A Gene for Autosomal Recessive Limb-Girdle Muscular Dystrophy in Manitoba Hutterites Maps to Chromosome Region 9q31-q33: Evidence for Another Limb-Girdle Muscular Dystrophy Locus

SummaryCharacterized by proximal muscle weakness and wasting, limb-girdle muscular dystrophies (LGMDs) are a heterogeneous group of clinical disorders. Previous reports have documented either autosomal dominant or autosomal recessive modes of inheritance, with genetic linkage studies providing evidence for the existence of at least 12 distinct loci. Gene products have been identified for five genes responsible for autosomal recessive forms of the disorder. We performed a genome scan using pooled DNA from a large Hutterite kindred in which the affected members display a mild form of autosomal recessive LGMD. A total of 200 markers were used to screen pools of DNA from patients and their siblings. Linkage between the LGMD locus and D9S302 (maximum LOD score 5.99 at recombination fraction .03) was established. Since this marker resides within the chromosomal region known to harbor the gene causing Fukuyama congenital muscular dystrophy (FCMD), we expanded our investigations, to include additional markers in chromosome region 9q31-q34.1. Haplotype analysis revealed five recombinations that place the LGMD locus distal to the FCMD locus. The LGMD locus maps close to D9S934 (maximum multipoint LOD score 7.61) in a region that is estimated to be ∼4.4 Mb (Genetic Location Database composite map). On the basis of an inferred ancestral recombination, the gene may lie in a 300-kb region between D9S302 and D9S934. Our results provide compelling evidence that yet another gene is involved in LGMD; we suggest that it be named “LGMD2H.

A View from the Past Into our Collective Future: The Oncofertility Consortium Vision Statement

Today, male and female adult and pediatric cancer patients, individuals transitioning between gender identities, and other individuals facing health extending but fertility limiting treatments can look forward to a fertile future. This is, in part, due to the work of members associated with the Oncofertility Consortium. The Oncofertility Consortium is an international, interdisciplinary initiative originally designed to explore the urgent unmet need associated with the reproductive future of cancer survivors. As the strategies for fertility management were invented, developed or applied, the individuals for who the program offered hope, similarly expanded. As a community of practice, Consortium participants share information in an open and rapid manner to addresses the complex health care and quality-of-life issues of cancer, transgender and other patients. To ensure that the organization remains contemporary to the needs of the community, the field designed a fully inclusive mechanism for strategic planning and here present the findings of this process. This interprofessional network of medical specialists, scientists, and scholars in the law, medical ethics, religious studies and other disciplines associated with human interventions, explore the relationships between health, disease, survivorship, treatment, gender and reproductive longevity. The goals are to continually integrate the best science in the service of the needs of patients and build a community of care that is ready for the challenges of the field in the future

Encapsulated Three-Dimensional Culture Supports Development of Nonhuman Primate Secondary Follicles1

In vitro ovarian follicle cultures may provide fertility-preserving options to women facing premature infertility due to cancer therapies. An encapsulated three-dimensional (3-D) culture system utilizing biomaterials to maintain cell-cell communication and support follicle development to produce a mature oocyte has been developed for the mouse. We tested whether this encapsulated 3-D system would also support development of nonhuman primate preantral follicles, for which in vitro growth has not been reported. Three questions were investigated: Does the cycle stage at which the follicles are isolated affect follicle development? Does the rigidity of the hydrogel influence follicle survival and growth? Do follicles require luteinizing hormone (LH), in addition to follicle-stimulating hormone (FSH), for steroidogenesis? Secondary follicles were isolated from adult rhesus monkeys, encapsulated within alginate hydrogels, and cultured individually for ≤30 days. Follicles isolated from the follicular phase of the menstrual cycle had a higher survival rate (P < 0.05) than those isolated from the luteal phase; however, this difference may also be attributed to differing sizes of follicles isolated during the different stages. Follicles survived and grew in two hydrogel conditions (0.5% and 0.25% alginate). Follicle diameters increased to a greater extent (P < 0.05) in the presence of FSH alone than in FSH plus LH. Regardless of gonadotropin treatment, follicles produced estradiol, androstenedione, and progesterone by 14–30 days in vitro. Thus, an alginate hydrogel maintains the 3-D structure of individual secondary macaque follicles, permits follicle growth, and supports steroidogenesis for ≤30 days in vitro. This study documents the first use of the alginate system to maintain primate tissue architecture, and findings suggest that encapsulated 3-D culture will be successful in supporting the in vitro development of human follicles

