Clinical and genetic findings in TRPM1-related congenital stationary night blindness.

PURPOSE: Congenital stationary night blindness (CSNB) is a heterogeneous group of Mendelian retinal disorders that present in childhood. Biallelic variants altering the protein-coding region of the TRPM1 gene are one of the commonest causes of CSNB. Here, we report the clinical and genetic findings in 10 unrelated individuals with TRPM1-retinopathy. METHODS: Study subjects were recruited through a tertiary clinical ophthalmic genetic service at Manchester, UK. All participants underwent visual electrodiagnostic testing and panel-based genetic analysis. RESULTS: Study subjects had a median age of 8 years (range: 3-20 years). All probands were myopic and had electroretinographic findings in keeping with complete CSNB. Notably, three probands reported no night vision problems. Fourteen different disease-associated TRPM1 variants were detected. One individual was homozygous for the NM_001252024.2 (TRPM1):c.965 + 29G>A variant and a mini-gene assay highlighted that this change results in mis-splicing and premature protein termination. Additionally, two unrelated probands who had CSNB and mild neurodevelopmental abnormalities were found to carry a 15q13.3 microdeletion. This copy number variant encompasses seven genes, including TRPM1, and was encountered in the heterozygous state and in trans with a missense TRPM1 variant in each case. CONCLUSION: Our findings highlight the importance of comprehensive genomic analysis, beyond the exons and protein-coding regions of genes, for individuals with CSNB. When this characteristic retinal phenotype is accompanied by extraocular findings (including learning and/or behavioural difficulties), a 15q13.3 microdeletion should be suspected. Focused analysis (e.g. microarray testing) is recommended to look for large-scale deletions encompassing TRPM1 in patients with CSNB and neurodevelopmental abnormalities.

Bi-allelic mutation of CTNNB1 causes a severe form of syndromic microphthalmia, persistent foetal vasculature and vitreoretinal dysplasia.

BACKGROUND: Inherited vitreoretinopathies arise as a consequence of congenital retinal vascularisation abnormalities. They represent a phenotypically and genetically heterogeneous group of disorders that can have a major impact on vision. Several genes encoding proteins and effectors of the canonical Wnt/ß-catenin pathway have been associated and precise diagnosis, although difficult, is essential for proper clinical management including syndrome specific management where appropriate. This work aimed to investigate the molecular basis of disease in a single proband born to consanguineous parents, who presented with microphthalmia, persistent foetal vasculature, posterior lens vacuoles, vitreoretinal dysplasia, microcephaly, hypotelorism and global developmental delay, and was registered severely visually impaired by 5 months of age. METHODS: Extensive genomic pre-screening, including microarray comparative genomic hybridisation and sequencing of a 114 gene panel associated with cataract and congenital ophthalmic disorders was conducted by an accredited clinical laboratory. Whole exome sequencing (WES) was undertaken on a research basis and in vitro TOPflash transcriptional reporter assay was utilised to assess the impact of the putative causal variant. RESULTS: In the proband, WES revealed a novel, likely pathogenic homozygous mutation in the cadherin-associated protein beta-1 gene (CTNNB1), c.884C>G; p.(Ala295Gly), which encodes a co-effector molecule of the Wnt/ß-catenin pathway. The proband's parents were shown to be heterozygous carriers but ophthalmic examination did not detect any abnormalities. Functional assessment of the missense variant demonstrated significant reduction of ß-catenin activity. CONCLUSIONS: This is the first report of a biallelic disease-causing variation in CTNNB1. We conclude that this biallelic, transcriptional inactivating mutation of CTNNB1 causes a severe, syndromic form of microphthalmia, persistent foetal vasculature and vitreoretinal dysplasia that results in serious visual loss in infancy.

Clinical and genetic variability in children with partial albinism.

