Investigating the role of somatic sequencing platforms for phaeochromocytoma and paraganglioma in a large UK cohort.

OBJECTIVES: Phaeochromocytomas and paragangliomas (PPGL) are rare neuroendocrine tumours with malignant potential and a hereditary basis in almost 40% of patients. Germline genetic testing has transformed the management of PPGL enabling stratification of surveillance approaches, earlier diagnosis and predictive testing of at-risk family members. Recent studies have identified somatic mutations in a further subset of patients, indicating that molecular drivers at either a germline or tumour level can be identified in up to 80% of PPGL cases. The aim of this study was to investigate the clinical utility of somatic sequencing in a large cohort of patients with PPGL in the United Kingdom. DESIGN AND PATIENTS: Prospectively collected matched germline and tumour samples (development cohort) and retrospectively collected tumour samples (validation cohort) of patients with PPGL were investigated. MEASUREMENTS: Clinical characteristics of patients were assessed and tumour and germline DNA was analysed using a next-generation sequencing strategy. A screen for variants within 'mutation hotspots' in 68 human cancer genes was performed. RESULTS: Of 141 included patients, 45 (32%) had a germline mutation. In 37 (26%) patients one or more driver somatic variants were identified including 26 likely pathogenic or pathogenic variants and 19 variants of uncertain significance. Pathogenic somatic variants, observed in 25 (18%) patients, were most commonly identified in the VHL, NF1, HRAS and RET genes. Pathogenic somatic variants were almost exclusively identified in patients without a germline mutation (all but one), suggesting that somatic sequencing is likely to be most informative for those patients with negative germline genetic test results. CONCLUSIONS: Somatic sequencing may further stratify surveillance approaches for patients without a germline genetic driver and may also inform targeted therapeutic strategies for patients with metastatic disease.

Breast cancer in multiple endocrine neoplasia type 1 (MEN1).

SUMMARY: A 38-year-old female was identified as carrying a heterozygous pathogenic MEN1 variant (c.1304delG) through predictive genetic testing, following a diagnosis of familial hyperparathyroidism. Routine screening for parathyroid and pituitary disease was negative. However, cross-sectional imaging by CT revealed a 41 mm pancreatic tail mass. Biopsy via endoscopic ultrasound confirmed the lesion to be a well-differentiated (grade 1) pancreatic neuroendocrine tumour (pNET) with MIB1<1%. Biochemically, hyperinsulinaemic hypoglycaemia was confirmed following an overnight fast, which was subsequently managed by diet alone prior to definitive surgery. Pre-operative work-up with octreotide SPECT CT demonstrated avid tracer uptake in the pancreatic lesion and, unexpectedly, a focal area of uptake in the left breast. Further investigation, and subsequent mastectomy, confirmed ductal carcinoma in situ pT2 (23 mm) grade 1, N0 (ER positive; HER2 negative). Following mastectomy, our patient underwent a successful distal pancreatectomy to resect the pNET. Loss of heterozygosity (LOH) at the MEN1 locus was found in both the breast tumour and pNET, thereby in keeping with a 'two-hit' hypothesis of oncogenesis, a suggestive but non-definitive clue for causation. To obtain further support for a causative relationship between MEN1 and breast cancer, we undertook a detailed review of the published literature which overall supports the notion that breast cancer is a MEN1-related malignancy that presents at a younger age and histologically, is typically of ductal subtype. Currently, clinical guidance regarding breast cancer surveillance in MEN1 does not exist and further research is required to establish a clinical and cost-effective surveillance strategy). LEARNING POINTS: We describe a case of pNET and breast cancer diagnosed at a young age of 38 years in a patient who is heterozygous for a pathogenic MEN1 variant. Loss of the wild-type allele was seen in both breast tissue and pNET specimen. Breast cancer may be an under-recognised MEN1-associated malignancy that presents at a younger age than in the general population with a relative risk of 2-3. Further research is required to determine the cost-effectiveness of breast cancer surveillance approach at a younger age in MEN1 patients relative to the general population .

Evidence that autosomal recessive spastic cerebral palsy-1 (CPSQ1) is caused by a missense variant in HPDL.

