethanol significantly reduced LDH activity in 41.7% of the matched embryo pairs and significantly reduced SDH activity in 57.1% of the matched embryo pairs. Although some investigators have proposed that a disturbance in neural crest cell migration plays a major role in the pathogenesis of ethanol-induced cardiac malformations, this study provides evidence that the bulbar ridges of the embryonic heart are also potential target tissues for ethanol.

Cardioteratogenic dose of ethanol in the chick embryo results in albumin ethanol concentrations comparable to human blood alcohol levels.

Three-day-old chick embryos (Hamburger-Hamilton stages 18-19) were exposed to a dose of ethyl alcohol (0.32 ml of 50% ethanol) that causes cardiac malformations in 96.6% of the animals. Ethanol was administered into the air sac after 72-80 h of incubation. Samples of albumin at the opposite pole of the egg were drawn 0-50 h after treatment and quantitated for ethanol concentration with capillary gas-liquid chromatography. Ethanol concentrations in the albumin increased significantly (P < 0.05) at 2, 5 and 15 h after injection of ethanol, reached a maximum mean ethanol concentration at 20 h (217.3 +/- 23.5 mg dl-1), decreased significantly at 30 h to 175.4 +/- 27.5 mg dl-1, then increased again and stabilized at 40-50 h. Individual sample concentrations ranged from 0 mg dl-1 (at 0.5-2 h) to 286.5 mg dl-1 at 40 h. Ethanol concentrations in the albumin were comparable to human blood alcohol levels during intoxication (> 150 mg dl-1). Our results suggest that a potent cardioteratogenic dose of ethanol in the chick embryo is reasonable in terms of potential human embryo exposure.

Strain-dependent effect of ethanol on ventricular septal defect frequency in White Leghorn chick embryos.

We have tested the potential of a single dose of ethanol (0.20 ml 50% ethanol in chick Ringer's saline (CRS) administered into the air sac) to produce ventricular septal defect (VSD) in three distinct commercially available strains of White Leghorn chick embryo: stress-resistant Dekalb Delta strain, Hy-Vac SPF type V, and Hy-Vac SPF type L. Eggs were controlled for both size and developmental stage (Hamburger-Hamilton stage 18-19) at time of injection. The frequency of VSD in Dekalb Delta embryos was 4.0% (1/25), in Hy-Vac SPF type L embryos 9.1% (3/33), and in Hy-Vac SPF type V embryos 38.9% (14/36). Statistical analysis with the two-tailed Fisher Exact Test indicated that frequencies were not significantly different (P = 0.3215) when Dekalb Delta and Hy-Vac type L embryos were compared. However, the frequency of VSD for Hy-Vac type V embryos was significantly greater than that for either the Dekalb Delta or the Hy-Vac type L embryos (P < 0.005). All VSDs observed were located within the crista supraventricularis. When Hy-Vac SPF type V embryos were exposed to either 0.20 ml 50% ethanol in CRS or to 0.20 ml CRS (controls), ethanol-treated embryos showed a VSD incidence of 34.1% compared with a 3.6% incidence in the controls (P = 0.0017). These data suggest that ethanol is the cause of VSD in this strain. From the results of this study, we are led to conclude that different commercial strains of White Leghorn chick embryo show different susceptibilities to the induction of VSD by ethanol.

An interactive image processing system for the quantification of cardiac function in the embryonic chick.

This paper describes an interactive computer-based method for quantifying embryonic chick heart blood pool volumes that have been recorded as digitized image sequences. The primary motivation for the development of such a system is to be able to extract measurement data that is useful for evaluating potential drug-induced effects on intracardiac blood flow while minimizing variabilities in the measurements that typically occur when human evaluators extract the measurements visually from photographs. The processing that is used to achieve this goal can be divided into four basic steps: enhancing the contrast of the digitized embryonic chick heart image, extracting the shape of the blood pool in the embryonic chick heart ventricle from the enhanced image, quantifying the axial dimensions of the blood pool and calculating the required cardiac blood flow data. The technical details and the basic theory motivating each processing step are provided as well as an analysis of the results obtained by applying the method to actual image data.

A computer-assisted image processing method for determining relative cardiac function in the chick embryo.

The general objective of this study was to develop a noninvasive method for efficiently and reproducibly determining relative cardiac function parameters in the chick embryo. The specific objectives of the study were 1) to develop several methods for computer-assisted image processing and quantitation of relative intraventricular blood volumes in the 3-day-old embryonic chick heart and 2) to compare methods for precision and with a previously established manual processing method. Images of the embryonic chick heart in ovo were recorded on videocassette tape, digitized, and enhanced by computer-aided histogram equalization. The area occupied by blood within the common ventricle was extracted by region-growing and spurious region removal algorithms and defined by the determination of edge-pixel coordinates. Edge-pixel coordinates of the longitudinal and transverse axes of the common ventricular blood region were located by three different methods, the lengths of the axes calculated, and volumes computed from the equation for determining volume of a prolate spheroid. Twenty-five images of the embryonic heart were randomly selected and processed. Volumes were calculated with each of the three methods on six different occasions. A coefficient of variation was calculated for each method. The intraobserver mean coefficient of variation for each method was 7.4%. When a 2-way ANOVA was conducted, mean coefficients of variation did not differ significantly for the three methods. However, computer processing (in addition to significantly reducing the time required to generate data) reduced the coefficient of variation observed in manual processing by 56.5%.

Cardioteratogenic dose of ethanol in the chick embryo results in egg white concentrations comparable to human blood alcohol levels.

Three-day-old chick embryos were exposed to a dose of ethyl alcohol (0.32 ml of 50% ethanol) that we previously demonstrated produces cardiac malformations in 96.6% of the animals. Ethanol was administered into the air sac at 72-80 h of incubation. Samples of egg white were drawn at 2, 6 and 24 h after treatment and analyzed by capillary gas-liquid chromatography. Ethanol concentrations were significantly higher at 6 and 24 h after exposure than at 2 h (P less than 0.01), but there were no differences in mean concentrations between 6 and 24 h (P greater than 0.2). Furthermore, concentrations (43-303 mg dl-1) were comparable to human blood alcohol levels during intoxication. These results suggest that the cardioteratogenic doses of ethanol administered to chick embryos in a previous study are not excessive in terms of potential human embryo exposure.