Individuals who have ocular features of albinism and skin pigmentation in keeping with their familial background present a considerable diagnostic challenge. Timely diagnosis through genomic testing can help avert diagnostic odysseys and facilitates accurate genetic counselling and tailored specialist management. Here, we report the clinical and gene panel testing findings in 12 children with presumed ocular albinism. A definitive molecular diagnosis was made in 8/12 probands (67%) and a possible molecular diagnosis was identified in a further 3/12 probands (25%). TYR was the most commonly mutated gene in this cohort (75% of patients, 9/12). A disease-causing TYR haplotype comprised of two common, functional polymorphisms, TYR c.[575 C > A;1205 G > A] p.[(Ser192Tyr);(Arg402Gln)], was found to be particularly prevalent. One participant had GPR143-associated X-linked ocular albinism and another proband had biallelic variants in SLC38A8, a glutamine transporter gene associated with foveal hypoplasia and optic nerve misrouting without pigmentation defects. Intriguingly, 2/12 individuals had a single, rare, likely pathogenic variant in each of TYR and OCA2 - a significant enrichment compared to a control cohort of 4046 individuals from the 100,000 genomes project pilot dataset. Overall, our findings highlight that panel-based genetic testing is a clinically useful test with a high diagnostic yield in children with partial/ocular albinism.

Molecular findings from 537 individuals with inherited retinal disease.

BACKGROUND: Inherited retinal diseases (IRDs) are a clinically and genetically heterogeneous set of disorders, for which diagnostic second-generation sequencing (next-generation sequencing, NGS) services have been developed worldwide. METHODS: We present the molecular findings of 537 individuals referred to a 105-gene diagnostic NGS test for IRDs. We assess the diagnostic yield, the spectrum of clinical referrals, the variant analysis burden and the genetic heterogeneity of IRD. We retrospectively analyse disease-causing variants, including an assessment of variant frequency in Exome Aggregation Consortium (ExAC). RESULTS: Individuals were referred from 10 clinically distinct classifications of IRD. Of the 4542 variants clinically analysed, we have reported 402 mutations as a cause or a potential cause of disease in 62 of the 105 genes surveyed. These variants account or likely account for the clinical diagnosis of IRD in 51% of the 537 referred individuals. 144 potentially disease-causing mutations were identified as novel at the time of clinical analysis, and we further demonstrate the segregation of known disease-causing variants among individuals with IRD. We show that clinically analysed variants indicated as rare in dbSNP and the Exome Variant Server remain rare in ExAC, and that genes discovered as a cause of IRD in the post-NGS era are rare causes of IRD in a population of clinically surveyed individuals. CONCLUSIONS: Our findings illustrate the continued powerful utility of custom-gene panel diagnostic NGS tests for IRD in the clinic, but suggest clear future avenues for increasing diagnostic yields.

Explaining Mendelian inheritance in genetic consultations: an IPR study of counselor and counselee experiences.

The explanation of Mendelian inheritance is a key component of most genetic counselling consultations, yet no evidence base exists for this area of practice. This qualitative study used Interpersonal Process Recall (IPR) to explore how information about X-linked inheritance is provided and received in genetic counseling. Twelve consultations involving two senior genetic counselors and 21 counselees were videotaped. Section(s) of videotape featuring the explanation were subsequently played back separately to both counselees and counselors and their responses and reflections recorded. All interviews were fully transcribed and analysed using the constant comparison method. A personalized diagram, drawn "live" by the counselor during the consultation was recalled by counselees as being central to their understanding of the "bottom line". This helped bridge the gap between scientific information and their family experience and did not appear to require a baseline understanding of genetic concepts such as genes or chromosomes. Counselors reflected on the diagram's positive impact on the way they sequenced, paced and tailored the explanation. A positive counselor-counselee relationship was vital even during this educative exchange: for counselees to feel at ease discussing complex genetic information and to help gauge counselee understanding.

The molecular genetics of breast cancer: the contribution of comparative genomic hybridization.

