Development and application of a weighted change score to evaluate interventions for vasomotor symptoms in patients with breast cancer using regression trees: a cohort study.

PURPOSE: Vasomotor symptoms (VMS) are common among individuals with breast cancer (BC) and poorly managed symptoms are associated with reduced quality of life, treatment discontinuation, and poorer breast cancer outcomes. Direct comparisons among therapies are limited, as prior studies evaluating VMS interventions have utilized heterogeneous change measures which may not fully assess the perceived impact of change in VMS severity. METHODS: We performed a prospective study where BC patients chose one of four categories of interventions to manage VMS. Change in VMS severity at 6 weeks was assessed using the validated Hot Flush Rating Scale (HFRS). A novel weighted change score integrating baseline symptom severity and directionality of change was computed to maximize the correlation between the change score and a perceived treatment effectiveness score. Variables influencing change in VMS severity were included in a regression tree to model factors influencing the weighted change score. RESULTS: 100 baseline and follow-up questionnaires assessing VMS were completed by 88 patients. Correlations between treatment effectiveness and VMS outcomes strengthened following adjustment for baseline symptoms. Patients with low VMS severity at baseline did not perceive change in treatment effectiveness. Intervention category was predictive of change in HFRS at 6 weeks. CONCLUSION: Baseline symptom severity and the directionality of change (improvement or deterioration of symptoms) influenced the perception of clinically meaningful change in VMS severity. Future interventional studies utilizing the weighted change score should target moderate-high baseline severity patients.

Evaluation of a nurse-led glaucoma assessment clinic for non-complex patients.

AIMS AND OBJECTIVES: The purpose of this study was to assess the safety, feasibility and effectiveness of a nurse-led glaucoma assessment clinic service. BACKGROUND: Glaucoma is a group of serious, irreversible optic neuropathies that progressively damage the optic nerve resulting in blindness. Over 64.3 million people globally are affected by glaucoma with numbers projected to increase to 111.8 million by 2040. Glaucoma is a major public health concern that requires the development of innovative models of care to meet current and future health care demands. METHODS/DESIGN: A mixed method design was used to evaluate the assessment of non-complex glaucoma patients attending a new nurse-led clinic. Under an ophthalmologist's supervision, the glaucoma nurse completed 100 hof clinical training and assessment to ensure they were competent in completing and interpreting the required glaucoma assessment protocols. Interrater reliability between the glaucoma nurse and ophthalmology doctor was undertaken. Glaucoma patient waitlist appointment data were also compared before and after the introduction of the nurse-led clinics. This study adhered to the SQUIRE checklist for the reporting of excellence with quality improvement projects. PATIENT CONTRIBUTION: Patients contributed to the evaluation of this new nurse-led service by providing follow-up feedback on their experience. RESULTS: Agreement between clinicians for appropriate follow-up appointment times was high with 93% (n = 315) agreement achieved. Furthermore in 297 (87.5%) cases, both clinicians agreed on the patient being referred to the doctor for follow-up review. Glaucoma consultations were shown to increase from 3,115 appointments in 2019/20 to 3,504 appointments in 2020/21 after the introduction of the nurse-led clinic. The nurse-led clinics accounted for 14.5% (n = 512) of clinic appointments. CONCLUSIONS: The introduction of the nurse-led glaucoma assessment clinic service allowed patients to be reviewed safely, efficiently and satisfactorily. This new service subsequently allowed for more complex glaucoma patients to be seen by the ophthalmologists. RELEVANCE TO CLINICAL PRACTICE: Findings revealed that suitably trained glaucoma nurses are able to clinically assess and safely monitor stable non-complex glaucoma patients. Highlights the need for appropriate investment in clinical training and supervision to ensure that glaucoma assessment nurses are adequately prepared to undertake this new practice role.

Delivery of cancer care via an outpatient telephone support line: a cross-sectional study of oncology nursing perspectives on quality and challenges.

