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.

Methylation analysis and diagnostics of Beckwith-Wiedemann syndrome in 1,000 subjects.

BACKGROUND: Beckwith-Wiedemann syndrome (BWS), a congenital overgrowth disorder with variable expressivity and a predisposition to tumorigenesis, results from disordered expression and/or function of imprinted genes at chromosome 11p15.5. There are no generally agreed clinical diagnostic criteria, with molecular studies commonly performed to confirm diagnosis. In particular, methylation status analysis at two 11p15.5 imprinting control centres (IC1 and IC2) detects up to 80% of BWS cases (though low-level mosaicism may not be detected). In order to evaluate the relationship between the clinical presentation of suspected BWS and IC1/2 methylation abnormalities we reviewed the results of >1,000 referrals for molecular diagnostic testing. RESULTS: Out of 1,091 referrals, 507 (46.5%) had a positive diagnostic test for BWS. The frequency of tumours was 3.4% in those with a molecular diagnosis of BWS. Previously reported genotype-phenotype associations with paternal uniparental disomy, IC1, and IC2 epimutation groups were confirmed and potential novel associations detected. Predictive values of previously described clinical diagnostic criteria were compared and, although there were differences in their sensitivity and specificity, receiver operating characteristic (ROC) analysis demonstrated that these were not optimal in predicting 11p15.5 methylation abnormalities. Using logistic regression, we identified clinical features with the best predictive value for a positive methylation abnormality. Furthermore, we developed a weighted scoring system (sensitivity 75.9%, and specificity 81.8%) to prioritise patients presenting with the most common features of BWS, and ROC analysis demonstrated superior performance (area under the curve 0.85, 95% CI 0.83 to 0.87) compared to previous criteria. CONCLUSIONS: We suggest that this novel tool will facilitate selection of patients with suspected BWS for routine diagnostic testing and so improve the diagnosis of the disorder.

Epimutation profiling in Beckwith-Wiedemann syndrome: relationship with assisted reproductive technology.

BACKGROUND: Beckwith-Wiedemann syndrome (BWS) is a congenital overgrowth disorder associated with abnormalities in 11p15.5 imprinted genes. The most common cause is loss of methylation (epimutation) at the imprinting control centre 2 (IC2/KvDMR1). Most IC2 epimutations occur sporadically but an association with conception after assisted reproductive technologies (ART) has been reported. A subgroup of IC2 epimutation cases also harbour epimutations at other imprinting centres (ICs) outside of 11p15.5. We have investigated the relationship between these multiple epimutation cases (ME+), history of ART and clinical phenotype in a cohort of 187 BWS IC2 epimutation patients. RESULTS: Methylation analysis at PLAGL1, MEST and IGF2R ICs demonstrated an over-representation of patients with abnormally low methylation (8.5%, 12% and 6% respectively). At IGF2R some patients (2%) had gain of methylation but this was also detected in controls. Though there were no significant correlations between the methylation index (MIs) at the three ICs tested, a subset of patients appeared to be susceptible to multiple epimutations (ME+) and 21.2% of ME + patients had been conceived by ART compared to 4.5% (P = 0.0033) without additional epimutations. Methylation array profiling (Illumina Goldengate®) of patients and controls (excluding 11p15.5 loci) demonstrated significant differences between patients and controls. No significant associations were found between aspects of the BWS phenotype and individual epimutations but we describe a case presenting with a post-ART BWS-like phenotype in which molecular analysis demonstrated loss of paternal allele methylation at the 11p15.5 IC1 locus (IC1 regulates imprinting of IGF2 and H19). Loss of paternal allele methylation at the IC1 is the molecular finding associated with Silver-Russell syndrome whereas BWS is associated with gain of maternal allele methylation at IC1. Further analysis demonstrated epimutations at PLAGL1 and MEST consistent with the hypothesis that the presence of multiple epimutations may be of clinical relevance. CONCLUSIONS: These findings suggest that the ME + subgroup of BWS patients are preferentially, but not exclusively, associated with a history of ART and that, though at present, there are no clear epigenotype-phenotype correlations for ME + BWS patients, non-11p15.5 IC epimutations can influence clinical phenotype.

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.

Loss-of-function mutations in RAB18 cause Warburg micro syndrome.