A subset of individuals diagnosed with cerebral palsy will have an underlying genetic diagnosis. Previously, a missense variant in GAD1 was described as a candidate mutation in a single family diagnosed with autosomal recessive spastic cerebral palsy-1 (CPSQ1; OMIM 603513). Following the ascertainment of a further branch of the CPSQ1 kindred, we found that the previously reported GAD1 variant did not segregate with the neurological disease phenotype in the recently ascertained branch of the kindred. Following genetic linkage studies to map autozygous regions and whole-exome sequencing, a missense variant (c.527 T > C; p. Leu176Pro, rs773333490) in the HPDL gene was detected and found to segregate with disease status in both branches of the kindred. HPDL encodes a 371-amino acid protein (4-Hydroxyphenylpyruvate Dioxygenase Like) that localizes to mitochondria but whose function is uncertain. Recently, biallelic loss of function variants and missense substitution-causing variants in HPDL were reported to cause a childhood onset progressive spastic movement disorder with a variable presentation. These findings suggest that HPDL-related neurological disease may mimic spastic cerebral palsy and that GAD1 should not be included in diagnostic gene panels for inherited cerebral palsy.

Investigating the clinical, pathological and molecular profile of oncocytic adrenocortical neoplasms: a case series and literature review.

Background: Malignant oncocytic adrenocortical neoplasms (OANs) are rare tumours with a distinctive biological behaviour compared to conventional adrenocortical carcinoma (ACC). The current prognostic systems overestimate the malignant potential of these tumours, and guidance for surveillance and treatment strategies are lacking. Aim: To evaluate the utility of clinical, pathological and molecular markers in predicting the biological behaviour and outcomes of malignant OANs. Methods: A retrospective clinicopathological review of 10 histologically confirmed OANs was carried out. Whole exome sequencing (WES) of germline and paired tumour samples was performed for four of the ten OAN cases and compared to WES data from five cases of conventional ACC and data from The Cancer Genome Atlas. We reviewed all the cases of malignant OAN reported in the literature and compared to our case series. Results: Eight (80%) tumours were classified as malignant, one borderline and one benign (Lin-Weiss-Bisceglia criteria, LWB). The malignant OAN were larger tumours and had higher MIB index and Helsinki scores. Molecular profiling identified a pathogenic germline variant in MSH6 in an individual in the OAN group. The OAN samples had a lower mutation burden compared to the ACC samples. Somatic driver variants were identified in OAN and ACC samples including a pathogenic missense variant in CTNNB1. Conclusion: In this study, the LWB classification demonstrated sensitivity for the differentiation of benign from malignant OAN. Molecular profiling identified dysregulation in DNA repair and Wnt signalling pathways in both OAN and ACC samples, suggesting a molecular overlap between OAN and conventional ACC.

SDHC epi-mutation testing in gastrointestinal stromal tumours and related tumours in clinical practice.

The enzyme succinate dehydrogenase (SDH) functions in the citric acid cycle and loss of function predisposes to the development of phaeochromocytoma/paraganglioma (PPGL), wild type gastrointestinal stromal tumour (wtGIST) and renal cell carcinoma. SDH-deficient tumours are most commonly associated with a germline SDH subunit gene (SDHA/B/C/D) mutation but can also be associated with epigenetic silencing of the SDHC gene. However, clinical diagnostic testing for an SDHC epimutation is not widely available. The objective of this study was to investigate the indications for and the optimum diagnostic pathways for the detection of SDHC epimutations in clinical practice. SDHC promoter methylation analysis of 32 paraffin embedded tumours (including 15 GIST and 17 PPGL) was performed using a pyrosequencing technique and correlated with SDHC gene expression. SDHC promoter methylation was identified in 6 (18.7%) tumours. All 6 SDHC epimutation cases presented with SDH deficient wtGIST and 3/6 cases had multiple primary tumours. No case of constitutional SDHC promoter hypermethylation was detected. Whole genome sequencing of germline DNA from three wtGIST cases with an SDHC epimutation, did not reveal any causative sequence anomalies. Herein, we recommend a diagnostic workflow for the detection of an SDHC epimutation in a service setting.

