Molecular genetic analysis of autosomal dominant cerebellar ataxia with retinal degeneration (ADCA type II) caused by CAG triplet repeat expansion

Autosomal dominant cerebellar ataxia with retinal degeneration (ADCAII) was previously mapped by linkage analysis studies to chromosome 3p12- p21.1 (SCA7). Positional cloning efforts have recently identified a novel gene, SCA7 , containing a translated CAG repeat, expanded in SCA7 patients. We cloned the SCA7 gene from a yeast artificial chromosome (YAC) clone contig spanning the SCA7 candidate region. Using a combination of genomic sequencing and cosmid-based exon trapping, two expressed sequence tags were identified. Sequencing of the corresponding cDNA clones and RT-PCR analysis identified the full- length SCA7 cDNA. Together, our sequence data defined the intron/exon boundaries of the first two coding exons of the SCA7 gene, with the first exon containing the expanded CAG repeat. Further, sequence comparison with the published SCA7 cDNA identified one additional putative exon in the 5'-UTR region of the SCA7 gene. The SCA7 gene was mapped on the YAC contig in the 2.5 cM interval between D3S1600 and D3S1287. In one extended Belgian SCA7 pedigree the expanded alleles ranged from 38 to at least 55 repeats with allele lengths being inversely correlated with onset age of ADCAII symptoms. The SCA7 repeats increased in length in successive generations. Normal alleles had from four to 18 repeats, with 10 repeats being the most common allele.      

Severe maternal psychopathology and infant-mother attachment

Eighty-two mother-infant dyads, comprising women with psychiatric disorder and individually matched controls, were followed up over the children's 1st year of life. The mothers with mental illness consisted of two subgroups: first, 25 severely mentally ill mothers who had been admitted to a psychiatric unit with their infants; and second, 16 mothers from a community sample meeting research diagnostic criteria for unipolar, nonpsychotic depression. With the exception of six dyads in the in-patient group, observations were made of the mother-infant interaction and the quality of the infant-mother attachment relationship at 12 months. The nature and course of the mothers' illness was also documented. Although few residual symptoms of maternal mental illness were detected at 1 year postpartum, interactional disturbances were evident among the case group dyads. A strong association was revealed between infant-mother attachment quality and maternal diagnosis; a manic episode of illness in the postpartum period was related to security in the attachment relationship, and psychotic or nonpsychotic depression was related to insecurity. Concurrent patterns of mother-infant interaction provided support for this finding.     

Monocyte counting: discrepancies in results obtained with different automated instruments.

To determine the accuracy of several methods for measuring the monocyte count, the results obtained by a number of different automated cell counters were analysed. Considerable discrepancies occurred for monocyte counts obtained in normal blood among the counters. The results of a visual monocyte count on a total of 800 leucocytes were used as the reference method. The technique of measuring the monocyte count by using dual staining with monoclonal antibodies CD45 and CD14 provided the closest agreement with the reference method. Six other automated counting systems were assessed. Two of these systems (Coulter VCS and Technicon H1) gave results, which, although under-estimating monocytosis, correlated well with the results obtained by the reference technique. A third system (Toa Sysmex NE-8000) gave unreliable results. Three of the automated systems evaluated measured a "third population"--that is, monocytes together with other leucocytes. One of these systems (Ortho ELT 1500), overestimated the count, as expected, but correlated well with the reference method. The second of these "third population counters" (Coulter S Plus IV) correlated moderately well with the reference monocytosis, while the Toa Sysmex E-5000 correlated poorly. It is clear that problems exist in the evaluation of different instruments for counting monocytes. An accurate and reliable reference method is a pre-requisite to evaluate this aspect of cell counters. As the visual method is too cumbersome a different reference method would be useful. Based on the results of this study, it is suggested that the technique using fluorescence labelled monoclonal antibodies should be regarded as an acceptable alternative.     

Malignant large cell calcifying Sertoli cell tumor of the testis

A 45-year old man presented with a slow-growing, unilateral beige testicular mass, with a diameter of 4 cm. The testosterone, FSH, LH, estradiol and betahCG serum levels were within normal limits, and there were no associated hormonal syndromes. The patient was treated with inguinal orchidectomy. Microscopically, the tumor was composed of nests of cells with large eosinophilic, slightly granular cytoplasm. There was only a mild degree of atypia and no mitotic activity. The tumor extended into the rete testis. There were intratumoral calcifications, and in the vicinity of the tumor, there was intratubular growth. Although this case is histologically similar to the three previously reported cases of clinically benign large cell calcifying Sertoli cell tumor of the testis with rete testis involvement, the current patient developed right sided para-aortic lymph node metastases 18 months after the initial diagnosis.     

Physiological reactivity to stress and parental support: comparison of clinical and non‐clinical adolescents

An Alarm Stress Task was developed to study affect regulation in the context of parent–child interactions in adolescents (mean age = 12.72, standard deviation = 2.06) with (n = 20) and without (n = 20) mental health problems. Changes in heart rate (HR), preejection period (PEP) and respiratory sinus arrhythmia (RSA) were used as indicators of affect regulation. HR increased, and PEP and RSA decreased significantly in reaction to a suggested failure on a simple task, indicating that this procedure induced affective arousal in adolescents. During reunion with the parent, RSA increased significantly. Support seeking on reunion was associated with stronger parasympathetic reactivity during stress and reunion, consistent with the model that the parasympathetic system is involved when affect is regulated by social engagement. Quality of parent–adolescent interactive behaviour was overall lower in the clinical sample. Individual and relationship‐based processes of affect regulation may be simultaneously assessed, highlighting the continuing importance of the parent–child relationship in adolescence for affect regulation and mental health. Copyright © 2008 John Wiley & Sons, Ltd.     