RATIONALE: Patient support lines (PSLs) assist in triaging clinical problems, addressing patient queries, and navigating a complex multi-disciplinary oncology team. While providing support and training to the nursing staff who operate these lines is key, there is limited data on their experience and feedback. METHODS: We conducted a cross-sectional study of oncology nurses' (ONs') perspectives on the provision of care via PSLs at a tertiary referral cancer center via an anonymous, descriptive survey. Measures collected included nursing and patient characteristics, nature of questions addressed, perceived patient and nursing satisfaction with the service, common challenges faced, and initiatives to improve the patient and nursing experience. The survey was delivered online, with electronic data collection, and analysis is reported descriptively. RESULTS: Seventy-one percent (30/42) of eligible ONs responded to the survey. The most common disease site, stage, and symptom addressed by PSLs were breast cancer, metastatic disease, and pain, respectively. The most common reported issue was treatment-related toxicity (96.7%, 29/30). Sixty-seven percent (20/30) of respondents were satisfied with the care provided by the service; however, many areas for potential improvement were identified. Fifty-nine percent (17/29) of respondents recommended redefining PSLs' responsibilities for improved use, with 75% (6/8) ONs identifying high call volumes due to inappropriate questions as a barrier to care. Sixty percent (18/30) of ONs reported having hospital-specific management plans for common issues would improve the care provided by the PSL. CONCLUSION: Despite high rates of satisfaction with the care provided by the PSL, our study identified several important areas for improvement which we feel warrant further investigation.

Using machine learning to predict individual patient toxicities from cancer treatments.

PURPOSE: Machine learning (ML) is a powerful tool for interrogating datasets and learning relationships between multiple variables. We utilized a ML model to identify those early breast cancer (EBC) patients at highest risk of developing severe vasomotor symptoms (VMS). METHODS: A gradient boosted decision model utilizing cross-sectional survey data from 360 EBC patients was created. Seventeen patient- and treatment-specific variables were considered in the model. The outcome variable was based on the Hot Flush Night Sweats (HFNS) Problem Rating Score, and individual scores were dichotomized around the median to indicate individuals with high and low problem scores. Model accuracy was assessed using the area under the receiver operating curve, and conditional partial dependence plots were constructed to illustrate relationships between variables and the outcome of interest. RESULTS: The model area under the ROC curve was 0.731 (SD 0.074). The most important variables in the model were as follows: the number of hot flashes per week, age, the prescription, or use of drug interventions to manage VMS, whether patients were asked about VMS in routine follow-up visits, and the presence or absence of changes to breast cancer treatments due to VMS. A threshold of 17 hot flashes per week was identified as being more predictive of severe VMS. Patients between the ages of 49 and 63 were more likely to report severe symptoms. CONCLUSION: Machine learning is a unique tool for predicting severe VMS. The use of ML to assess other treatment-related toxicities and their management requires further study.

Vasomotor symptoms in early breast cancer-a "real world" exploration of the patient experience.

BACKGROUND: Despite the frequency of vasomotor symptoms (VMS) in patients with early breast cancer (EBC), their optimal management remains unknown. A patient survey was performed to determine perspectives on this important clinical challenge. METHODS: Patients with EBC experiencing VMS participated in an anonymous survey. Patients reported on the frequency and severity of VMS using the validated Hot Flush Rating Scale (HFRS) and ranked their most bothersome symptoms. Respondents were also asked to determine endpoints that defined effective treatment of VMS and report on the effectiveness of previously tried interventions. RESULTS: Responses were received from 373 patients, median age 56 years (range 23-83), who experienced an average of 5.0 hot flashes per day (SD 6.57). Patients reported the most bothersome symptoms to be feeling hot/sweating (155/316, 49%) and sleeping difficulties (86/316, 27%). Fifty-five percent (201/365) of patients would consider a treatment to be effective if it reduced night-time awakenings. While 68% of respondents were interested in trying interventions from their healthcare team to manage VMS, only 18% actually did so. Of the 137 patients who had tried an intervention for VMS, pharmacological treatments, exercise, and relaxation strategies were more likely to be effective, while therapies such as melatonin and black cohosh were deemed less effective. CONCLUSION: VMS are a common and bothersome problem for EBC patients, with a minority receiving interventions to manage these symptoms. Further research is needed to identify patient-centered strategies for managing these distressing symptoms.