MethylCal: Bayesian calibration of methylation levels.

Bisulfite amplicon sequencing has become the primary choice for single-base methylation quantification of multiple targets in parallel. The main limitation of this technology is a preferential amplification of an allele and strand in the PCR due to methylation state. This effect, known as 'PCR bias', causes inaccurate estimation of the methylation levels and calibration methods based on standard controls have been proposed to correct for it. Here, we present a Bayesian calibration tool, MethylCal, which can analyse jointly all CpGs within a CpG island (CGI) or a Differentially Methylated Region (DMR), avoiding 'one-at-a-time' CpG calibration. This enables more precise modeling of the methylation levels observed in the standard controls. It also provides accurate predictions of the methylation levels not considered in the controlled experiment, a feature that is paramount in the derivation of the corrected methylation degree. We tested the proposed method on eight independent assays (two CpG islands and six imprinting DMRs) and demonstrated its benefits, including the ability to detect outliers. We also evaluated MethylCal's calibration in two practical cases, a clinical diagnostic test on 18 patients potentially affected by Beckwith-Wiedemann syndrome, and 17 individuals with celiac disease. The calibration of the methylation levels obtained by MethylCal allows a clearer identification of patients undergoing loss or gain of methylation in borderline cases and could influence further clinical or treatment decisions.

Genome-wide association study of extreme high bone mass: Contribution of common genetic variation to extreme BMD phenotypes and potential novel BMD-associated genes.

BACKGROUND: Generalised high bone mass (HBM), associated with features of a mild skeletal dysplasia, has a prevalence of 0.18% in a UK DXA-scanned adult population. We hypothesized that the genetic component of extreme HBM includes contributions from common variants of small effect and rarer variants of large effect, both enriched in an extreme phenotype cohort. METHODS: We performed a genome-wide association study (GWAS) of adults with either extreme high or low BMD. Adults included individuals with unexplained extreme HBM (n = 240) from the UK with BMD Z-scores ≥+3.2, high BMD females from the Anglo-Australasian Osteoporosis Genetics Consortium (AOGC) (n = 1055) with Z-scores +1.5 to +4.0 and low BMD females also part of AOGC (n = 900), with Z-scores -1.5 to -4.0. Following imputation, we tested association between 6,379,332 SNPs and total hip and lumbar spine BMD Z-scores. For potential target genes, we assessed expression in human osteoblasts and murine osteocytes. RESULTS: We observed significant enrichment for associations with established BMD-associated loci, particularly those known to regulate endochondral ossification and Wnt signalling, suggesting that part of the genetic contribution to unexplained HBM is polygenic. Further, we identified associations exceeding genome-wide significance between BMD and four loci: two established BMD-associated loci (5q14.3 containing MEF2C and 1p36.12 containing WNT4) and two novel loci: 5p13.3 containing NPR3 (rs9292469; minor allele frequency [MAF] = 0.33%) associated with lumbar spine BMD and 11p15.2 containing SPON1 (rs2697825; MAF = 0.17%) associated with total hip BMD. Mouse models with mutations in either Npr3 or Spon1 have been reported, both have altered skeletal phenotypes, providing in vivo validation that these genes are physiologically important in bone. NRP3 regulates endochondral ossification and skeletal growth, whilst SPON1 modulates TGF-ß regulated BMP-driven osteoblast differentiation. Rs9292469 (downstream of NPR3) also showed some evidence for association with forearm BMD in the independent GEFOS sample (n = 32,965). We found Spon1 was highly expressed in murine osteocytes from the tibiae, femora, humeri and calvaria, whereas Npr3 expression was more variable. CONCLUSION: We report the most extreme-truncate GWAS of BMD performed to date. Our findings, suggest potentially new anabolic bone regulatory pathways that warrant further study.

Germline pathogenic variants in PALB2 and other cancer-predisposing genes in families with hereditary diffuse gastric cancer without CDH1 mutation: a whole-exome sequencing study.

