Genomic multidisciplinary teams: A model for navigating genetic mainstreaming and precision medicine.

AIM: Recent rapid advances in genomics are revolutionising patient diagnosis and management of genetic conditions. However, this has led to many challenges in service provision, education and upskilling requirements for non-genetics health-care professionals and remuneration for genomic testing. In Australia, Medicare funding with a Paediatric genomic testing item for patients with intellectual disability or syndromic features has attempted to address this latter issue. The Sydney Children's Hospitals Network - Westmead (SCHN-W) Clinical Genetics Department established Paediatric and Neurology genomic multidisciplinary team (MDT) meetings to address the Medicare-specified requirement for discussion with clinical genetics, and increasing genomic testing advice requests. METHODS: This SCHN-W genomic MDT was evaluated with two implementation science frameworks - the RE-AIM (Reach, Effectiveness, Adoption, Implementation, Maintenance) and GMIR - Genomic Medicine Integrative Research frameworks. Data from June 2020 to July 2022 were synthesised and evaluated, as well as process mapping of the MDT service. RESULTS: A total of 205 patients were discussed in 34 MDT meetings, facilitating 148 genomic tests, of which 73 were Medicare eligible. This was equivalent to 26% of SCHN-W genetics outpatient activity, and 13% of all Medicare-funded paediatric genomic testing in NSW. 39% of patients received a genetic diagnosis. CONCLUSION: The genomic MDT facilitated increased genomic testing at a tertiary paediatric centre and is an effective model for mainstreaming and facilitating precision medicine. However, significant implementation issues were identified including cost and sustainability, as well as the high level of resourcing that will be required to scale up this approach to other areas of medicine.

Narrowing the diagnostic gap: Genomes, episignatures, long-read sequencing, and health economic analyses in an exome-negative intellectual disability cohort.

PURPOSE: Genome sequencing (GS)-specific diagnostic rates in prospective tightly ascertained exome sequencing (ES)-negative intellectual disability (ID) cohorts have not been reported extensively. METHODS: ES, GS, epigenetic signatures, and long-read sequencing diagnoses were assessed in 74 trios with at least moderate ID. RESULTS: The ES diagnostic yield was 42 of 74 (57%). GS diagnoses were made in 9 of 32 (28%) ES-unresolved families. Repeated ES with a contemporary pipeline on the GS-diagnosed families identified 8 of 9 single-nucleotide variations/copy-number variations undetected in older ES, confirming a GS-unique diagnostic rate of 1 in 32 (3%). Episignatures contributed diagnostic information in 9% with GS corroboration in 1 of 32 (3%) and diagnostic clues in 2 of 32 (6%). A genetic etiology for ID was detected in 51 of 74 (69%) families. Twelve candidate disease genes were identified. Contemporary ES followed by GS cost US$4976 (95% CI: $3704; $6969) per diagnosis and first-line GS at a cost of $7062 (95% CI: $6210; $8475) per diagnosis. CONCLUSION: Performing GS only in ID trios would be cost equivalent to ES if GS were available at $2435, about a 60% reduction from current prices. This study demonstrates that first-line GS achieves higher diagnostic rate than contemporary ES but at a higher cost.

Genotype-phenotype correlations in paediatric and adolescent phaeochromocytoma and paraganglioma: a cross-sectional study.