Legal Privilege Legislation: Consequences for Patient Safety.

Increasing awareness of the extent of preventable harm from healthcare has led to efforts to improve patient safety through a variety of efforts, including legislation. Extending legal privilege to quality and safety reviews leads to further harm for many patients, families and healthcare providers. The intentional isolation, silencing and exclusion after the incident undermines trust, prevents learning and impedes an opportunity to heal and recover for all those directly involved. Our case study examines Section 51 of British Columbia's Evidence Act (1996) and concludes that amending this legislation is an urgent and necessary step toward trauma-informed care.

Developing patient-centred strategies to optimize the management of vasomotor symptoms in breast cancer patients: a survey of health care providers.

PURPOSE: Vasomotor symptoms (VMS) such as hot flashes and night sweats are common in breast cancer patients and can affect both quality of life and treatment adherence. However, there is limited practical data to guide clinicians in the optimal selection of therapeutic strategies. A survey of health care providers was performed to better understand perspectives and prescribing practices for managing this problem. METHODS: Canadian health care providers who treat patients with early stage breast cancer (EBC) participated in an anonymous electronic survey. Participants provided their perspectives on the prevalence and severity of VMS among patients with EBC, outlined their management strategies, and provided feedback on the perceived efficacy of interventions for VMS. RESULTS: Responses were received from 65 providers including breast oncologists (36/65, 55%) and nurses with oncology expertise (29/65, 45%). Seventy-seven percent of participants reported regularly asking patients about VMS, and most indicated that bothersome VMS occurred in the majority of patients. Health care providers cited hot flash severity and sleep disruption as the most important issues for patients. The most common first- and second-line interventions recommended were lifestyle modifications (n = 32/65, 49.2%) and pharmacologic strategies (n = 27/65, 41.5%), respectively. Most respondents felt that interventions, including pharmacologic, over-the-counter, and complementary therapies, were only "somewhat effective". Overall, half of respondents (n = 35/65, 54%) reported being "confident" in managing VMS. CONCLUSION: Given the variability of treatment recommendations, and health care provider uncertainty around the benefits of therapies for VMS, more 'real-world' trials are needed to optimize patient care.

A randomized trial comparing vascular access strategies for patients receiving chemotherapy with trastuzumab for early-stage breast cancer.

PURPOSE: Trastuzumab-based chemotherapy is usually administered through either a peripherally inserted central catheter (PICC) or a totally implanted vascular access device (PORT). As the most effective type of access is unknown, a feasibility trial, prior to conducting a large pragmatic trial, was undertaken. METHODS: The trial methodology utilized the integrated consent model incorporating oral consent. Patients receiving trastuzumab-based neo/adjuvant chemotherapy for early-stage breast cancer were randomized to a PICC or PORT insertion. Feasibility was reflected through a combination of endpoints; however, the a priori definition of feasibility was > 25% of patients approached agreed to randomization and > 25% of physicians approached patients. Secondary outcomes included rates of line-associated complications such as thrombotic events requiring anticoagulation, line infections or phlebitis. RESULTS: During the study period, 4/15 (26.7%) medical oncologists approached patients about study participation. Of 59 patients approached, 56 (94.9%) agreed to randomization, 29 (51.8%) were randomized to PICC and 27 (48.2%) to PORT access. Overall, 17.2% (5/29) and 14.8% (4/27) of patients had at least one line-associated complication in the PICC and PORT arms respectively. The study was terminated early due to slow accrual. CONCLUSION: The study met its feasibility endpoints with respect to patient and physician engagement. However, the slow rate of accrual (56 patients in 2 years) means that conducting a large pragmatic trial would require additional strategies to make such a study possible. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02632435.