BACKGROUND: Germline pathogenic variants in the E-cadherin gene (CDH1) are strongly associated with the development of hereditary diffuse gastric cancer. There is a paucity of data to guide risk assessment and management of families with hereditary diffuse gastric cancer that do not carry a CDH1 pathogenic variant, making it difficult to make informed decisions about surveillance and risk-reducing surgery. We aimed to identify new candidate genes associated with predisposition to hereditary diffuse gastric cancer in affected families without pathogenic CDH1 variants. METHODS: We did whole-exome sequencing on DNA extracted from the blood of 39 individuals (28 individuals diagnosed with hereditary diffuse gastric cancer and 11 unaffected first-degree relatives) in 22 families without pathogenic CDH1 variants. Genes with loss-of-function variants were prioritised using gene-interaction analysis to identify clusters of genes that could be involved in predisposition to hereditary diffuse gastric cancer. FINDINGS: Protein-affecting germline variants were identified in probands from six families with hereditary diffuse gastric cancer; variants were found in genes known to predispose to cancer and in lesser-studied DNA repair genes. A frameshift deletion in PALB2 was found in one member of a family with a history of gastric and breast cancer. Two different MSH2 variants were identified in two unrelated affected individuals, including one frameshift insertion and one previously described start-codon loss. One family had a unique combination of variants in the DNA repair genes ATR and NBN. Two variants in the DNA repair gene RECQL5 were identified in two unrelated families: one missense variant and a splice-acceptor variant. INTERPRETATION: The results of this study suggest a role for the known cancer predisposition gene PALB2 in families with hereditary diffuse gastric cancer and no detected pathogenic CDH1 variants. We also identified new candidate genes associated with disease risk in these families. FUNDING: UK Medical Research Council (Sackler programme), European Research Council under the European Union's Seventh Framework Programme (2007-13), National Institute for Health Research Cambridge Biomedical Research Centre, Experimental Cancer Medicine Centres, and Cancer Research UK.

Translating in vivo metabolomic analysis of succinate dehydrogenase deficient tumours into clinical utility.

PURPOSE: Mutations in the mitochondrial enzyme succinate dehydrogenase (SDH) subunit genes are associated with a wide spectrum of tumours including phaeochromocytoma and paraganglioma (PPGL) 1, 2, gastrointestinal stromal tumours (GIST) 3, renal cell carcinoma (RCC) 4 and pituitary adenomas5. SDH-related tumorigenesis is believed to be secondary to accumulation of the oncometabolite succinate. Our aim was to investigate the potential clinical applications of MRI spectroscopy (1H-MRS) in a range of suspected SDH-related tumours. PATIENTS AND METHODS: Fifteen patients were recruited to this study. Respiratory-gated single-voxel 1H-MRS was performed at 3T to quantify the content of succinate at 2.4 ppm and choline at 3.22 ppm. RESULTS: A succinate peak was seen in six patients, all of whom had a germline SDHx mutation or loss of SDHB by immunohistochemistry. A succinate peak was also detected in two patients with a metastatic wild-type GIST (wtGIST) and no detectable germline SDHx mutation but a somatic epimutation in SDHC. Three patients without a tumour succinate peak retained SDHB expression, consistent with SDH functionality. In six cases with a borderline or absent peak, technical difficulties such as motion artefact rendered 1H-MRS difficult to interpret. Sequential imaging in a patient with a metastatic abdominal paraganglioma demonstrated loss of the succinate peak after four cycles of [177Lu]-DOTATATE, with a corresponding biochemical response in normetanephrine. CONCLUSIONS: This study has demonstrated the translation into clinical practice of in vivo metabolomic analysis using 1H-MRS in patients with SDH-deficient tumours. Potential applications include non-invasive diagnosis and disease stratification, as well as monitoring of tumour response to targeted treatments.

Clinical and Molecular Features of Renal and Pheochromocytoma/Paraganglioma Tumor Association Syndrome (RAPTAS): Case Series and Literature Review.

