Frameshift mutation hotspot identified in Smith-Magenis syndrome: case report and review of literature

Smith-Magenis syndrome (SMS) is a complex syndrome involving intellectual disabilities, sleep disturbance, behavioural problems, and a variety of craniofacial, skeletal, and visceral anomalies. While the majority of SMS cases harbor an ~3.5 Mb common deletion on 17p11.2 that encompasses the retinoic acid induced-1 (RAI1) gene, some patients carry small intragenic deletions or point mutations in RAI1. We present data on two cases of Smith-Magenis syndrome with mutation of RAI1. Both cases are phenotypically consistent with SMS and RAI1 mutation but also have other anomalies not previously reported in SMS, including spontaneous pneumothoraces. These cases also illustrate variability in the SMS phenotype not previously shown for RAI1 mutation cases, including hearing loss, absence of self-abusive behaviours, and mild global delays. Sequencing of RAI1 revealed mutation of the same heptameric C-tract (CCCCCCC) in exon 3 in both cases (c.3103delC one case and and c.3103insC in the other), resulting in frameshift mutations. Of the seven reported frameshift mutations occurring in poly C-tracts in RAI1, four cases (~57%) occur at this heptameric C-tract. Collectively, these results indicate that this heptameric C-tract is a preferential hotspot for single nucleotide insertion/deletions (SNindels) and therefore, should be considered a primary target for analysis in patients suspected for mutations in RAI1. We expect that as more patients are sequenced for mutations in RAI1, the incidence of frameshift mutations in this hotspot will become more evident

Severe neurocognitive and growth disorders due to variation in THOC2, an essential component of nuclear mRNA export machinery

Highly conserved TREX-mediated mRNA export is emerging as a key pathway in neuronal development and differentiation. TREX subunit variants cause neurodevelopmental disorders (NDDs) by interfering with mRNA export from the cell nucleus to the cytoplasm. Previously we implicated four missense variants in the X-linked THOC2 gene in intellectual disability (ID). We now report an additional six affected individuals from five unrelated families with two de novo and threematernally inherited pathogenic or likely pathogenic variants in THOC2 extending the genotypic and phenotypic spectrum. These comprise three rare missense THOC2 variants that affect evolutionarily conserved amino acid residues and reduce protein stability and two with canonical splice-site THOC2 variants that result in C-terminally truncated THOC2 proteins.We present detailed clinical assessment and functional studies on a de novo variant in a female with an epileptic encephalopathy and discuss an additional four families with rare variants in THOC2 with supportive evidence for pathogenicity. Severe neurocognitive features, including movement and seizure disorders, were observed in this cohort. Taken together our data show that even subtle alterations to the canonical molecular pathways such asmRNAexport, otherwise essential for cellular life, can be compatible with life, but lead to NDDs in human

A randomized controlled trial of a decision aid for women considering genetic testing for breast and ovarian cancer risk

PURPOSE: To measure the effectiveness of a tailored decision aid (DA) designed to help women make informed decisions about genetic testing for breast/ovarian cancer risk. METHODS: A total of 145 women were randomized to receive the DA or a control pamphlet at the end of their first genetic counseling consultation. Of these, 120 (82.8%) completed two questionnaires, 1 week and 6 months post-consultation. RESULTS: While the DA had no effect on informed choice, post-decisional regret or actual genetic testing decision, the trial showed that women who received the DA had higher knowledge levels and felt more informed about genetic testing than women who received the control pamphlet (chi(2)(2) = 6.82; P = 0.033; chi(2)(1) = 4.86; P = 0.028 respectively). The DA also helped women who did not have blood drawn at their first consultation to clarify their values with regards to genetic testing (chi(2)(1) = 5.27; P = 0.022). Women who received the DA were less likely to share the information with other family members than women in the control condition (chi(2)(1) = 8.78; P = 0.003). CONCLUSIONS: Decision aids are an effective decision-support strategy for women considering genetic testing for breast/ovarian cancer risk, and are most effective before the patient has made a decision, which is generally at the point of having blood drawn

Homologous recombination DNA repair defects in PALB2- associated breast cancers

Abstract: Mono-allelic germline pathogenic variants in the Partner And Localizer of BRCA2 (PALB2) gene predispose to a high-risk of breast cancer development, consistent with the role of PALB2 in homologous recombination (HR) DNA repair. Here, we sought to define the repertoire of somatic genetic alterations in PALB2-associated breast cancers (BCs), and whether PALB2-associated BCs display bi-allelic inactivation of PALB2 and/or genomic features of HR-deficiency (HRD). Twenty-four breast cancer patients with pathogenic PALB2 germline mutations were analyzed by whole-exome sequencing (WES, n = 16) or targeted capture massively parallel sequencing (410 cancer genes, n = 8). Somatic genetic alterations, loss of heterozygosity (LOH) of the PALB2 wild-type allele, large-scale state transitions (LSTs) and mutational signatures were defined. PALB2-associated BCs were found to be heterogeneous at the genetic level, with PIK3CA (29%), PALB2 (21%), TP53 (21%), and NOTCH3 (17%) being the genes most frequently affected by somatic mutations. Bi-allelic PALB2 inactivation was found in 16 of the 24 cases (67%), either through LOH (n = 11) or second somatic mutations (n = 5) of the wild-type allele. High LST scores were found in all 12 PALB2-associated BCs with bi-allelic PALB2 inactivation sequenced by WES, of which eight displayed the HRD-related mutational signature 3. In addition, bi-allelic inactivation of PALB2 was significantly associated with high LST scores. Our findings suggest that the identification of bi-allelic PALB2 inactivation in PALB2-associated BCs is required for the personalization of HR-directed therapies, such as platinum salts and/or PARP inhibitors, as the vast majority of PALB2-associated BCs without PALB2 bi-allelic inactivation lack genomic features of HRD