A multicentre, randomized pilot trial comparing vascular access strategies for early stage breast cancer patients receiving non-trastuzumab containing chemotherapy.

PURPOSE: All vascular access strategies foradministering chemotherapy in early stage breast cancer (EBC) are associated with risks and benefits. As the most effective type of access is unknown a feasibility trial, prior to conducting a large pragmatic trial, was undertaken. METHODS: The trial methodology utilized broad eligibility criteria and the integrated consent model incorporating oral consent. EBC patients receiving non-trastuzumab-containing chemotherapy were randomized to peripheral access or central line insertion. The a priori definition of feasibility was: > 25% of patients approached agreed to randomisation and > 25% of physicians approached patients. Secondary outcomes included rates of line-associated complications. RESULTS: Of 159 patients approached, 150 (94.3%) agreed to randomisation, 77 (51.3%) were randomized to peripheral and 73 (48.7%) to central access. 6/26 (23.1%) of medical oncologists approached patients. Rates of complications per chemotherapy cycles in the peripheral vs central access groups with risk difference (RD) (95% CI) were: thrombotic events requiring anticoagulation [1 (0.3%) vs. 3 (1.0%), RD - 0.7(- 1.9,0.5)], line infections [0 (0%) vs. 1 (0.3%), RD - 0.3(- 0.9,0.3)], phlebitis [2 (0.6%) vs. 0 (0%), RD 0.3(- 0.3,0.8)], and tissue infiltrations [4 (1.1%) vs. 1 (0.3%), RD 0.8(- 0.4,2.1)]. Overall, 8.0% (6/75) and 7.7% (5/65) of patients had at least one of these complications in the peripheral and central access arms respectively [RD - 0.9(- 9.4,7.6)]. The study was terminated early due to slow accrual. CONCLUSION: While meeting its a priori feasibility criteria for patient engagement, the slow accrual means that conducting a large pragmatic trial would require overcoming the barriers to physician recruitment. TRIAL REGISTRATION: NCT02688998.

Non-invasive prenatal diagnosis of Duchenne and Becker muscular dystrophies by relative haplotype dosage.

OBJECTIVE: Development of an accurate and affordable test for the non-invasive prenatal diagnosis of Duchenne and Becker muscular dystrophies (DMD/BMD) to implement in clinical practice. METHOD: Cell-free DNA was extracted from maternal blood and prepared for massively parallel sequencing on an Illumina MiSeq by targeted capture enrichment of single nucleotide polymorphisms (SNPs) across the dystrophin gene on chromosome X. Sequencing data were analysed by relative haplotype dosage. RESULTS: Seven healthy pregnant donors and two pregnant DMD carriers all bearing a male fetus were recruited through the non-invasive prenatal diagnosis for single gene disorders study. Non-invasive prenatal diagnosis testing was conducted by relative haplotype dosage analysis for X-linked disorders where the genomic DNA from the chorionic villus sampling (for healthy pregnant donors) or from the proband (for pregnant DMD carriers) was used to identify the reference haplotype. Results for all patients showed a test accuracy of 100%, when the calculated fetal fraction was >4% and correlated with known outcomes. A recombination event was also detected in a DMD patient. CONCLUSION: Our new test for NIPD of DMD/BMD has been shown to be accurate and reliable during initial stages of validation. It is also feasible for implementation into clinical service.

Comparison of the clinical scoring systems in Silver-Russell syndrome and development of modified diagnostic criteria to guide molecular genetic testing.