Context: The co-occurrence of pheochromocytoma (PC) and renal tumors was linked to the inherited familial cancer syndrome von Hippel-Lindau (VHL) disease more than six decades ago. Subsequently, other shared genetic causes of predisposition to renal tumors and to PC, paraganglioma (PGL), or head and neck paraganglioma (HNPGL) have been described, but case series of non-VHL-related cases of renal tumor and pheochromocytoma/paraganglioma tumor association syndrome (RAPTAS) are rare. Objective: To determine the clinical and molecular features of non-VHL RAPTAS by literature review and characterization of a case series. Design: A review of the literature was performed and a retrospective study of referrals for investigation of genetic causes of RAPTAS. Results: Literature review revealed evidence of an association, in addition to VHL disease, between germline mutations in SDHB, SDHC, SDHD, TMEM127, and MAX genes and RAPTAS [defined here as the co-occurrence of tumors from both classes (PC/PGL/HNPGL and renal tumors) in the same individual or in first-degree relatives]. In both the literature review and our case series of 22 probands with non-VHL RAPTAS, SDHB mutations were the most frequent cause of non-VHL RAPTAS. A genetic cause was identified in 36.3% (8/22) of kindreds. Conclusion: Renal tumors and PC/PGL/HNPGL tumors share common molecular features and their co-occurrence in an individual or family should prompt genetic investigations. We report a case of MAX-associated renal cell carcinoma and confirm the role of TMEM127 mutations with renal cell carcinoma predisposition.

SDHA related tumorigenesis: a new case series and literature review for variant interpretation and pathogenicity.

PURPOSE: To evaluate the role of germline SDHA mutation analysis by (1) comprehensive literature review, (2) description of novel germline SDHA mutations and (3) in silico structural prediction analysis of missense substitutions in SDHA. PATIENTS AND METHODS: A systematic literature review and a retrospective review of the molecular and clinical features of patients identified with putative germline variants in UK molecular genetic laboratories was performed. To evaluate the molecular consequences of SDHA missense variants, a novel model of the SDHA/B/C/D complex was generated and the structural effects of missense substitutions identified in the literature, our UK novel cohort and a further 32 "control missense variants" were predicted by the mCSM computational platform. These structural predictions were correlated with the results of tumor studies and other bioinformatic predictions. RESULTS: Literature review revealed reports of 17 different germline SDHA variants in 47 affected individuals from 45 kindreds. A further 10 different variants in 15 previously unreported cases (seven novel variants in eight patients) were added from our UK series. In silico structural prediction studies of 11 candidate missense germline mutations suggested that most (63.7%) would destabilize the SDHA protomer, and that most (78.1%) rare SDHA missense variants present in a control data set (ESP6500) were also associated with impaired protein stability. CONCLUSION: The clinical spectrum of SDHA-associated neoplasia differs from that of germline mutations in other SDH-subunits. The interpretation of the significance of novel SDHA missense substitutions is challenging. We recommend that multiple investigations (e.g. tumor studies, metabolomic profiling) should be performed to aid classification of rare missense variants before genetic testing results are used to influence clinical management.

Nephroblastomatosis or Wilms tumor in a fourth patient with a somatic PIK3CA mutation.

Wilms tumor and nephroblastomatosis are associated with syndromic conditions including hemihyperplasia. Hemihyperplasia is genetically heterogeneous and may be the result of genomic abnormalities seen in Beckwith-Wiedemann syndrome, mosaic chromosome or genomic abnormalities, or somatic point mutations. Somatic missense mutations affecting the PI3K-AKT-MTOR pathway result in segmental overgrowth and are present in numerous benign and malignant tumors. Here, we report a fourth patient with asymmetric overgrowth due to a somatic PIK3CA mutation who had nephroblastomatosis or Wilms tumor. Similar to two of three reported patients with a somatic PIK3CA mutation and renal tumors, he shared a PIK3CA mutation affecting codon 1047, presented at birth with asymmetric overgrowth, and had fibroadipose overgrowth. Codon 1047 is most commonly affected by somatic mutations in PIK3CA-related overgrowth spectrum (PROS). While the fibroadipose overgrowth phenotype appears to be common in individuals with PIK3CA mutations at codon 1047, individuals with a clinical diagnosis of Klippel-Trenaunay syndrome or isolated lymphatic malformation also had mutations affecting this amino acid. Screening for Wilms tumor in individuals with PROS-related hemihyperplasia may be considered and, until the natural history is fully elucidated in larger cohort studies, may follow guidelines for Beckwith-Wiedemann syndrome, or isolated hemihyperplasia. It is not known if the specific PIK3CA mutation, the mosaic distribution, or the clinical presentation affect the Wilms tumor or nephroblastomatosis risk in individuals with PROS. © 2016 Wiley Periodicals, Inc.