Phaeochromocytoma (PC) and paraganglioma (PGL) syndromes associated with germline pathogenic variants are associated with high morbidity and mortality. Establishing genotype-phenotype correlations within a young population is challenging due to their rare occurrence. OBJECTIVE: To describe genotype-phenotype correlations in paediatric and adolescent patients diagnosed with PC/PGL. To establish the incidence of PC/PGL in a young population and prevalence of germline pathogenic variants within this group. STUDY DESIGN: We conducted a cross-sectional study of patients diagnosed with a PC/PGL aged 0-21 years old who were reviewed within Familial Cancer Services within New South Wales and the Australian Capital Territory, Australia. RESULTS: A germline pathogenic variant was detected in 80% (24/30) of patients; SDHB: n=12, VHL: n=11, and MAX: n=1. Only patients harbouring a germline pathogenic variant reported a family history of syndromic tumours, those with apparently sporadic disease did not (62.5% versus 0%, p=0.02). All patients with VHL presented with an adrenal tumour compared with 25% of those with SDHB (100% versus 25%, p=0.01). Occurrence of multiple primary PC/PGL was seen in patients with VHL however was absent in patients with SDHB (36% versus 0%, p=0.03). Incidence rate of paediatric PC/PGL was 0.45 cases per million person years. CONCLUSIONS: PC/PGL diagnosed in children and adolescents were strongly associated with germline pathogenic variants in VHL or SDHB. These patients should be referred to specialist services for family counselling and genetic testing along followed by investigations for the detection of bilateral, multifocal or metastatic disease, and lifelong surveillance for recurrent disease.

Study of How Adiposity in Pregnancy has an Effect on outcomeS (SHAPES): protocol for a prospective cohort study.

INTRODUCTION: Maternal obesity increases the risk of multiple maternal and infant pregnancy complications, such as gestational diabetes and pre-eclampsia. Current UK guidelines use body mass index (BMI) to identify which women require additional care due to increased risk of complications. However, BMI may not accurately predict which women will develop complications during pregnancy as it does not determine amount and distribution of adipose tissue. Some adiposity measures (eg, waist circumference, ultrasound measures of abdominal visceral fat) can better identify where body fat is stored, which may be useful in predicting those women who need additional care. METHODS AND ANALYSIS: This prospective cohort study (SHAPES, Study of How Adiposity in Pregnancy has an Effect on outcomeS) aims to evaluate the prognostic performance of adiposity measures (either alone or in combination with other adiposity, sociodemographic or clinical measures) to estimate risk of adverse pregnancy outcomes. Pregnant women (n=1400) will be recruited at their first trimester ultrasound scan (11+2-14+1 weeks') at Newcastle upon Tyne National Health Service Foundation Trust, UK. Early pregnancy adiposity measures and clinical and sociodemographic data will be collected. Routine data on maternal and infant pregnancy outcomes will be collected from routine hospital records. Regression methods will be used to compare the different adiposity measures with BMI in terms of their ability to predict pregnancy complications. If no individual measure performs better than BMI, multivariable models will be developed and evaluated to identify the most parsimonious model. The apparent performance of the developed model will be summarised using calibration, discrimination and internal validation analyses. ETHICS AND DISSEMINATION: Ethical favourable opinion has been obtained from the North East: Newcastle & North Tyneside 1 Research Ethics Committee (REC reference: 22/NE/0035). All participants provide informed consent to take part in SHAPES. Planned dissemination includes peer-reviewed publications and additional dissemination appropriate to target audiences, including policy briefs for policymakers, media/social-media coverage for public and conferences for research TRIAL REGISTRATION NUMBER: ISRCTN82185177.

DNA methylation episignatures are sensitive and specific biomarkers for detection of patients with KAT6A/KAT6B variants.

Accurate diagnosis for patients living with neurodevelopmental disorders is often met with numerous challenges, related to the ambiguity of findings and lack of specificity in genetic variants leading to pathology. Genome-wide DNA methylation analysis has been used to develop highly sensitive and specific 'episignatures' as biomarkers capable of differentiating and classifying complex neurodevelopmental disorders. In this study we describe distinct episignatures for KAT6A syndrome, caused by pathogenic variants in the lysine acetyltransferase A gene (KAT6A), and for the two neurodevelopmental disorders associated with lysine acetyl transferase B (KAT6B). We demonstrate the ability of our models to differentiate between highly overlapping episignatures, increasing the ability to effectively identify and diagnose these conditions.

Whole Genome Sequencing, Focused Assays and Functional Studies Increasing Understanding in Cryptic Inherited Retinal Dystrophies.