Identification of Claudin 1 Transcript Variants in Human Invasive Breast Cancer

<div><p>Background</p><p>The claudin 1 tight junction protein, solely responsible for the barrier function of epithelial cells, is frequently down regulated in invasive human breast cancer. The underlying mechanism is largely unknown, and no obvious mutations in the claudin 1 gene (<i>CLDN1</i>) have been identified to date in breast cancer. Since many genes have been shown to undergo deregulation through splicing and mis-splicing events in cancer, the current study was undertaken to investigate the occurrence of transcript variants for <i>CLDN1</i> in human invasive breast cancer.</p><p>Methods</p><p>RT-PCR analysis of <i>CLDN1</i> transcripts was conducted on RNA isolated from 12 human invasive breast tumors. The PCR products from each tumor were resolved by agarose gel electrophoresis, cloned and sequenced. Genomic DNA was also isolated from each of the 12 tumors and amplified using PCR <i>CLDN1</i> specific primers. Sanger sequencing and single nucleotide polymorphism (SNP) analyses were conducted.</p><p>Results</p><p>A number of <i>CLDN1</i> transcript variants were identified in these breast tumors. All variants were shorter than the classical <i>CLDN1</i> transcript. Sequence analysis of the PCR products revealed several splice variants, primarily in exon 1 of <i>CLDN1</i>; resulting in truncated proteins. One variant, V1, resulted in a premature stop codon and thus likely led to nonsense mediated decay. Interestingly, another transcript variant, V2, was not detected in normal breast tissue samples. Further, sequence analysis of the tumor genomic DNA revealed SNPs in 3 of the 4 coding exons, including a rare missense SNP (rs140846629) in exon 2 which represents an Ala124Thr substitution. To our knowledge this is the first report of <i>CLDN1</i> transcript variants in human invasive breast cancer. These studies suggest that alternate splicing may also be a mechanism by which claudin 1 is down regulated at both the mRNA and protein levels in invasive breast cancer and may provide novel insights into how <i>CLDN1</i> is reduced or silenced in human breast cancer.</p></div

Development of a Population-Based Newborn Screening Method for Severe Combined Immunodeficiency in Manitoba, Canada

The incidence of Severe Combined Immunodeficiency (SCID) in Manitoba, (1/15,000), is at least three to four times higher than the national average and that reported from other jurisdictions. It is overrepresented in two population groups: Mennonites (ZAP70 founder mutation) and First Nations of Northern Cree ancestry (IKBKB founder mutation). We have previously demonstrated that in these two populations the most widely utilized T-cell receptor excision circle (TREC) assay is an ineffective newborn screening test to detect SCID as these patients have normal numbers of mature T-cells. We have developed a semi-automated, closed tube, high resolution DNA melting procedure to simultaneously genotype both of these mutations from the same newborn blood spot DNA extract used for the TREC assay. Parallel analysis of all newborn screening specimens utilizing both TREC analysis and the high-resolution DNA procedure should provide as complete ascertainment as possible of SCID in the Manitoba population

Identification of <i>CLDN1</i> transcript variants in invasive human breast cancer.

<p>PCR analysis was carried out on reverse transcribed RNA from 12 breast tumors using primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163387#pone.0163387.t001" target="_blank">Table 1</a>) flanking the coding regions of <i>CLDN1</i>, <i>3</i>, and <i>4</i>. The expected full length cDNA products (representing the classical transcripts) for <i>CLDN1</i> and <i>CLDN4</i> (700 bases and 706 bases respectively) was evident in all tumors. However, no <i>CLDN3</i> transcript was detected in some of the breast tumors (middle panel; lanes 4, 10, 11). Colored arrows indicate <i>CLDN1</i> PCR products which were verified by Sanger sequencing: yellow arrow, transcript variant 1 (V1) is 615 bp; red arrow, transcript variant 2 (V2) is 440bp; blue arrow, transcript variant 3 (V3) is 362 bp; and green arrow, transcript variant 4 (V4) is 217 bp. In the lower panel, the product indicated by the black arrow, was a non-specific band and had no sequence homology to <i>CLDN4</i>.</p