Genetic testing for BRCA1 and BRCA2 in the Province of Ontario

In 2001, genetic testing for BRCA1 and BRCA2 was introduced in Ontario, for women at high‐risk of breast or ovarian cancer. To date over 30,000 individuals have been tested throughout Ontario. Testing was offered to all Ontario residents who were eligible under any of 13 criteria. We report the results of tests conducted at Mount Sinai Hospital from 2007 to 2014. A total of 4726 individuals were tested, 764 (16.2%) were found to carry a pathogenic variant (mutation). Among 3684 women and men who underwent testing without a known familial BRCA mutation, 331 (9.0%) were found to carry a mutation. Among 1042 women and men tested for a known family mutation, 433 (41.6%) were positive. There were 603 female mutation carriers, of these, 303 were affected with breast or ovarian cancer (50%) and 16 with another cancer (2.3%). Of 284 unaffected female carriers, 242 (85%) were tested for a known family mutation and 42 (15%) were the first person in the family to be tested. By placing greater emphasis on recruiting unaffected female relatives of known mutation carriers for testing, greater than one‐half of newly identified carriers will be unaffected.     

Comparison of latex agglutination with enzyme immunoassay for detection of rotavirus in fecal specimens

Human rotaviruses are the most important etiologic agents of acquired diarrhea in infants and young children worldwide. Early diagnosis is essentialfor effective patient treatment. The latex agglutination (LA) assays for rotavirus diagnosis are rapid, inexpensive, and the most widely used to screen specimens. The performance of the LA Rotagen (Biokit S.A., Barcelona, Spain) was evaluated for rotavirus detection infecal samples of outpatients with acute gastroenteritis. This assay was compared with the enzyme immunoassay (EIA) EIARA (Bio-Manguinhos, Rio de Janeiro, Brazil). From January to October 2000, 285 fecal specimens were analyzed. Forty-four samples (15.4%) were reactive, 214 (75.4%) were nonreactive, and 27 (9.5%) were indeterminate by LA. All LA-positive samples were positive by EIA, and 2 LA-negative samples were positive by EIA. Of specimens indeterminate by LA, 67% were positive by EIA. The sensitivity, specificity, and accuracy of LA were 69%, 100%, and 93%, respectively. These results indicate that assay is as sensitive and specific as the EIA, and it could be applied on a large scale for screening stool specimens in suspected rotavirus diarrhea. However, the indeterminate results must be confirmed by other methods, such as EIA.     

Detection of chromosomes and estimation of aneuploidy in human spermatozoa using fluorescence in-situ hybridization

The development and application of fluorescence in-situ hybridization (FISH) has opened the way for comprehensive studies on numerical chromosome abnormalities in human spermatozoa. FISH can be rapidly applied to large numbers of spermatozoa and thus overcomes the major limitation of karyotyping spermatozoa after penetration of zona-free hamster oocytes. The simultaneous hybridization of two or more chromosome-specific probes to spermatozoa and subsequent detection of the bound probes using different fluorescent detection systems enables two or more chromosomes to be localized simultaneously in the same spermatozoon and provides a technique for undertaking reasonable estimates of aneuploidy. The most commonly used probes are those which bind to the centromeric region of specific chromosomes. Most studies to date have concentrated on estimating aneuploidy in spermatozoa from normospermic men, although reports are beginning to appear on aneuploidy in spermatozoa from subfertile and infertile men. Multi- probe FISH studies have generally reported disomy (hyperhaploidy) estimates of 0.05-0.2% per chromosome. There is preliminary evidence that some chromosomes such as X, Y and 21 are predisposed towards higher rates of non-disjunction during spermatogenesis. There are also suggestions of inter-donor variability in aneuploidy frequencies for specific chromosomes, although this requires confirmation in larger studies. While FISH is clearly a powerful technique that has many applications in reproductive medicine, it must also be realized that it does have limitations and the technology itself is still evolving and has yet to be fully validated on spermatozoa.      

Identification of MEN1 gene mutations in sporadic carcinoid tumors of the lung

Lung carcinoids occur sporadically and rarely in association with multiple endocrine neoplasia type 1 (MEN1). There are no well defined genetic abnormalities known to occur in these tumors. We studied 11 sporadic lung carcinoids for loss of heterozygosity (LOH) at the locus of the MEN1 gene on chromosome 11q13, and for mutations of the MEN1 gene using dideoxy fingerprinting. Additionally, a lung carcinoid from a MEN1 patient was studied. In four of 11 (36%) sporadic tumors, both copies of the MEN1 gene were inactivated. All four tumors showed the presence of a MEN1 gene mutation and loss of the other allele. Observed mutations included a 1 bp insertion, a 1 bp deletion, a 13 bp deletion and a single nucleotide substitution affecting a donor splice site. Each mutation predicts truncation or potentially complete loss of menin. The remaining seven tumors showed neither the presence of a MEN1 gene mutation nor 11q13 LOH. The tumor from the MEN1 patient showed LOH at chromosome 11q13 and a complex germline MEN1 gene mutation. The data implicate the MEN1 gene in the pathogenesis of sporadic lung carcinoids, representing the first defined genetic alteration in these tumors.      