The inherited retinal dystrophies (IRDs) are a clinically and genetically complex group of disorders primarily affecting the rod and cone photoreceptors or other retinal neuronal layers, with emerging therapies heralding the need for accurate molecular diagnosis. Targeted capture and panel-based strategies examining the partial or full exome deliver molecular diagnoses in many IRD families tested. However, approximately one in three families remain unsolved and unable to obtain personalised recurrence risk or access to new clinical trials or therapy. In this study, we investigated whole genome sequencing (WGS), focused assays and functional studies to assist with unsolved IRD cases and facilitate integration of these approaches to a broad molecular diagnostic clinical service. The WGS approach identified variants not covered or underinvestigated by targeted capture panel-based clinical testing strategies in six families. This included structural variants, with notable benefit of the WGS approach in repetitive regions demonstrated by a family with a hybrid gene and hemizygous missense variant involving the opsin genes, OPN1LW and OPN1MW. There was also benefit in investigation of the repetitive GC-rich ORF15 region of RPGR. Further molecular investigations were facilitated by focused assays in these regions. Deep intronic variants were identified in IQCB1 and ABCA4, with functional RNA based studies of the IQCB1 variant revealing activation of a cryptic splice acceptor site. While targeted capture panel-based methods are successful in achieving an efficient molecular diagnosis in a proportion of cases, this study highlights the additional benefit and clinical value that may be derived from WGS, focused assays and functional genomics in the highly heterogeneous IRDs.

Deleterious variants in CRLS1 lead to cardiolipin deficiency and cause an autosomal recessive multi-system mitochondrial disease.

Mitochondrial diseases are a group of inherited diseases with highly varied and complex clinical presentations. Here, we report four individuals, including two siblings, affected by a progressive mitochondrial encephalopathy with biallelic variants in the cardiolipin biosynthesis gene CRLS1. Three affected individuals had a similar infantile presentation comprising progressive encephalopathy, bull's eye maculopathy, auditory neuropathy, diabetes insipidus, autonomic instability, cardiac defects and early death. The fourth affected individual presented with chronic encephalopathy with neurodevelopmental regression, congenital nystagmus with decreased vision, sensorineural hearing loss, failure to thrive and acquired microcephaly. Using patient-derived fibroblasts, we characterized cardiolipin synthase 1 (CRLS1) dysfunction that impaired mitochondrial morphology and biogenesis, providing functional evidence that the CRLS1 variants cause mitochondrial disease. Lipid profiling in fibroblasts from two patients further confirmed the functional defect demonstrating reduced cardiolipin levels, altered acyl-chain composition and significantly increased levels of phosphatidylglycerol, the substrate of CRLS1. Proteomic profiling of patient cells and mouse Crls1 knockout cell lines identified both endoplasmic reticular and mitochondrial stress responses, and key features that distinguish between varying degrees of cardiolipin insufficiency. These findings support that deleterious variants in CRLS1 cause an autosomal recessive mitochondrial disease, presenting as a severe encephalopathy with multi-systemic involvement. Furthermore, we identify key signatures in cardiolipin and proteome profiles across various degrees of cardiolipin loss, facilitating the use of omics technologies to guide future diagnosis of mitochondrial diseases.

A new era of genetic testing in congenital heart disease: A review.

Genetic and genomic testing in pediatric CHD is becoming increasingly routine, and can have important psychosocial, clinical and reproductive implications. In this paper we highlight important challenges and considerations when providing genetics consults and testing in pediatric CHD and illustrate the role of a dedicated CHD genetics clinic. Key lessons include that a) a genetic diagnosis can have clinical utility that justifies testing early in life, b) adequate genetic counselling is crucial to ensure families are supported, understand the range of possible results, and are prepared for new or unexpected health information, and c) further integration of the clinical genetics and cardiology workflows will be required to effectively manage the burgeoning information arising from genetic testing. Our experience demonstrates that a dedicated CHD genetics clinic is a valuable addition to a multidisciplinary team providing care to children with CHD.

A craniosynostosis massively parallel sequencing panel study in 309 Australian and New Zealand patients: findings and recommendations.