Somatic activating mutations in Pik3ca cause sporadic venous malformations in mice and humans.

Venous malformations (VMs) are painful and deforming vascular lesions composed of dilated vascular channels, which are present from birth. Mutations in the TEK gene, encoding the tyrosine kinase receptor TIE2, are found in about half of sporadic (nonfamilial) VMs, and the causes of the remaining cases are unknown. Sclerotherapy, widely accepted as first-line treatment, is not fully efficient, and targeted therapy for this disease remains underexplored. We have generated a mouse model that faithfully mirrors human VM through mosaic expression of Pik3ca(H1047R), a constitutively active mutant of the p110α isoform of phosphatidylinositol 3-kinase (PI3K), in the embryonic mesoderm. Endothelial expression of Pik3ca(H1047R)resulted in endothelial cell (EC) hyperproliferation, reduction in pericyte coverage of blood vessels, and decreased expression of arteriovenous specification markers. PI3K pathway inhibition with rapamycin normalized EC hyperproliferation and pericyte coverage in postnatal retinas and stimulated VM regression in vivo. In line with the mouse data, we also report the presence of activating PIK3CA mutations in human VMs, mutually exclusive with TEK mutations. Our data demonstrate a causal relationship between activating Pik3ca mutations and the genesis of VMs, provide a genetic model that faithfully mirrors the normal etiology and development of this human disease, and establish the basis for the use of PI3K-targeted therapies in VMs.

Mutations in Known Monogenic High Bone Mass Loci Only Explain a Small Proportion of High Bone Mass Cases.

High bone mass (HBM) can be an incidental clinical finding; however, monogenic HBM disorders (eg, LRP5 or SOST mutations) are rare. We aimed to determine to what extent HBM is explained by mutations in known HBM genes. A total of 258 unrelated HBM cases were identified from a review of 335,115 DXA scans from 13 UK centers. Cases were assessed clinically and underwent sequencing of known anabolic HBM loci: LRP5 (exons 2, 3, 4), LRP4 (exons 25, 26), SOST (exons 1, 2, and the van Buchem's disease [VBD] 52-kb intronic deletion 3'). Family members were assessed for HBM segregation with identified variants. Three-dimensional protein models were constructed for identified variants. Two novel missense LRP5 HBM mutations ([c.518C>T; p.Thr173Met], [c.796C>T; p.Arg266Cys]) were identified, plus three previously reported missense LRP5 mutations ([c.593A>G; p.Asn198Ser], [c.724G>A; p.Ala242Thr], [c.266A>G; p.Gln89Arg]), associated with HBM in 11 adults from seven families. Individuals with LRP5 HBM (∼prevalence 5/100,000) displayed a variable phenotype of skeletal dysplasia with increased trabecular BMD and cortical thickness on HRpQCT, and gynoid fat mass accumulation on DXA, compared with both non-LRP5 HBM and controls. One mostly asymptomatic woman carried a novel heterozygous nonsense SOST mutation (c.530C>A; p.Ser177X) predicted to prematurely truncate sclerostin. Protein modeling suggests the severity of the LRP5-HBM phenotype corresponds to the degree of protein disruption and the consequent effect on SOST-LRP5 binding. We predict p.Asn198Ser and p.Ala242Thr directly disrupt SOST binding; both correspond to severe HBM phenotypes (BMD Z-scores +3.1 to +12.2, inability to float). Less disruptive structural alterations predicted from p.Arg266Cys, p.Thr173Met, and p.Gln89Arg were associated with less severe phenotypes (Z-scores +2.4 to +6.2, ability to float). In conclusion, although mutations in known HBM loci may be asymptomatic, they only account for a very small proportion (∼3%) of HBM individuals, suggesting the great majority are explained by either unknown monogenic causes or polygenic inheritance.