Warburg Micro syndrome and Martsolf syndrome are heterogenous autosomal-recessive developmental disorders characterized by brain, eye, and endocrine abnormalities. Previously, identification of mutations in RAB3GAP1 and RAB3GAP2 in both these syndromes implicated dysregulation of the RAB3 cycle (which controls calcium-mediated exocytosis of neurotransmitters and hormones) in disease pathogenesis. RAB3GAP1 and RAB3GAP2 encode the catalytic and noncatalytic subunits of the hetrodimeric enzyme RAB3GAP (RAB3GTPase-activating protein), a key regulator of the RAB3 cycle. We performed autozygosity mapping in five consanguineous families without RAB3GAP1/2 mutations and identified loss-of-function mutations in RAB18. A c.71T > A (p.Leu24Gln) founder mutation was identified in four Pakistani families, and a homozygous exon 2 deletion (predicted to result in a frameshift) was found in the fifth family. A single family whose members were compound heterozygotes for an anti-termination mutation of the stop codon c.619T > C (p.X207QextX20) and an inframe arginine deletion c.277_279 del (p.Arg93 del) were identified after direct gene sequencing and multiplex ligation-dependent probe amplification (MLPA) of a further 58 families. Nucleotide binding assays for RAB18(Leu24Gln) and RAB18(Arg93del) showed that these mutant proteins were functionally null in that they were unable to bind guanine. The clinical features of Warburg Micro syndrome patients with RAB3GAP1 or RAB3GAP2 mutations and RAB18 mutations are indistinguishable, although the role of RAB18 in trafficking is still emerging, and it has not been linked previously to the RAB3 pathway. Knockdown of rab18 in zebrafish suggests that it might have a conserved developmental role. Our findings imply that RAB18 has a critical role in human brain and eye development and neurodegeneration.

Mutation analysis of HIF prolyl hydroxylases (PHD/EGLN) in individuals with features of phaeochromocytoma and renal cell carcinoma susceptibility.

Germline mutations in the von Hippel-Lindau disease (VHL) and succinate dehydrogenase subunit B (SDHB) genes can cause inherited phaeochromocytoma and/or renal cell carcinoma (RCC). Dysregulation of the hypoxia-inducible factor (HIF) transcription factors has been linked to VHL and SDHB-related RCC; both HIF dysregulation and disordered function of a prolyl hydroxylase domain isoform 3 (PHD3/EGLN3)-related pathway of neuronal apoptosis have been linked to the development of phaeochromocytoma. The 2-oxoglutarate-dependent prolyl hydroxylase enzymes PHD1 (EGLN2), PHD2 (EGLN1) and PHD3 (EGLN3) have a key role in regulating the stability of HIF-α subunits (and hence expression of the HIF-α transcription factors). A germline PHD2 mutation has been reported in association with congenital erythrocytosis and recurrent extra-adrenal phaeochromocytoma. We undertook mutation analysis of PHD1, PHD2 and PHD3 in two cohorts of patients with features of inherited phaeochromocytoma (n=82) and inherited RCC (n=64) and no evidence of germline mutations in known susceptibility genes. No confirmed pathogenic mutations were detected suggesting that mutations in these genes are not a frequent cause of inherited phaeochromocytoma or RCC.

Novel TSHR mutations in consanguineous families with congenital nongoitrous hypothyroidism.

OBJECTIVE: Nonsyndromic autosomal recessively inherited nongoitrous congenital hypothyroidism (CHNG) can be caused by mutations in TSHR, PAX8, TSHB and NKX2-5. We aimed to investigate mutational frequencies of these genes and genotype/phenotype correlations in consanguineous families with CHNG. DESIGN: Because consanguinity in individuals with a presumptive genetic condition is often an indicator of an autosomal recessive inheritance and allows firmer correlations to be established between genotype and phenotype, we planned to execute our study in consanguineous families. PATIENTS: Hundred and thirty-nine children with CHNG phenotype born to consanguineous families. MEASUREMENTS: First, we investigated cases for evidence of linkage to the four known CHNG genes by microsatellite marker analysis. Mutation analysis by direct sequencing was then performed in those cases in whom linkage to the relevant candidate gene could not be excluded. In addition, in silico analysis of the predicted structural effects of TSHR mutations was performed and related to the mutation-specific disease phenotype. RESULTS: Homozygous germline TSHR mutations were detected in six families (5%), but no mutations were detected in PAX8, TSHB and NKX2-5. Four of TSHR mutations had not previously been described. Genotype-phenotype correlations were established and found to be related to the predicted structural effects of the mutations. CONCLUSIONS: Known causative genes account for the development of CHNG only in a minority of cases, and our cohort should provide a powerful resource to identify novel causative genes and to delineate the extent of locus heterogeneity in autosomal recessively inherited CHNG.