PURPOSE: The craniosynostoses are characterized by premature fusion of one or more cranial sutures. The relative contribution of previously reported genes to craniosynostosis in large cohorts is unclear. Here we report on the use of a massively parallel sequencing panel in individuals with craniosynostosis without a prior molecular diagnosis. METHODS: A 20-gene panel was designed based on the genes' association with craniosynostosis, and clinically validated through retrospective testing of an Australian and New Zealand cohort of 233 individuals with craniosynostosis in whom previous testing had not identified a causative variant within FGFR1-3 hot-spot regions or the TWIST1 gene. An additional 76 individuals were tested prospectively. RESULTS: Pathogenic or likely pathogenic variants in non-FGFR genes were identified in 43 individuals, with diagnostic yields of 14% and 15% in retrospective and prospective cohorts, respectively. Variants were identified most frequently in TCF12 (N = 22) and EFNB1 (N = 8), typically in individuals with nonsyndromic coronal craniosynostosis or TWIST1-negative clinically suspected Saethre-Chotzen syndrome. Clinically significant variants were also identified in ALX4, EFNA4, ERF, and FGF10. CONCLUSION: These findings support the clinical utility of a massively parallel sequencing panel for craniosynostosis. TCF12 and EFNB1 should be included in genetic testing for nonsyndromic coronal craniosynostosis or clinically suspected Saethre-Chotzen syndrome.

NAD Deficiency, Congenital Malformations, and Niacin Supplementation.

BACKGROUND: Congenital malformations can be manifested as combinations of phenotypes that co-occur more often than expected by chance. In many such cases, it has proved difficult to identify a genetic cause. We sought the genetic cause of cardiac, vertebral, and renal defects, among others, in unrelated patients. METHODS: We used genomic sequencing to identify potentially pathogenic gene variants in families in which a person had multiple congenital malformations. We tested the function of the variant by using assays of in vitro enzyme activity and by quantifying metabolites in patient plasma. We engineered mouse models with similar variants using the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system. RESULTS: Variants were identified in two genes that encode enzymes of the kynurenine pathway, 3-hydroxyanthranilic acid 3,4-dioxygenase (HAAO) and kynureninase (KYNU). Three patients carried homozygous variants predicting loss-of-function changes in the HAAO or KYNU proteins (HAAO p.D162*, HAAO p.W186*, or KYNU p.V57Efs*21). Another patient carried heterozygous KYNU variants (p.Y156* and p.F349Kfs*4). The mutant enzymes had greatly reduced activity in vitro. Nicotinamide adenine dinucleotide (NAD) is synthesized de novo from tryptophan through the kynurenine pathway. The patients had reduced levels of circulating NAD. Defects similar to those in the patients developed in the embryos of Haao-null or Kynu-null mice owing to NAD deficiency. In null mice, the prevention of NAD deficiency during gestation averted defects. CONCLUSIONS: Disruption of NAD synthesis caused a deficiency of NAD and congenital malformations in humans and mice. Niacin supplementation during gestation prevented the malformations in mice. (Funded by the National Health and Medical Research Council of Australia and others.).

Disorders of sex development: insights from targeted gene sequencing of a large international patient cohort.

BACKGROUND: Disorders of sex development (DSD) are congenital conditions in which chromosomal, gonadal, or phenotypic sex is atypical. Clinical management of DSD is often difficult and currently only 13% of patients receive an accurate clinical genetic diagnosis. To address this we have developed a massively parallel sequencing targeted DSD gene panel which allows us to sequence all 64 known diagnostic DSD genes and candidate genes simultaneously. RESULTS: We analyzed DNA from the largest reported international cohort of patients with DSD (278 patients with 46,XY DSD and 48 with 46,XX DSD). Our targeted gene panel compares favorably with other sequencing platforms. We found a total of 28 diagnostic genes that are implicated in DSD, highlighting the genetic spectrum of this disorder. Sequencing revealed 93 previously unreported DSD gene variants. Overall, we identified a likely genetic diagnosis in 43% of patients with 46,XY DSD. In patients with 46,XY disorders of androgen synthesis and action the genetic diagnosis rate reached 60%. Surprisingly, little difference in diagnostic rate was observed between singletons and trios. In many cases our findings are informative as to the likely cause of the DSD, which will facilitate clinical management. CONCLUSIONS: Our massively parallel sequencing targeted DSD gene panel represents an economical means of improving the genetic diagnostic capability for patients affected by DSD. Implementation of this panel in a large cohort of patients has expanded our understanding of the underlying genetic etiology of DSD. The inclusion of research candidate genes also provides an invaluable resource for future identification of novel genes.