How to use Chlamydia antibody testing in subfertility patients

Screening for tubal factor subfertility by means of Chlamydia antibody testing (CAT) was introduced into the initial work-up of subfertile couples several years ago. The results reported, however, are heterogeneous, and no uniformity exists in cut-off levels of titres, or in definitions of tubal factor subfertility. We performed a prospective cohort study to evaluate the implications of varying the definitions of tubal pathology and of modifying the cut-off levels on the clinical impact of CAT in predicting tubal factor subfertility. In 227 consecutive patients who attended our fertility clinic, the Chlamydia IgG antibody titre was determined and related to tuboperitoneal abnormalities at laparoscopy as a reference standard. According to received operating characteristic (ROC) curve analysis, a titre of 16 is the optimum cut-off level. Increasing the cut-off level improves specificity and positive likelihood ratio (LR+), at the expense of sensitivity and negative LR (LR-). Changing the definition of tubal factor subfertility from unspecified tuboperitoneal abnormalities into extensive adhesions and/or bilateral distal tubal occlusion improves LR+, LR- and kappa significantly. We conclude that CAT is more accurate in predicting severe distal tubal pathology than unspecified tuboperitoneal abnormalities. Although from a statistical point of view a titre of 16 is the optimum cut-off level, from a clinical point of view 32 or 64 may be preferable, depending on the aim of screening and the inception cohort.      

Trisomy 7 mosaicism, maternal uniparental heterodisomy 7 and Hirschsprung's disease in a child with Silver-Russell syndrome

Prenatal trisomy 7 is usually a cell culture artifact in amniocytes with normal diploid karyotype at birth and normal fetal outcome. In the same way, true prenatal trisomy 7 mosaicism usually results in a normal child except when trisomic cells persist after birth or when trisomy rescue leads to maternal uniparental disomy, which is responsible for 5.5-7% of patients with Silver-Russell syndrome (SRS). We report here on the unusual association of SRS and Hirschsprung's disease (HSCR) in a patient with maternal uniparental heterodisomy 7 and trisomy 7 mosaicism in intestine and skin fibroblasts. HSCR may be fortuitous given its frequency, multifactorial inheritance and genetic heterogeneity. However, the presence of the trisomy 7 mosaicism in intestine as well as in skin fibroblasts suggests that SRS and HSCR might possibly be related. Such an association might result from either an increased dosage of a nonimprinted gene due to trisomy 7 mosaicism in skin fibroblasts (leading to SRS) and in intestine (leading to HSCR), or from an overexpression, through genomic imprinting, of maternally expressed imprinted allele(s) in skin fibroblasts and intestine or from a combination of trisomy 7 mosaicism and genomic imprinting. This report suggests that the SRS phenotype observed in maternal uniparental disomy 7 (mUPD(7)) patients might also result from an undetected low level of trisomy 7 mosaicism. In order to validate this hypothesis, we propose to perform a conventional and molecular cytogenetic analysis in different tissues every time mUPD7 is displayed.     

Preimplantation genetic diagnosis for Charcot-Marie-Tooth disease type 1A

Charcot-Marie-Tooth (CMT) disease is the 'common' name for a range of hereditary peripheral neuropathies. CMT1 is the most common form and is transmitted in an autosomal dominant manner. CMT1A maps to chromosome 17p11.2 and is caused, in the majority of cases, by a 1.5 Mb DNA duplication, that includes the peripheral myelin protein 22 (PMP) gene. This paper reports on preimplantation genetic diagnosis (PGD) for CMT1A in five couples. The CMT1A duplication was detected by fluorescent PCR analysis using polymorphic (CA)n markers localized within the duplication. Single-cell PCR on blastomeres allowed genetic analysis of embryos obtained after ICSI. Only healthy unaffected embryos were transferred to the uterus. PCR experiments with single EBV-transformed lymphoblasts or with research blastomeres allowed the evaluation of amplification efficiencies, as well as contamination and allele drop-out (ADO) rates for each PCR protocol. Three simplex PCR protocols (using one primer pair) and two duplex PCR protocols (using two primer pairs) were developed for CMT1A. Additionally, a protocol using all three primer pairs in triplex was also established. Thirteen clinical ICSI-PGD cycles were performed for five couples (12 simplex PCR cycles and one duplex PCR cycle), resulting in seven embryo transfers. Three singleton pregnancies ensued in two couples and three healthy babies were delivered. This report describes different fluorescent PCR-based tests which allow efficient and accurate single-cell level detection of the CMT1A duplication. On the basis of the presence of the healthy allele of the affected parent-to-be (and/or absence of the affected one), healthy embryos can be selected for transfer. The assays are suitable for PGD for other couples who present with the same CMT1A duplication [depending on their informativity for the (CA)n markers available] as described here.     

Improved detection of rhinoviruses by nucleic acid sequence-based amplification after nucleotide sequence determination of the 5' noncoding regions of additional rhinovirus strains

The isothermal nucleic acid sequence-based amplification (NASBA) system was applied for the detection of rhinoviruses using primers targeted at the 5' noncoding region (5' NCR) of the viral genome. The nucleotide sequence of the 5' NCRs of 34 rhinovirus isolates was determined to map the most conserved regions and design more appropriate primers and probes. The assay amplified RNA extracted from 30 rhinovirus reference strains and 88 rhinovirus isolates, it did not amplify RNA from 49 enterovirus isolates and other respiratory viruses. The assay allows one to discriminate between group A and B rhinoviruses. Sensitivities for the detection of group B and group A rhinoviruses was 20 and 200 50% tissue culture infective doses, respectively.