BACKGROUND: About half of all children with a clinical diagnosis of Silver-Russell syndrome (SRS) have a detectable molecular genetic abnormality (maternal uniparental disomy of chromosome upd(7)mat or hypomethylation of H19 differentially methylated region (DMR). The selection of children for molecular genetic testing can be difficult for non-specialists because of the broad phenotypic spectrum of SRS and the tendency of the facial features to mitigate during late childhood. Several clinical scoring systems for SRS have been developed by specialist researchers, but the utility of these for guiding molecular genetic testing in routine clinical practice has not been established. OBJECTIVES: To evaluate the utility of four published clinical scoring systems for genetic testing in a cohort of patients referred to a clinical service laboratory. PATIENTS: Individuals with suspected SRS referred for molecular genetic testing of H19 DMR methylation status or upd(7)mat. RESULTS: 36 of 139 (25.9%) patients referred for testing had a genetic abnormality identified. Comparison of four published clinical scoring systems demonstrated that all included subjective criteria that could be difficult for the general clinician to assess. We developed a novel, simplified, scoring system utilising four objective, easily measured parameters that performed similarly to the most sensitive and specific published scoring system. DISCUSSION: Effective utilisation of genetic testing by clinicians without specialist clinical genetics training will be facilitated by the development of targeted testing protocols that are based on robust objective clinical features and are designed for use in a busy clinical practice rather than a research setting.

Mutation spectrum in RAB3GAP1, RAB3GAP2, and RAB18 and genotype-phenotype correlations in warburg micro syndrome and Martsolf syndrome.

Warburg Micro syndrome and Martsolf syndrome (MS) are heterogeneous autosomal-recessive developmental disorders characterized by brain, eye, and endocrine abnormalities. Causative biallelic germline mutations have been identified in RAB3GAP1, RAB3GAP2, or RAB18, each of which encode proteins involved in membrane trafficking. This report provides an up to date overview of all known disease variants identified in 29 previously published families and 52 new families. One-hundred and forty-four Micro and nine Martsolf families were investigated, identifying mutations in RAB3GAP1 in 41% of cases, mutations in RAB3GAP2 in 7% of cases, and mutations in RAB18 in 5% of cases. These are listed in Leiden Open source Variation Databases, which was created by us for all three genes. Genotype-phenotype correlations for these genes have now established that the clinical phenotypes in Micro syndrome and MS represent a phenotypic continuum related to the nature and severity of the mutations present in the disease genes, with more deleterious mutations causing Micro syndrome and milder mutations causing MS. RAB18 has not yet been linked to the RAB3 pathways, but mutations in all three genes cause an indistinguishable phenotype, making it likely that there is some overlap. There is considerable genetic heterogeneity for these disorders and further gene identification will help delineate these pathways.

Evaluation of SDHB, SDHD and VHL gene susceptibility testing in the assessment of individuals with non-syndromic phaeochromocytoma, paraganglioma and head and neck paraganglioma.

OBJECTIVES: Research studies have reported that about a third of individuals with phaeochromocytoma/paraganglioma (PPGL) have an inherited predisposition, although the frequency of specific mutations can vary between populations. We evaluated VHL, SDHB and SDHD mutation testing in cohorts of patients with non-syndromic PPGL and head and neck paraganglioma (HNPGL). DESIGN: Prospective, observational evaluation of NHS practice. PATIENTS: Individuals with PPGL/HNPGL referred to a supraregional genetics testing service over a 10-year period. MEASUREMENTS: Clinical (age, tumour site, malignancy, etc.), mutation frequencies and characteristics. RESULTS: A total of 501 probands with PPGL (n = 413) or HNPGL (n = 88) were studied. Thirty-one percent of patients with PPGL presented had a pathogenic mutation in SDHB, SDHD or VHL. Mutation detection rates were highest in those with a positive family history (62%), malignancy (53%), multiple tumours (33%) or PGL (44%). Twenty-eight percent of individuals with a single sporadic phaeochromocytoma had a mutation. Overall, 63% of patients with HNPGL had a mutation (92% of those with a family history, 89% of those with multicentric tumours and 34% of those with a single sporadic HNPGL). Penetrance was calculated in 121 SDHB mutation-positive probands and 187 of their mutation-positive relatives. Most relatives were asymptomatic and lifetime penetrance in non-proband SDHB mutation carriers was <50%. CONCLUSIONS: Practice-based evaluations of genetic testing in PPGL reveal high mutation detection rates. Although clinical criteria can be used to prioritize mutation testing, mutations were detected in 'low risk groups' indicating a need for comprehensive and inexpensive genetic testing strategies for PPGL and HNPGL.