Autosomal glycogenosis of liver and muscle due to phosphorylase kinase deficiency is caused by mutations in the phosphorylase kinase beta subunit (PHKB)

Glycogen storage disease due to phosphorylase kinase deficiency occurs in several variants that differ in mode of inheritance and tissue- specificity. This heterogeneity is suspected to be largely due to mutations affecting different subunits and isoforms of phosphorylase kinase. The gene of the ubiquitously expressed beta subunit, PHKB, was a candidate for involvement in autosomally transmitted phosphorylase kinase deficiency of liver and muscle. To identify such mutations, the complete PHKB coding sequence was amplified by RT-PCR of RNA isolated from blood samples of patients and analyzed by direct sequencing of PCR products. The characterization of mutations was complemented by PCR of genomic DNA. In one female and four male patients, we identified five independent nonsense mutations (Y418ter; R428ter; Y974H+E975ter; Q656ter in two cases), one single-base insertion in codon N421, one splice-site mutation affecting exon 31, and a large deletion involving the loss of exon 8. Although these severe translation-disrupting mutations occur in constitutively expressed sequences of the only known beta subunit gene of phosphorylase kinase, PHKB, they are associated with a surprisingly mild clinical phenotype, affecting virtually only the liver, and relatively high residual enzyme activity of approximately 10%.      

De novo duplication of MECP2 in a girl with mental retardation and no obvious dysmorphic features

Makrythanasis P, Moix I, Gimelli S, Fluss J, Aliferis K, Antonarakis SE, Morris MA, Béna F, Bottani A. De novo duplication of MECP2 in a girl with mental retardation and no obvious dysmorphic features. Loss‐of‐function mutations of MECP2 are responsible for Rett syndrome (RTT), an X‐linked neurodevelopmental disorder affecting mainly girls. The availability of MECP2 testing has led to the identification of such mutations in girls with atypical RTT features and the recognition of milder forms. Furthermore, duplication of the entire gene has recently been described in boys with mental retardation and recurrent infections. We describe a girl with a heterozygous de novo MECP2 duplication. The patient, at the age of 19, has mental retardation with no autistic features. She is friendly but gets frequently anxious. She has neither dysmorphic features nor malformations. Her motor development was delayed with walking at 20 months. Speech is fluid with good pronunciation but is simple and repetitive. Diagnosis was made after single‐strand conformation analysis (SSCA) and multiplex ligation‐dependent probe amplification (MLPA) analysis of MECP2. Array comparative genomic hybridization (aCGH) analysis showed a duplication of 29 kb including MECP2 and part of IRAK1. Fluorescent in situ hybridization (FISH) has revealed that the duplicated region is inserted near the telomere of the short arm of chromosome 10. X‐chromosome inactivation in leukocyte DNA was not skewed. We conclude that it is likely that this MECP2 duplication is responsible for the mental retardation in this patient. This case broadens the phenotypic spectrum of MECP2 abnormalities with consequent implication in diagnosis and genetic counselling of girls with non‐syndromic mental retardation.     

Prostatic involvement by transitional cell carcinoma in patients with bladder cancer and its prognostic significance

To study the importance of prostatic involvement by transitional cell carcinoma (TCC) in patients with bladder cancer, we examined the entire prostates by whole-mount sections from 214 radical cystoprostatectomy specimens for detailed patterns of involvement by TCC and correlated the results with lymph node metastasis and patients' survival. Prostatic involvement by TCC was detected in 69 (32%) of 214 cases. Among them, 30 (43%) patients had carcinoma in situ (CIS) and the other 39 (57%) were invasive TCC. Carcinoma in situ occurred in either prostatic urethra (n = 6, 20%) or, more commonly, in prostatic ducts/acini (n = 14, 47%), and in a combination of prostatic urethra and ducts (n = 10, 33%). Ten (26%) of the invasive TCC resulted from direct penetration from the primary tumor in the bladder, and the remaining 29 (72%) cases arose from prostatic urethra/ducts, of which 11, 13, and 5 invaded the lamina propria, prostatic stroma, and periprostatic or seminal vesical tissue, respectively. Both prostatic TCC involvement and nodal metastasis were highly significant prognostic factors for patients' survival and the survival significance of prostatic TCC involvement still existed regardless of lymph node status. Furthermore, the presence of prostatic CIS and degrees of prostatic invasion are associated with nodal metastasis and survival. Patients with prostatic CIS or urethral lamina propria invasion had a similar, but higher incidence of lymph node metastasis and lower long-term and 5-year survival than those patients without prostatic involvement. Similarly, prostatic stromal invasion and periprostatic/seminal vesical invasion had a similar, but much higher nodal metastasis and worse survival than patients with only prostatic CIS or urethral lamina propria invasion. In summary, presence of prostatic TCC involvement and levels of involvement are significant prognostic factors in patients with bladder cancer.