Robert M. W. Hofstra

Evidence Based Selection of Housekeeping Genes

For accurate and reliable gene expression analysis, normalization of gene expression data against housekeeping genes (reference or internal control genes) is required. It is known that commonly used housekeeping genes (e.g. ACTB, GAPDH, HPRT1, and B2M) vary considerably under different experimental conditions and therefore their use for normalization is limited. We performed a meta-analysis of 13,629 human gene array samples in order to identify the most stable expressed genes. Here we show novel candidate housekeeping genes (e.g. RPS13, RPL27, RPS20 and OAZ1) with enhanced stability among a multitude of different cell types and varying experimental conditions. None of the commonly used housekeeping genes were present in the top 50 of the most stable expressed genes. In addition, using 2,543 diverse mouse gene array samples we were able to confirm the enhanced stability of the candidate novel housekeeping genes in another mammalian species. Therefore, the identified novel candidate housekeeping genes seem to be the most appropriate choice for normalizing gene expression data.

Geographical and ethnic variation of the 677C>T allele of 5,10 methylenetetrahydrofolate reductase ( MTHFR ): findings from over 7000 newborns from 16 areas world wide

Since its biochemical characterisation in 19911 and its genetic identification in 1995,2 677C>T allele (T allele) of the 5,10 methylenetetrahydrofolate reductase ( MTHFR ) gene has been a focus of increasing interest from researchers world wide. The expanding spectrum of common conditions linked with the 677C>T allele now includes certain adverse birth outcomes (including birth defects), pregnancy complications, cancers, adult cardiovascular diseases, and psychiatric disorders.3–8 Although several of these associations remain unconfirmed or controversial,4 their scope is such that it becomes of interest to explore the geographical and ethnic distribution of the allele and associated genotypes.9 Accurate information on such distribution can contribute to studies of gene-disease associations (by providing reference population data) and population genetics (by highlighting geographical and ethnic variations suggestive of evolutionary pressures),10 as well as help to evaluate health impact (by allowing estimates of population attributable fraction). Current population data, however, show gaps and even for some ethnic groups or large geographical areas (for example, China) few data are available.3 Our aim was to supplement the available data by collecting a large and diverse sample of newborns from different geographical areas and ethnic groups, and to examine international variations in the distribution of the 677C>T allele. We present findings relating to more than 7000 newborns from 16 areas around the world. The study was conducted under the auspices of the International Clearinghouse for Birth Defect Monitoring Systems (ICBDMS) and was coordinated through its head office, the International Center on Birth Defects (ICBD). ### Sample selection Participating programmes, in consultation with the coordinating group, identified a population sampling approach that would be simple yet minimise sampling bias with respect to the MTHFR genotype. We made an explicit attempt to sample systematically the newborn population. Details of each programme’s approach are listed below, and further …

Plakophilin-2 Mutations Are the Major Determinant of Familial Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy

Background— Mutations in the plakophilin-2 gene (PKP2) have been found in patients with arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVC). Hence, genetic screening can potentially be a valuable tool in the diagnostic workup of patients with ARVC. Methods and Results— To establish the prevalence and character of PKP2 mutations and to study potential differences in the associated phenotype, we evaluated 96 index patients, including 56 who fulfilled the published task force criteria. In addition, 114 family members from 34 of these 56 ARVC index patients were phenotyped. In 24 of these 56 ARVC patients (43%), 14 different (11 novel) PKP2 mutations were identified. Four different mutations were found more than once; haplotype analyses revealed identical haplotypes in the different mutation carriers, suggesting founder mutations. No specific genotype–phenotype correlations could be identified, except that negative T waves in V2 and V3 occurred more often in PKP2 mutation carriers (P<0.05). Of the 34 index patients whose family members were phenotyped, 23 familial cases were identified. PKP2 mutations were identified in 16 of these 23 ARVC index patients (70%) with familial ARVC. On the other hand, no PKP2 mutations at all were found in 11 probands without additional affected family members (P<0.001). Conclusions— PKP2 mutations can be identified in nearly half of the Dutch patients fulfilling the ARVC criteria. In familial ARVC, even the vast majority (70%) is caused by PKP2 mutations. However, nonfamilial ARVC is not related to PKP2. The high yield of mutational analysis in familial ARVC is unique in inherited cardiomyopathies.