RASA1 mutations and associated phenotypes in 68 families with capillary malformation-arteriovenous malformation.

Capillary malformation-arteriovenous malformation (CM-AVM) is an autosomal-dominant disorder, caused by heterozygous RASA1 mutations, and manifesting multifocal CMs and high risk for fast-flow lesions. A limited number of patients have been reported, raising the question of the phenotypic borders. We identified new patients with a clinical diagnosis of CM-AVM, and patients with overlapping phenotypes. RASA1 was screened in 261 index patients with: CM-AVM (n = 100), common CM(s) (port-wine stain; n = 100), Sturge-Weber syndrome (n = 37), or isolated AVM(s) (n = 24). Fifty-eight distinct RASA1 mutations (43 novel) were identified in 68 index patients with CM-AVM and none in patients with other phenotypes. A novel clinical feature was identified: cutaneous zones of numerous small white pale halos with a central red spot. An additional question addressed in this study was the "second-hit" hypothesis as a pathophysiological mechanism for CM-AVM. One tissue from a patient with a germline RASA1 mutation was available. The analysis of the tissue showed loss of the wild-type RASA1 allele. In conclusion, mutations in RASA1 underscore the specific CM-AVM phenotype and the clinical diagnosis is based on identifying the characteristic CMs. The high incidence of fast-flow lesions warrants careful clinical and radiologic examination, and regular follow-up.

Whole-exome-sequencing identifies mutations in histone acetyltransferase gene KAT6B in individuals with the Say-Barber-Biesecker variant of Ohdo syndrome.

Say-Barber-Biesecker-Young-Simpson syndrome (SBBYSS or Ohdo syndrome) is a multiple anomaly syndrome characterized by severe intellectual disability, blepharophimosis, and a mask-like facial appearance. A number of individuals with SBBYSS also have thyroid abnormalities and cleft palate. The condition usually occurs sporadically and is therefore presumed to be due in most cases to new dominant mutations. In individuals with SBBYSS, a whole-exome sequencing approach was used to demonstrate de novo protein-truncating mutations in the highly conserved histone acetyltransferase gene KAT6B (MYST4/MORF)) in three out of four individuals sequenced. Sanger sequencing was used to confirm truncating mutations of KAT6B, clustering in the final exon of the gene in all four individuals and in a further nine persons with typical SBBYSS. Where parental samples were available, the mutations were shown to have occurred de novo. During mammalian development KAT6B is upregulated specifically in the developing central nervous system, facial structures, and limb buds. The phenotypic features seen in the Qkf mouse, a hypomorphic Kat6b mutant, include small eyes, ventrally placed ears and long first digits that mirror the human phenotype. This is a further example of how perturbation of a protein involved in chromatin modification might give rise to a multisystem developmental disorder.

Role of human herpes virus 6 in corneal inflammation alone or with human herpesviruses.

PURPOSE: : The purpose of this study was to determine the association of human herpes virus 6 (HHV-6) and/or other human herpesviruses in corneal inflammation using polymerase chain reaction (PCR). METHODS: : We collected tear films, conjunctival smears, and a corneal button of inflamed cornea, and the presence of HHV-6 and other herpesviruses in these samples were assessed by a nested PCR. RESULTS: : In tear films collected from 3 of 9 patients with dendritic keratitis, HHV-6 DNA was positive twice, together with herpes simplex virus (HSV) or varicella zoster virus DNA most often, during the acute phase of the disease. Two other patients in this group were either positive for HSV-1 and varicella zoster virus or for HSV-1 and Epstein-Barr virus DNA but negative for HHV-6. When another 12 patients' smear samples from corneal ulcer or keratouveitis were examined, 9 were positive for HHV-6 DNA. Of these, 4 were positive for HSV-1 simultaneously, whereas the remaining 5 patients were negative for HSV-1. One patient's smear was positive for HSV-1 but not for HHV-6. In the corneal button, both HSV and HHV-6 DNAs were positive by nested PCR. HHV-6 was also positive by nested PCR in the conjunctival swab obtained from the contralateral inflamed eye of the patient. CONCLUSIONS: : In 22 patients with corneal inflammation, HHV-6 was positive in 14 of 22 patients and HSV-1 was found in 9 of those patients. These data indicated that the association of HHV-6 with disease was more frequent than with other herpesviruses and that HHV-6 may be another sole causative agent for corneal inflammation.