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.

Clinical and molecular genetic features of Beckwith-Wiedemann syndrome associated with assisted reproductive technologies.

BACKGROUND: Beckwith-Wiedemann syndrome (BWS) is a model imprinting disorder resulting from mutations or epigenetic events affecting imprinted genes at 11p15.5. Most BWS cases are sporadic and result from imprinting errors (epimutations) involving either of the two 11p15.5 imprinting control regions (IC1 and IC2). Previously, we and other reported an association between sporadic BWS and assisted reproductive technologies (ARTs). METHODS: In this study, we compared the clinical phenotype and molecular features of ART (IVF and ICSI) and non-ART children with sporadic BWS. A total of 25 patients with post-ART BWS were ascertained (12 after IVF and 13 after ICSI). RESULTS: Molecular genetic analysis revealed an IC2 epimutations (KvDMR1 loss of methylation) in 24 of the 25 children tested. Comparison of clinical features of children with post-ART BWS to those with non-ART BWS and IC2 defects revealed a lower frequency of exomphalos (43 versus 69%, P = 0.029) and a higher risk of neoplasia (two cases, P = 0.0014). As loss of methylation at imprinting control regions other than 11p15.5 might modify the phenotype of BWS patients with IC2 epimutations, we investigated differentially methylated regions (DMRs) at 6q24, 7q32 and 15q13 in post-ART and non-ART BWS IC2 cases (n = 55). Loss of maternal allele methylation at these DMRs occurred in 37.5% of ART and 6.4% of non-ART BWS IC2 defect cases. Thus, more generalized DMR hypomethylation is more frequent, but not exclusive to post-ART BWS. CONCLUSIONS: These findings provide further evidence that ART may be associated with disturbed normal genomic imprinting in a subset of children.

Beckwith Weidemann syndrome: a behavioral phenotype-genotype study.

Neurobehavioral defects have been reported in human imprinting disorders such as Prader-Willi syndrome and Angelman syndrome and imprinted genes are often implicated in neurodevelopment processes. Beckwith-Wiedemann syndrome (BWS) is a classical human imprinting disorder characterized by prenatal and postnatal overgrowth and variable developmental anomalies. As neurodevelopmental aspects of BWS have not previously been studied in detail, we undertook a questionnaire based neurobehavioral survey of 87 children with BWS. A greater than expected proportion of children demonstrated abnormal scores on measures of emotional and behavioral difficulties. In addition, 6.8% of children had been diagnosed with an autistic spectrum disorder (ASD). 4/6 BWS children with ASD had normal chromosomes and ASD occurred in children with UPD and imprinting center 2 defects. These findings suggest a potential role for the 11p15.5 imprinted gene cluster in ASD and indicate a need for further investigations of neurobehavioral phenotypes in BWS.

A survey of assisted reproductive technology births and imprinting disorders.

BACKGROUND: Genomic imprinting is an epigenetic process in which allele-specific gene expression is dependent on the parental inheritance. Although only a minority of human genes are imprinted, those that have been identified to date have been preferentially implicated in prenatal growth and neurodevelopment. Mutations or epimutations in imprinted genes or imprinting control centres are associated with imprinting disorders such as Angelman syndrome (AS) and Beckwith-Wiedemann syndrome (BWS). Recently, an increased frequency of assisted reproductive technology (ART) conceptions has been reported in children with BWS and AS. However, the risk of imprinting disorders in ART children is unknown. METHODS: We undertook a survey of 2492 children born after ART in the Republic of Ireland and Central England with the aim of detecting cases (both clinically diagnosed and previously unrecognized) of BWS and AS in this cohort. The response rate to an initial questionnaire was 61%, corresponding to data for 1524 children. After evaluation of the questionnaire, 70 children were invited for a detailed clinical assessment, and 47 accepted (response rate of 67%). RESULTS: In this entire cohort, we detected one case of BWS and no cases of AS. We did not find evidence that there exists a significant group of ART children with unrecognized milder forms of AS or BWS. CONCLUSIONS: Although previous studies have suggested an increased relative risk of BWS and AS after ART, our findings suggest that the absolute risk of imprinting disorders in children conceived by ART is small (<1%). Precise risk estimates of risk are difficult to define because of the rarity of the conditions and incomplete response rates to the questionnaire and clinical examination invitations. Hence further investigations are indicated to (i) refine the absolute and relative risks of imprinting disorders after ART and (ii) ensure that changes in ART protocols are not associated with increased frequencies of epigenetic changes and imprinting disorders in children born after ART.