Development and validation of a targeted gene sequencing panel for application to disparate cancers

Next generation sequencing has revolutionised genomic studies of cancer, having facilitated the development of precision oncology treatments based on a tumour’s molecular profile. We aimed to develop a targeted gene sequencing panel for application to disparate cancer types with particular focus on tumours of the head and neck, plus test for utility in liquid biopsy. The final panel designed through Roche/Nimblegen combined 451 cancer-associated genes (2.01 Mb target region). 136 patient DNA samples were collected for performance and application testing. Panel sensitivity and precision were measured using well-characterised DNA controls (n = 47), and specificity by Sanger sequencing of the Aryl Hydrocarbon Receptor Interacting Protein (AIP) gene in 89 patients. Assessment of liquid biopsy application employed a pool of synthetic circulating tumour DNA (ctDNA). Library preparation and sequencing were conducted on Illumina-based platforms prior to analysis with our accredited (ISO15189) bioinformatics pipeline. We achieved a mean coverage of 395x, with sensitivity and specificity of >99% and precision of >97%. Liquid biopsy revealed detection to 1.25% variant allele frequency. Application to head and neck tumours/cancers resulted in detection of mutations aligned to published databases. In conclusion, we have developed an analytically-validated panel for application to cancers of disparate types with utility in liquid biopsy

The FANCM:p.Arg658* truncating variant is associated with risk of triple-negative breast cancer

Abstract: Breast cancer is a common disease partially caused by genetic risk factors. Germline pathogenic variants in DNA repair genes BRCA1, BRCA2, PALB2, ATM, and CHEK2 are associated with breast cancer risk. FANCM, which encodes for a DNA translocase, has been proposed as a breast cancer predisposition gene, with greater effects for the ER-negative and triple-negative breast cancer (TNBC) subtypes. We tested the three recurrent protein-truncating variants FANCM:p.Arg658*, p.Gln1701*, and p.Arg1931* for association with breast cancer risk in 67,112 cases, 53,766 controls, and 26,662 carriers of pathogenic variants of BRCA1 or BRCA2. These three variants were also studied functionally by measuring survival and chromosome fragility in FANCM−/− patient-derived immortalized fibroblasts treated with diepoxybutane or olaparib. We observed that FANCM:p.Arg658* was associated with increased risk of ER-negative disease and TNBC (OR = 2.44, P = 0.034 and OR = 3.79; P = 0.009, respectively). In a country-restricted analysis, we confirmed the associations detected for FANCM:p.Arg658* and found that also FANCM:p.Arg1931* was associated with ER-negative breast cancer risk (OR = 1.96; P = 0.006). The functional results indicated that all three variants were deleterious affecting cell survival and chromosome stability with FANCM:p.Arg658* causing more severe phenotypes. In conclusion, we confirmed that the two rare FANCM deleterious variants p.Arg658* and p.Arg1931* are risk factors for ER-negative and TNBC subtypes. Overall our data suggest that the effect of truncating variants on breast cancer risk may depend on their position in the gene. Cell sensitivity to olaparib exposure, identifies a possible therapeutic option to treat FANCM-associated tumors

The ARID1B spectrum in 143 patients: from nonsyndromic intellectual disability to Coffin–Siris syndrome

Purpose: Pathogenic variants in ARID1B are one of the most frequent causes of intellectual disability (ID) as determined by large-scale exome sequencing studies. Most studies published thus far describe clinically diagnosed Coffin–Siris patients (ARID1B-CSS) and it is unclear whether these data are representative for patients identified through sequencing of unbiased ID cohorts (ARID1B-ID). We therefore sought to determine genotypic and phenotypic differences between ARID1B-ID and ARID1B-CSS. In parallel, we investigated the effect of different methods of phenotype reporting. Methods: Clinicians entered clinical data in an extensive web-based survey. Results: 79 ARID1B-CSS and 64 ARID1B-ID patients were included. CSS-associated dysmorphic features, such as thick eyebrows, long eyelashes, thick alae nasi, long and/or broad philtrum, small nails and small or absent fifth distal phalanx and hypertrichosis, were observed significantly more often (p < 0.001) in ARID1B-CSS patients. No other significant differences were identified. Conclusion: There are only minor differences between ARID1B-ID and ARID1B-CSS patients. ARID1B-related disorders seem to consist of a spectrum, and patients should be managed similarly. We demonstrated that data collection methods without an explicit option to report the absence of a feature (such as most Human Phenotype Ontology-based methods) tended to underestimate gene-related features