De novo mutations of SETBP1 cause Schinzel-Giedion syndrome.

Schinzel-Giedion syndrome is characterized by severe mental retardation, distinctive facial features and multiple congenital malformations; most affected individuals die before the age of ten. We sequenced the exomes of four affected individuals (cases) and found heterozygous de novo variants in SETBP1 in all four. We also identified SETBP1 mutations in eight additional cases using Sanger sequencing. All mutations clustered to a highly conserved 11-bp exonic region, suggesting a dominant-negative or gain-of-function effect.

Epidemiology and course of disease in childhood uveitis.

PURPOSE: To describe the disease characteristics and visual outcome of pediatric uveitis. DESIGN: Retrospective, longitudinal observation. PARTICIPANTS: Five hundred twenty-seven pediatric uveitis patients from the National Eye Institute, University of Illinois, Chicago, and Oregon Health Sciences University. METHODS: Retrospective chart review. MAIN OUTCOME MEASURES: Demographics, uveitis disease characteristics, complications, treatments, and visual outcomes were determined at baseline and at 1-, 3-, 5-, and 10-year time points. RESULTS: The patient population was 54% female; 62.4% white, 12.5% black, 2.7% Asian, 2.1% multiracial, and 14.61% Hispanic. Median age at diagnosis was 9.4 years. The leading diagnoses were idiopathic uveitis (28.8%), juvenile idiopathic arthritis-associated uveitis (20.9%), and pars planitis (17.1%). Insidious onset (58%) and persistent duration (75.3%) were most common. Anterior uveitis was predominant (44.6%). Complications were frequent, and cystoid macular edema (odds ratio [OR] 2.94; P = 0.006) and hypotony (OR, 4.54; P = 0.026) had the most significant visual impact. Ocular surgery was performed in 18.9% of patients. The prevalence of legal blindness was 9.23% at baseline, 6.52% at 1 year, 3.17% at 3 years, 15.15% at 5 years, and 7.69% at 10 years. Posterior uveitis and panuveitis had more severe vision loss. Hispanic ethnicity was associated with a higher prevalence of infectious uveitis and vision loss at baseline. CONCLUSIONS: The rate and spectrum of vision threatening complications of pediatric uveitis are significant. Prospective studies using standard outcome measures and including diverse populations are needed to identify children most at risk.

Corneal verticillata after dual anti-epidermal growth factor receptor and anti-vascular endothelial growth factor receptor 2 therapy (vandetanib) for anaplastic astrocytoma.

PURPOSE: To describe a patient with a history of epithelial basement membrane dystrophy who developed symptomatic corneal verticillata after vandetanib therapy for anaplastic astrocytoma. METHODS: Retrospective interventional case report. RESULTS: A 48-year-old female patient with a history of anaplastic astrocytoma status post resection, external beam radiation, and chemotherapy presented with glare symptoms, decreased contrast sensitivity, and increased lacrimation after approximately 12 months of therapy with the anti-epidermal growth factor receptor (EGFR) and anti-vascular endothelial growth factor receptor 2 protein tyrosine kinase inhibitor, vandetanib. Ophthalmic examination revealed diffuse corneal verticillata and fine subepithelial opacities. Schirmer 1 testing was normal bilaterally. Therapy with carboxymethylcellulose, 5% sodium chloride ointment, and a decrease in the dose of vandetanib led to an improvement in the patient's ophthalmic symptoms despite persistence of the corneal findings. The patient remained under surveillance for tumor recurrence. CONCLUSIONS: Vandetanib (ZD6474), a protein tyrosine kinase inhibitor with dual anti-EGFR and anti-vascular endothelial growth factor receptor 2 action, may have contributed to the formation of corneal verticillata in our patient. Inhibition of EGFR, which is involved with corneal epithelial cell migration and wound healing, may play a role in the pathogenesis underlying corneal vortex keratopathy and ocular surface conditions with significant epithelial turnover.