Association of hereditary nonpolyposis colorectal cancer-related tumors displaying low microsatellite instability with MSH6 germline mutations

Hereditary nonpolyposis colorectal cancer (HNPCC) (Amsterdam criteria) is often caused by mutations in mismatch repair (MMR) genes, and tumors of patients with HNPCC show microsatellite instability (MSI-high phenotype). Germline mutations of MMR genes have rarely been found in families that have HNPCC or suspected HNPCC and that do not show microsatellite instability (MSI-low phenotype). Therefore, an MSI-high phenotype is often used as an inclusion criterion for mutation testing of MMR genes. Correction of base-base mismatches is the major function of MSH6. Since mismatches present with an MSI-low phenotype, we assumed that the phenotype in patients with HNPCC-related tumors might be associated with MSH6 germline mutations. We divided 36 patients with suspected HNPCC into an MSI-low group (n=18) and an MSI-high group (n=18), on the basis of the results of MSI testing. Additionally, three unrelated patients from Amsterdam families with MSI-low tumors were investigated. All patients were screened for MSH2, MLH1, and MSH6 mutations. Four presumably causative MSH6 mutations were detected in the patients (22%) who had suspected HNPCC and MSI-low tumors. Furthermore, we detected one frameshift mutation in one of the three patients with HNPCC and MSI-low tumors. In the MSI-high group, one MSH6 missense mutation was found, but the same patient also had an MLH1 mutation, which may explain the MSI-high phenotype. These results suggest that MSH6 may be involved in a substantial proportion of patients with HNPCC or suspected HNPCC and MSI-low tumors. Our data emphasize that an MSI-low phenotype cannot be considered an exclusion criterion for mutation testing of MMR genes in general.

Molecular and Clinical Characteristics of MSH6 Variants: An Analysis of 25 Index Carriers of a Germline Variant

The MSH6 gene is one of the mismatch-repair genes involved in hereditary nonpolyposis colorectal cancer (HNPCC). Three hundred sixteen individuals who were known or suspected to have HNPCC were analyzed for MSH6 germline mutations. For 25 index patients and 8 relatives with MSH6 variants, molecular and clinical features are described. For analysis of microsatellite instability (MSI), the five consensus markers were used. Immunohistochemical analysis of the MLH1, MSH2, and MSH6 proteins was performed. Five truncating MSH6 mutations, of which one was detected seven times, were found in 12 index patients, and 10 MSH6 variants with unknown pathogenicity were found in 13 index patients. Fourteen (54%) of 26 colorectal cancers (CRCs) and endometrial cancers showed no, or only weak, MSI. Twelve of 18 tumors of truncating-mutation carriers and 3 of 17 tumors of missense-mutation carriers showed loss of MSH6 staining. Six of the families that we studied fulfilled the original Amsterdam criteria; most families with MSH6, however, were only suspected to have HNPCC. In families that did not fulfill the revised Amsterdam criteria, the prevalence of MSH6 variants is about the same as the prevalence of those in MLH1/MSH2. Endometrial cancer and/or atypical hyperplasia were diagnosed in 8 of 12 female carriers of MSH6 truncating mutations. Most CRCs were localized distally in the colon. Although, molecularly, missense variants are labeled as doubtfully pathogenic, clinical data disclose a great resemblance between missense-variant carriers and truncating-mutation carriers. We conclude that, in all patients suspected to have HNPCC, MSH6-mutation analysis should be considered. Neither MSI nor immunohistochemistry should be a definitive selection criterion for MSH6-mutation analysis.

Trans-eQTLs Reveal That Independent Genetic Variants Associated with a Complex Phenotype Converge on Intermediate Genes, with a Major Role for the HLA

For many complex traits, genetic variants have been found associated. However, it is still mostly unclear through which downstream mechanism these variants cause these phenotypes. Knowledge of these intermediate steps is crucial to understand pathogenesis, while also providing leads for potential pharmacological intervention. Here we relied upon natural human genetic variation to identify effects of these variants on trans-gene expression (expression quantitative trait locus mapping, eQTL) in whole peripheral blood from 1,469 unrelated individuals. We looked at 1,167 published trait- or disease-associated SNPs and observed trans-eQTL effects on 113 different genes, of which we replicated 46 in monocytes of 1,490 different individuals and 18 in a smaller dataset that comprised subcutaneous adipose, visceral adipose, liver tissue, and muscle tissue. HLA single-nucleotide polymorphisms (SNPs) were 10-fold enriched for trans-eQTLs: 48% of the trans-acting SNPs map within the HLA, including ulcerative colitis susceptibility variants that affect plausible candidate genes AOAH and TRBV18 in trans. We identified 18 pairs of unlinked SNPs associated with the same phenotype and affecting expression of the same trans-gene (21 times more than expected, P<10−16). This was particularly pronounced for mean platelet volume (MPV): Two independent SNPs significantly affect the well-known blood coagulation genes GP9 and F13A1 but also C19orf33, SAMD14, VCL, and GNG11. Several of these SNPs have a substantially higher effect on the downstream trans-genes than on the eventual phenotypes, supporting the concept that the effects of these SNPs on expression seems to be much less multifactorial. Therefore, these trans-eQTLs could well represent some of the intermediate genes that connect genetic variants with their eventual complex phenotypic outcomes.