Comparative genomic hybridization (CGH) has been the technique of choice over the last 10 years for mapping DNA copy number changes in human tumors. Here we review the literature to demonstrate how CGH has contributed to the comprehension of molecular aspects of breast tumorigenesis. At least two distinct molecular pathways of breast cancer have been characterized that show a strong correlation with histological grade. It seems that grade I invasive ductal carcinomas (IDCs) arise from well-differentiated ductal carcinoma in situ (DCIS), whereas grade III IDCs come from poorly differentiated DCIS. In addition, dedifferentiation from a low- to a high-grade breast cancer has proven an unlikely phenomenon. CGH has been instrumental in dissecting distinct molecular pathways toward breast malignancy and in establishing a direct relationship between genotype and clinical pathological features.

Molecular evolution of breast cancer.

Molecular analysis of invasive breast cancer and its precursors has furthered our understanding of breast cancer progression. In the past few years, new multi-step pathways of breast cancer progression have been delineated through genotypic-phenotypic correlations. Nuclear grade, more than any other pathological feature, is strongly associated with the number and pattern of molecular genetic abnormalities in breast cancer cells. Thus, there are two distinct major pathways to the evolution of low- and high-grade invasive carcinomas: whilst the former consistently show oestrogen receptor (ER) and progesterone receptor (PgR) positivity and 16q loss, the latter are usually ER/PgR-negative and show Her-2 overexpression/amplification and complex karyotypes. The boundaries between the evolutionary pathways of well-differentiated/low-grade ductal and lobular carcinomas have been blurred, with changes in E-cadherin expression being one of the few distinguishing features between the two. In addition, lesions long thought to be precursors of breast carcinomas, such as hyperplasia of usual type, are currently considered mere risk indicators, whilst columnar cell lesions are now implicated as non-obligate precursors of atypical ductal hyperplasia (ADH) and well-differentiated ductal carcinoma in situ (DCIS). However, only through the combination of comprehensive morphological analysis and cutting-edge molecular tools can this knowledge be translated into clinical practice and patient management.

Distribution and significance of 14-3-3sigma, a novel myoepithelial marker, in normal, benign, and malignant breast tissue.

14-3-3sigma is a candidate tumour suppressor gene transactivated by p53 in response to DNA damage. In gene expression analysis of normal luminal and myoepithelial cells, 14-3-3sigma was preferentially expressed by myoepithelial cells. This study has analysed the immunohistochemical distribution and subcellular localization of 14-3-3sigma in normal breast tissue and in a large series of benign and malignant breast lesions on whole tissue sections and by tissue microarray. Immunohistochemistry demonstrated that 14-3-3sigma was consistently expressed in the cytoplasmic compartment and occasionally in the nuclei of myoepithelial cells arranged as a continuous layer around normal ducts and lobular units, whereas luminal epithelial, stromal, endothelial, pericytic, lipomatous, and neural cells showed no staining. Myoepithelial cells of benign proliferations and pre-invasive lesions were consistently positive for 14-3-3sigma. Strong expression of 14-3-3sigma was evident in one case of ductal carcinoma in situ (5.5%) and in 105/554 invasive cancers (18.9%). Survival data were available for 452 patients with invasive breast carcinoma. 14-3-3sigma cytoplasmic subcellular localization was a statistically significant prognostic factor for the whole series of invasive carcinomas, as well as for those positive for oestrogen (ER) or progesterone receptors (PR). This analysis demonstrates the utility of 14-3-3sigma as a new adjunct antibody for characterization of myoepithelial cells and myoepithelial lesions and it may be a novel prognostic factor for breast cancer patients.

The diagnosis and management of pre-invasive breast disease: pathology of atypical lobular hyperplasia and lobular carcinoma in situ.

The term lobular neoplasia refers to a spectrum of lesions featuring atypical lobular hyperplasia and lobular carcinoma in situ (LCIS). The histopathological characteristics of these lesions are well documented. What is less well understood is the management implications of a patient diagnosed with LCIS; treatment regimes vary and are somewhat controversial. LCIS is now considered a risk factor and a non-obligate precursor for the subsequent development of invasive cancer.