The genetics of osteoporosis.

INTRODUCTION: Osteoporosis is the commonest metabolic bone disease worldwide. The clinical hallmark of osteoporosis is low trauma fracture, with the most devastating being hip fracture, resulting in significant effects on both morbidity and mortality. SOURCES OF DATA: Data for this review have been gathered from the published literature and from a range of web resources. AREAS OF AGREEMENT: Genome-wide association studies in the field of osteoporosis have led to the identification of a number of loci associated with both bone mineral density and fracture risk and further increased our understanding of disease. AREAS OF CONTROVERSY: The early strategies for mapping osteoporosis disease genes reported only isolated associations, with replication in independent cohorts proving difficult. Neither candidate gene or linkage studies showed association at genome-wide level of significance. GROWING POINTS: The advent of massive parallel sequencing technologies has proved extremely successful in mapping monogenic diseases and thus leading to the utilization of this new technology in complex disease genetics. AREAS TIMELY FOR DEVELOPING RESEARCH: The identification of novel genes and pathways will potentially lead to the identification of novel therapeutic options for patients with osteoporosis.

Germline FH mutations presenting with pheochromocytoma.

CONTEXT: At least a third of the patients with pheochromocytoma (PCC) or paraganglioma (PGL) harbor an underlying germline mutation in a known PCC/PGL gene. Mutations in genes (SDHB, SDHD, SDHC, and SDHA) encoding a component of the tricarboxylic acid cycle, succinate dehydrogenase (SDH), are a major cause of inherited PCC and PGL. SDHB mutations are also, albeit less frequently, associated with inherited renal cell carcinoma. Inactivation of SDH and another tricarboxylic acid cycle component, fumarate hydratase (FH), have both been associated with abnormalities of cellular metabolism, responsible for the activation of hypoxic gene response pathways and epigenetic alterations (eg, DNA methylation). However, the clinical phenotype of germline mutations in SDHx genes and FH is usually distinct, with FH mutations classically associated with hereditary cutaneous and uterine leiomyomatosis and renal cell carcinoma, although recently an association with PCC/PGL has been reported. OBJECTIVE AND DESIGN: To identify potential novel PCC/PGL predisposition genes, we initially undertook exome resequencing studies in a case of childhood PCC, and subsequently FH mutation analysis in a further 71 patients with PCC, PGL, or head and neck PGL. RESULTS: After identifying a candidate FH missense mutation in the exome study, we then detected a further candidate missense mutation (p.Glu53Lys) by candidate gene sequencing. In vitro analyses demonstrated that both missense mutations (p.Cys434Tyr and p.Glu53Lys) were catalytically inactive. CONCLUSIONS: These findings 1) confirm that germline FH mutations may present, albeit rarely with PCC or PGL; and 2) extend the clinical phenotype associated with FH mutations to pediatric PCC.

Multicentric carpotarsal osteolysis is caused by mutations clustering in the amino-terminal transcriptional activation domain of MAFB.

Multicentric carpotarsal osteolysis (MCTO) is a rare skeletal dysplasia characterized by aggressive osteolysis, particularly affecting the carpal and tarsal bones, and is frequently associated with progressive renal failure. Using exome capture and next-generation sequencing in five unrelated simplex cases of MCTO, we identified previously unreported missense mutations clustering within a 51 base pair region of the single exon of MAFB, validated by Sanger sequencing. A further six unrelated simplex cases with MCTO were also heterozygous for previously unreported mutations within this same region, as were affected members of two families with autosomal-dominant MCTO. MAFB encodes a transcription factor that negatively regulates RANKL-induced osteoclastogenesis and is essential for normal renal development. Identification of this gene paves the way for development of novel therapeutic approaches for this crippling disease and provides insight into normal bone and kidney development.