Denaturing high performance liquid chromatography detection of SDHB, SDHD, and VHL germline mutations in pheochromocytoma.

BACKGROUND: Pheochromocytomas are neuroendocrine tumors of chromaffin cell origin which arise from the adrenal medulla and less commonly the extra-adrenal sympathetic paraganglia. Pheochromocytomas are component tumors of the familial syndromes multiple endocrine neoplasia Type 2, von Hippel Lindau disease, Neurofibromatosis Type 1, and the pheochromocytoma/paraganglioma syndromes caused by mutations in the RET, VHL, NF1, SDHB, and SDHD genes, respectively. The aim of this study was to evaluate denaturing high performance liquid chromatography (dHPLC) as a screening tool for the detection of germline mutations within VHL, SDHB, and SDHD in pheochromocytoma patients. METHODS: Polymerase chain reaction of all exons of VHL, SDHB, and SDHD genes was performed on leukocyte DNA extracted from stored blood samples of 74 unrelated patients treated for pheochromocytoma. After dHPLC analysis, all samples demonstrating variance were selected for sequencing. RESULTS: Of the 74 patients, 12 mutations and 16 polymorphisms were identified by dHPLC and confirmed on sequencing. More specifically, a total of 5 mutations and 15 polymorphisms were detected in SDHB and 7 mutations and 1 polymorphism were identified in VHL. No SDHD mutations or polymorphisms were identified. By sequencing only dHPLC variants, the total amount of DNA sequencing required was reduced by approximately 88%. CONCLUSIONS: dHPLC is an effective screening tool for the detection of germline mutations in SDHB, SDHD, and VHL and has application for diagnostic germline mutation analysis in pheochromocytoma patients.

Brain involvement in muscular dystrophies with defective dystroglycan glycosylation.

OBJECTIVE: To assess the range and severity of brain involvement, as assessed by magnetic resonance imaging, in 27 patients with mutations in POMT1 (4), POMT2 (9), POMGnT1 (7), Fukutin (4), or LARGE (3), responsible for muscular dystrophies with abnormal glycosylation of dystroglycan (dystroglycanopathies). METHODS: Blinded review of magnetic resonance imaging brain scans from 27 patients with mutations in 1 of these 5 genes. RESULTS: Brain magnetic resonance images were normal in 3 of 27 patients; in another 5, only nonspecific abnormalities (ventricular dilatation, periventricular white matter abnormalities, or both) were seen. The remaining 19 patients had a spectrum of structural defects, ranging from complete lissencephaly in patients with Walker-Warburg syndrome to isolated cerebellar involvement. Cerebellar cysts and/or dysplasia and hypoplasia were the predominant features in four patients. Polymicrogyria (11/27) was more severe in the frontoparietal regions in 6, and had an occipitofrontal gradient in 2. Pontine clefts, with an unusual appearance to the corticospinal tracts, were seen in five patients with a muscle-eye-brain-like phenotype, three patients with POMGnT1, one with LARGE, and one with POMT2 mutations. Prominent cerebellar cysts were always seen with POMGnT1 mutations, but rarely seen in POMT1 and POMT2. Brainstem and pontine abnormalities were common in patients with POMT2, POMGnT1, and LARGE mutations. INTERPRETATION: Our results expand the spectrum of brain involvement associated with mutations in LARGE, POMGnT1, POMT1, and POMT2. Pontine clefts were visible in some dystroglycanopathy patients. Infratentorial structures were often affected in isolation, highlighting their susceptibility to involvement in these conditions.