Factor V Leiden and adverse pregnancy outcome

Research Doctorate - Doctor of Philosophy (PhD)Intrauterine fetal death, fetal growth restriction (FGR) and pre-eclampsia are major causes of fetal and maternal morbidity and mortality; and placental insufficiency is frequently proposed as the most important underlying mechanism. Given the importance of establishing and maintaining an adequate placental circulation, hereditary thrombophilias are postulated as a possible cause of placental insufficiency. Despite initial reports supporting an association between factor V Leiden (fVL) and adverse pregnancy outcomes, a number of other studies yielded conflicting results. A systematic review and meta-analysis of the literature up to January 2003 was undertaken to address the question of whether the common maternal fVL genotype is associated with an increased risk of adverse pregnancy outcomes (pre-eclampsia, fetal growth restriction and fetal loss). Subsequent meta-analyses were also evaluated. To address the shortfalls observed in the large number of small and possibility underpowered case-control studies, a decision was made to undertake a large nested case-control study within the Avon Longitudinal Study of Parents and Children (ALSPAC) cohort. The aim of this study was to evaluate the association between: 1) maternal fVL and intrauterine fetal death, fetal growth restriction and pre-eclampsia; 2) fetal fVL and intrauterine fetal death, fetal growth restriction; 3) maternal prothrombin gene variant (PGV) and intrauterine fetal death, fetal growth restriction and pre-eclampsia and 4) fetal PGV and intrauterine fetal death, fetal growth restriction and pre-eclampsia. Data from other published cohort studies was combined by meta-analysis to increase the power of detecting an association. Overall, the results of the study within the large ALSPAC cohort show no statistically significant association between maternal/fetal fVL or PGV, either alone or in combination with birth weight <10th centile. Furthermore, the FGR meta-analysis which pooled the results of this cohort study and other cohort studies found no evidence of an effect of maternal fVL on FGR. Given the size of the pooled sample, there was 80% power to detect an OR of 1.09, indicating that if an effect of fVL on FGR was missed by this meta-analysis, it would be quite small. The results of this study within the large ALSPAC cohort show no statistically significant association between maternal or fetal fVL or PGV, either alone or in combination with pre-eclampsia. However, increasing the power by combining this study with other cohort studies by meta-analysis revealed a positive association between maternal fVL and pre-eclampsia with an OR of 1.49 (95% CI 1.13-1.96 p=0.003). A narrative review was subsequently published examining the translation from statistical association to change in clinical practice with respect to fVL and adverse pregnancy outcomes. The thesis concludes with a discussion of management in different clinical scenarios relating to fVL and adverse pregnancy outcomes, and the identification of future priority research areas

Lessons from the skin: cutaneous features of familial cancer

As the molecular basis of disease continues to be elucidated, familial cancer syndromes, which consist of a range of neoplastic and non-neoplastic features, are emerging. The usual pathway of referral to a genetics clinic or familial cancer centre is via an oncologist, when high-risk features that suggest a possible hereditary basis for the presenting cancer are recognised. Traditionally, these high-risk features include more than two family members with similar cancers over two or more generations, a young age of onset, and more than one synchronous or metachronous tumour. These features are effective in ascertaining a substantial proportion of families with hereditary breast and ovarian cancer due to a BRCA mutation, or the more common bowel-cancer predisposition syndromes, such as hereditary non-polyposis colon cancer and familial adenomatous polyposis. However, there are a range of familial cancer syndromes that are not easily detected and that can remain undiagnosed when history and examination are not extended to include non-malignant features. The identification of cutaneous signs associated with rare familial-cancer syndromes provides individuals and their families with the opportunity to undertake early surveillance for malignant and non-malignant complications that might in time be shown to improve outcomes

The virtual consultation: practitioners' experiences of genetic counseling by videoconferencing in Australia

Objective: Videoconferencing for clinical genetics services, or telegenetics, is becoming an increasingly utilized method of delivering genetic counseling to rural areas; however, there has been little qualitative exploration of the practitioner's experience, particularly for hereditary breast/ovarian cancer counseling. Methods: Semistructured interviews were conducted with genetic practitioners (n=15) delivering telegenetics services in New South Wales, Australia. Interviews explored experiences, perceived aims of the service, satisfaction, and the advantages and disadvantages of the technology. Interviews were audiotaped, transcribed, and thematically analyzed. Results: All practitioners were highly satisfied with telegenetics. They perceived the advantages of videoconferencing as primarily increased efficiency and convenience for genetic clinicians, minimized travel for the patient, reduced costs, and increased access to rural areas. Disadvantages included the inhibition of rapport building between genetic clinician and patient and the difficulty in detecting nonverbal cues. Telegenetics was seen as a structured interaction that allowed less time for emotional exploration than a traditional face-to-face consultation. Technical disadvantages involved visual resolution, connection speed, and interruptions to voice transmission. Conclusion: Practitioners were satisfied with telegenetics and perceived the advantages as outweighing the disadvantages