CHRNG genotype-phenotype correlations in the multiple pterygium syndromes.

BACKGROUND: Germline mutations in the CHRNG gene that encodes the γ subunit of the embryonal acetylcholine receptor may cause the non-lethal Escobar variant (EVMPS) or the lethal form (LMPS) of multiple pterygium syndrome (MPS). In addition CHRNG mutations and mutations in other components of the embryonal acetylcholine receptor may present with fetal akinesia deformation sequence (FADS) without pterygia. METHODS: In order to elucidate further the role of CHRNG mutations in MPS/FADS, this study evaluated the results of CHRNG mutation analysis in 100 families with a clinical diagnosis of MPS/FADS. RESULTS: CHRNG mutations were identified in 11/41 (27%) of families with EVMPS and 5/59 (8%) with LMPS/FADS. Most patients with a detectable CHRNG mutation (21 of 24 (87.5%)) had pterygia but no CHRNG mutations were detected in the presence of central nervous system anomalies. DISCUSSION: The mutation spectrum was similar in EVMPS and LMPS/FADS kindreds and EVMPS and LMPS phenotypes were observed in different families with the same CHRNG mutation. Despite this intrafamilial variability, it is estimated that there is a 95% chance that a subsequent sibling will have the same MPS phenotype (EVMPS or LMPS) as the proband (though concordance is less for more distant relatives). Based on these findings, a molecular genetic diagnostic pathway for the investigation of MPS/FADS is proposed.

The Genetics of Inherited Cholestatic Disorders in Neonates and Infants: Evolving Challenges.

Many inherited conditions cause cholestasis in the neonate or infant. Next-generation sequencing methods can facilitate a prompt diagnosis in some of these cases; application of these methods in patients with liver diseases of unknown cause has also uncovered novel gene-disease associations and improved our understanding of physiological bile secretion and flow. By helping to define the molecular basis of certain cholestatic disorders, these methods have also identified new targets for therapy as well patient subgroups more likely to benefit from specific therapies. At the same time, sequencing methods have presented new diagnostic challenges, such as the interpretation of single heterozygous genetic variants. This article discusses those challenges in the context of neonatal and infantile cholestasis, focusing on difficulties in predicting variant pathogenicity, the possibility of other causal variants not identified by the genetic screen used, and phenotypic variability among patients with variants in the same genes. A prospective, observational study performed between 2010-2013, which sequenced six important genes (ATP8B1, ABCB11, ABCB4, NPC1, NPC2 and SLC25A13) in an international cohort of 222 patients with infantile liver disease, is given as an example of potential benefits and challenges that clinicians could face having received a complex genetic result. Further studies including large cohorts of patients with paediatric liver disease are needed to clarify the spectrum of phenotypes associated with, as well as appropriate clinical response to, single heterozygous variants in cholestasis-associated genes.

Design and validation of a metabolic disorder resequencing microarray (BRUM1).