PHOX2B mutations and polyalanine expansions correlate with the severity of the respiratory phenotype and associated symptoms in both congenital and late onset Central Hypoventilation syndrome

Congenital Central Hypoventilation syndrome (CCHS (MIM 209880)) is a rare disorder, with fewer than 200 patients currently reported worldwide, characterised by absence of adequate autonomic control of respiration with decreased sensitivity to hypercapnia and hypoxia, in the absence of neuromuscular or lung disease, or an identifiable brain stem lesion.1 Children with CCHS show an adequate ventilation while awake but hypoventilate during sleep. More severely affected children hypoventilate both when awake and during sleep.1 CCHS has been reported in association with several disorders, among which aganglionic megacolon (Hirschsprung disease, HSCR) and tumours of neural crest origin, reflecting a common molecular pathogenesis sustained by defects of one or more genes that control the correct development of neural crest derived cell lineages.1–3 A genetic aetiology has long been hypothesised for CCHS based on recurrence reported in siblings, in half siblings and in affected children born to women with CCHS.2–6 More recently, a generalised autonomic nervous system (ANS) imbalance has been observed among children with CCHS and an increased incidence of ANS dysfunctions (ANSD) reported among relatives of 56 patients with CCHS, as against relatives of 56 matched controls.7 A family transmission study has shown that the risk of developing an ANSD symptom including CCHS, regarded as the most severe expression of ANS imbalance, mainly depends on the genotype at a major locus, while significant residual variants could be due to additional minor genes, modifying loci effects or environmental factors.8 Genes involved in the ANS development, like the RET proto-oncogene, its ligand GDNF , the Endothelin 3 gene, the Brain Derived Neurotrophic Factor ( BDNF ) and the RNX genes, have been tested and a few mutations found, showing no cosegregation with the disease phenotype in CCHS families.9–13 The PHOX2B gene encodes a 314 amino acids …

C-elegans model identifies genetic modifiers of alpha-synuclein inclusion formation during aging

Inclusions in the brain containing α-synuclein are the pathological hallmark of Parkinsons disease, but how these inclusions are formed and how this links to disease is poorly understood. We have developed a C. elegans model that makes it possible to monitor, in living animals, the formation of α-synuclein inclusions. In worms of old age, inclusions contain aggregated α- synuclein, resembling a critical pathological feature. We used genome-wide RNA interference to identify processes involved in inclusion formation, and identified 80 genes that, when knocked down, resulted in a premature increase in the number of inclusions. Quality control and vesicle-trafficking genes expressed in the ER/Golgi complex and vesicular compartments were overrepresented, indicating a specific role for these processes in α-synuclein inclusion formation. Suppressors include aging-associated genes, such as sir-2.1/SIRT1 and lagr-1/LASS2. Altogether, our data suggest a link between α-synuclein inclusion formation and cellular aging, likely through an endomembrane-related mechanism. The processes and genes identified here present a framework for further study of the disease mechanism and provide candidate susceptibility genes and drug targets for Parkinsons disease and other α-synuclein related disorders.

Phospholamban R14del mutation in patients diagnosed with dilated cardiomyopathy or arrhythmogenic right ventricular cardiomyopathy: evidence supporting the concept of arrhythmogenic cardiomyopathy

To investigate whether phospholamban gene (PLN) mutations underlie patients diagnosed with either arrhythmogenic right ventricular cardiomyopathy (ARVC) or idiopathic dilated cardiomyopathy (DCM).

BRAF-V600E is not involved in the colorectal tumorigenesis of HNPCC in patients with functional MLH1 and MSH2 genes

Recently, it was shown that the oncogenic activation of BRAF, a member of the RAS/RAF family of kinases, by the V600E mutation is characteristic for sporadic colon tumors with microsatellite instability. Further, it was shown to associate with the silencing of the mismatch repair (MMR) gene MLH1 by hypermethylation. Moreover, BRAF mutations proved to be absent in tumors from hereditary nonpolyposis colorectal cancer syndrome (HNPCC) families with germline mutations in the MMR genes MLH1 and MSH2. These data suggest that the oncogenic activation of BRAF is involved only in sporadic colorectal tumorigenesis. In order to further support this hypothesis, we have extended the analysis of the BRAF gene to a different subset of HNPCC families without germline mutations in MLH1 and MSH2. BRAF-V600E mutations were analysed by automatic sequencing in 38 tumors from HNPCC families with germline mutations in the MSH6 gene and also in HNPCC (suspected) families that do not have mutations in the MMR genes MLH1, MSH2 and MSH6. All patients belong to different families. No mutations were detected in 14 tumors from HNPCC patients with germline mutations in MSH6. Further, no mutations of BRAF were found in tumors from 23 MMR-negative families, from which 13 fulfilled the Amsterdam criteria (HNPCC) and 10 were suspected for HNPCC as they were positive for the Bethesda criteria. Overall, our data reinforce the concept that BRAF is not involved in the colorectal tumorigenesis of HNPCC. The detection of a positive BRAF-V600E mutation in a colorectal cancer suggests a sporadic origin of the disease and the absence of germline alterations of MLH1, MSH2 and also of MSH6. These findings have a potential impact in the genetic testing for HNPCC diagnostics and suggest a potential use of BRAF as exclusion criteria for HNPCC or as a molecular marker of sporadic cancer.