Whole-exome re-sequencing in a family quartet identifies POP1 mutations as the cause of a novel skeletal dysplasia.

Recent advances in DNA sequencing have enabled mapping of genes for monogenic traits in families with small pedigrees and even in unrelated cases. We report the identification of disease-causing mutations in a rare, severe, skeletal dysplasia, studying a family of two healthy unrelated parents and two affected children using whole-exome sequencing. The two affected daughters have clinical and radiographic features suggestive of anauxetic dysplasia (OMIM 607095), a rare form of dwarfism caused by mutations of RMRP. However, mutations of RMRP were excluded in this family by direct sequencing. Our studies identified two novel compound heterozygous loss-of-function mutations in POP1, which encodes a core component of the RNase mitochondrial RNA processing (RNase MRP) complex that directly interacts with the RMRP RNA domains that are affected in anauxetic dysplasia. We demonstrate that these mutations impair the integrity and activity of this complex and that they impair cell proliferation, providing likely molecular and cellular mechanisms by which POP1 mutations cause this severe skeletal dysplasia.

Molecular diagnosis for heterogeneous genetic diseases with targeted high-throughput DNA sequencing applied to retinitis pigmentosa.

BACKGROUND: The genetic heterogeneity of many Mendelian disorders, such as retinitis pigmentosa which results from mutations in over 40 genes, is a major obstacle to obtaining a molecular diagnosis in clinical practice. Targeted high-throughput DNA sequencing offers a potential solution and was used to develop a molecular diagnostic screen for patients with retinitis pigmentosa. METHODS: A custom sequence capture array was designed to target the coding regions of all known retinitis pigmentosa genes and used to enrich these sequences from DNA samples of five patients. Enriched DNA was subjected to high-throughput sequencing singly or in pools, and sequence variants were identified by alignment of up to 10 million reads per sample to the normal reference sequence. Potential pathogenicity was assessed by functional predictions and frequency in controls. RESULTS AND CONCLUSIONS: Known homozygous PDE6B and compound heterozygous CRB1 mutations were detected in two patients. A novel homozygous missense mutation (c.2957A→T; p.N986I) in the cyclic nucleotide gated channel ß1 (CNGB1) gene predicted to have a deleterious effect and absent in 720 control chromosomes was detected in one case in which conventional genetic screening had failed to detect mutations. The detection of known and novel retinitis pigmentosa mutations in this study establishes high-throughput DNA sequencing with DNA pooling as an effective diagnostic tool for heterogeneous genetic diseases.

Tau and alpha-synuclein in susceptibility to, and dementia in, Parkinson's disease.

OBJECTIVE: Parkinson's disease (PD) is a neurodegenerative condition that typically presents as a movement disorder but is known to be associated with variable degrees of cognitive impairment including dementia. We investigated the genetic basis of susceptibility to and cognitive heterogeneity of this disease. METHODS: In 659 PD patients, 109 of which were followed up for 3.5 years from diagnosis, and 2,176 control subjects, we studied candidate genes involved in protein aggregation and inclusion body formation, the pathological hallmark of parkinsonism: microtubule-associated protein tau (MAPT), glycogen synthase kinase-3beta (GSK3B), and alpha-synuclein (SNCA). RESULTS: We observed that cognitive decline and the development of PD dementia are strongly associated (p = 10(-4)) with the inversion polymorphism containing MAPT. We also found a novel synergistic interaction between the MAPT inversion polymorphism and the single nucleotide polymorphism rs356219 from the 3' region of SNCA. In our data, carrying a risk genotype at either of these loci marginally increases the risk for development of PD, whereas carrying the combination of risk genotypes at both loci approximately doubles the risk for development of the disease (p = 3 x 10(-6)). INTERPRETATION: Our data support the hypothesis that tau and alpha-synuclein are involved in shared or converging pathways in the pathogenesis of PD, and suggest that the tau inversion influences the development of cognitive impairment and dementia in patients with idiopathic PD. These findings have potentially important implications for understanding the interface between tau and alpha-synuclein pathways in neurodegenerative disorders and for unraveling the biological basis for cognitive impairment and dementia in PD.