The molecular genetic diagnosis of inherited metabolic disorders is challenging. The diseases are rare, and most show locus heterogeneity. Hence, testing of the genes associated with IMDs is time consuming and often not easily available. We report a resequencing array that allows the simultaneous resequencing of up to 92 genes associated with IMDs. To validate the array, DNA samples from 51 patients with 52 different known variants (including point variants, small insertion, and deletions [indels]) in seven genes (C14ORF133, GAA, NPC1, NPC2, VPS33B, WFS1, and SLC19A2) were amplified by PCR and hybridized to the array. A further patient cohort with 48 different mutations in NPC1 were analyzed blind. Out of 76 point variants, 73 were identified using automated software analysis followed by manual review. Ten insertion and deletion variants were detected in the extra tiling using mutation specific probes, with 11 heterozygous deletions and 3 heterozygous insertions. In summary, we identified 96% (95% confidence interval [CI] 89-99%) of point variants added to the array, but the pickup rate reduced to 83% (95% CI 75-89%) when insertions/deletions were included. Although the methodology has strengths and weaknesses, application of this technique could expedite diagnosis in most patients with multilocus IMDs.

A new locus-specific database (LSDB) for mutations in the folliculin (FLCN) gene.

Birt-Hogg-Dubé syndrome (BHD) is an autosomal dominant condition characterised by the presence of facial fibrofolliculomas, pulmonary cysts which may be associated with spontaneous pneumothorax and renal tumours. Germline mutations in the gene Folliculin (FLCN) were first identified in BHD patients in 2002. In addition FLCN mutations have also been described in families with isolated primary spontaneous pneumothorax (PSP) and also familial clear cell renal carcinomas (FcRCC). We have established a locus-specific database based on the Leiden Open (source) Variation Database (LOVD) software. The version of the database contains 60 previously published mutations and 10 previously unpublished novel germline FLCN mutations. The mutations are comprised of deletions (44.3%), substitutions (35.7%), duplications (14.3%) and deletion/insertions (5.7%). The database is accessible online at http://www.lovd.nl/flcn.

Tumor risks and genotype-phenotype-proteotype analysis in 358 patients with germline mutations in SDHB and SDHD.

Succinate dehydrogenase B (SDHB) and D (SDHD) subunit gene mutations predispose to adrenal and extraadrenal pheochromocytomas, head and neck paragangliomas (HNPGL), and other tumor types. We report tumor risks in 358 patients with SDHB (n=295) and SDHD (n=63) mutations. Risks of HNPGL and pheochromocytoma in SDHB mutation carriers were 29% and 52%, respectively, at age 60 years and 71% and 29%, respectively, in SDHD mutation carriers. Risks of malignant pheochromocytoma and renal tumors (14% at age 70 years) were higher in SDHB mutation carriers; 55 different mutations (including a novel recurrent exon 1 deletion) were identified. No clear genotype-phenotype correlations were detected for SDHB mutations. However, SDHD mutations predicted to result in loss of expression or a truncated or unstable protein were associated with a significantly increased risk of pheochromocytoma compared to missense mutations that were not predicted to impair protein stability (most such cases had the common p.Pro81Leu mutation). Analysis of the largest cohort of SDHB/D mutation carriers has enhanced estimates of penetrance and tumor risk and supports in silicon protein structure prediction analysis for functional assessment of mutations. The differing effect of the SDHD p.Pro81Leu on HNPGL and pheochromocytoma risks suggests differing mechanisms of tumorigenesis in SDH-associated HNPGL and pheochromocytoma.

Multiplex ligation-dependent probe amplification (MLPA) analysis is an effective tool for the detection of novel intragenic PLA2G6 mutations: implications for molecular diagnosis.

Phospholipase associated neurodegeneration (PLAN) comprises a heterogeneous group of autosomal recessive neurological disorders caused by mutations in the PLA2G6 gene. Direct gene sequencing detects approximately 85% mutations in infantile neuroaxonal dystrophy. We report the novel use of multiplex ligation-dependent probe amplification (MLPA) analysis to detect novel PLA2G6 duplications and deletions. The identification of such copy number variants (CNVs) expands the PLAN mutation spectrum and may account for up to 12.5% of PLA2G6 mutations. MLPA should thus be employed to detect CNVs of PLA2G6 in patients who show clinical features of PLAN but in whom both disease-causing mutations cannot be identified on routine sequencing.