CAG Repeat Size Influences the Progression Rate of Spinocerebellar Ataxia Type 3.

OBJECTIVE: In spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD), the expanded cytosine adenine guanine (CAG) repeat in ATXN3 is the causal mutation, and its length is the main factor in determining the age at onset (AO) of clinical symptoms. However, the contribution of the expanded CAG repeat length to the rate of disease progression after onset has remained a matter of debate, even though an understanding of this factor is crucial for experimental data on disease modifiers and their translation to clinical trials and their design. METHODS: Eighty-two Dutch patients with SCA3/MJD were evaluated annually for 15 years using the International Cooperative Ataxia Rating Scale (ICARS). Using linear growth curve models, ICARS progression rates were calculated and tested for their relation to the length of the CAG repeat expansion and to the residual age at onset (RAO): The difference between the observed AO and the AO predicted on the basis of the CAG repeat length. RESULTS: On average, ICARS scores increased 2.57 points/year of disease. The length of the CAG repeat was positively correlated with a more rapid ICARS progression, explaining 30% of the differences between patients. Combining both the length of the CAG repeat and RAO as comodifiers explained up to 47% of the interpatient variation in ICARS progression. INTERPRETATION: Our data imply that the length of the expanded CAG repeat in ATXN3 is a major determinant of clinical decline, which suggests that CAG-dependent molecular mechanisms similar to those responsible for disease onset also contribute to the rate of disease progression in SCA3/MJD. ANN NEUROL 2021;89:66-73.

Sensitive Monogenic Noninvasive Prenatal Diagnosis by Targeted Haplotyping.

During pregnancy, cell-free DNA (cfDNA) in maternal blood encompasses a small percentage of cell-free fetal DNA (cffDNA), an easily accessible source for determination of fetal disease status in risk families through non-invasive procedures. In case of monogenic heritable disease, background maternal cfDNA prohibits direct observation of the maternally inherited allele. Non-invasive prenatal diagnostics (NIPD) of monogenic diseases therefore relies on parental haplotyping and statistical assessment of inherited alleles from cffDNA, techniques currently unavailable for routine clinical practice. Here, we present monogenic NIPD (MG-NIPD), which requires a blood sample from both parents, for targeted locus amplification (TLA)-based phasing of heterozygous variants selectively at a gene of interest. Capture probes-based targeted sequencing of cfDNA from the pregnant mother and a tailored statistical analysis enables predicting fetal gene inheritance. MG-NIPD was validated for 18 pregnancies, focusing on CFTR, CYP21A2, and HBB. In all cases we could predict the inherited alleles with >98% confidence, even at relatively early stages (8 weeks) of pregnancy. This prediction and the accuracy of parental haplotyping was confirmed by sequencing of fetal material obtained by parallel invasive procedures. MG-NIPD is a robust method that requires standard instrumentation and can be implemented in any clinic to provide families carrying a severe monogenic disease with a prenatal diagnostic test based on a simple blood draw.

NIPTeR: an R package for fast and accurate trisomy prediction in non-invasive prenatal testing.

BACKGROUND: Various algorithms have been developed to predict fetal trisomies using cell-free DNA in non-invasive prenatal testing (NIPT). As basis for prediction, a control group of non-trisomy samples is needed. Prediction accuracy is dependent on the characteristics of this group and can be improved by reducing variability between samples and by ensuring the control group is representative for the sample analyzed. RESULTS: NIPTeR is an open-source R Package that enables fast NIPT analysis and simple but flexible workflow creation, including variation reduction, trisomy prediction algorithms and quality control. This broad range of functions allows users to account for variability in NIPT data, calculate control group statistics and predict the presence of trisomies. CONCLUSION: NIPTeR supports laboratories processing next-generation sequencing data for NIPT in assessing data quality and determining whether a fetal trisomy is present. NIPTeR is available under the GNU LGPL v3 license and can be freely downloaded from https://github.com/molgenis/NIPTeR or CRAN.

PLS3 mutations in X-linked osteoporosis with fractures.

Plastin 3 (PLS3), a protein involved in the formation of filamentous actin (F-actin) bundles, appears to be important in human bone health, on the basis of pathogenic variants in PLS3 in five families with X-linked osteoporosis and osteoporotic fractures that we report here. The bone-regulatory properties of PLS3 were supported by in vivo analyses in zebrafish. Furthermore, in an additional five families (described in less detail) referred for diagnosis or ruling out of osteogenesis imperfecta type I, a rare variant (rs140121121) in PLS3 was found. This variant was also associated with a risk of fracture among elderly heterozygous women that was two times as high as that among noncarriers, which indicates that genetic variation in PLS3 is a novel etiologic factor involved in common, multi-factorial osteoporosis.

Unraveling the regulatory mechanisms underlying tissue-dependent genetic variation of gene expression.

It is known that genetic variants can affect gene expression, but it is not yet completely clear through what mechanisms genetic variation mediate this expression. We therefore compared the cis-effect of single nucleotide polymorphisms (SNPs) on gene expression between blood samples from 1,240 human subjects and four primary non-blood tissues (liver, subcutaneous, and visceral adipose tissue and skeletal muscle) from 85 subjects. We characterized four different mechanisms for 2,072 probes that show tissue-dependent genetic regulation between blood and non-blood tissues: on average 33.2% only showed cis-regulation in non-blood tissues; 14.5% of the eQTL probes were regulated by different, independent SNPs depending on the tissue of investigation. 47.9% showed a different effect size although they were regulated by the same SNPs. Surprisingly, we observed that 4.4% were regulated by the same SNP but with opposite allelic direction. We show here that SNPs that are located in transcriptional regulatory elements are enriched for tissue-dependent regulation, including SNPs at 3' and 5' untranslated regions (P = 1.84×10(-5) and 4.7×10(-4), respectively) and SNPs that are synonymous-coding (P = 9.9×10(-4)). SNPs that are associated with complex traits more often exert a tissue-dependent effect on gene expression (P = 2.6×10(-10)). Our study yields new insights into the genetic basis of tissue-dependent expression and suggests that complex trait associated genetic variants have even more complex regulatory effects than previously anticipated.

Contribution of rare and common variants determine complex diseases-Hirschsprung disease as a model.

Finding genes for complex diseases has been the goal of many genetic studies. Most of these studies have been successful by searching for genes and mutations in rare familial cases, by screening candidate genes and by performing genome wide association studies. However, only a small fraction of the total genetic risk for these complex genetic diseases can be explained by the identified mutations and associated genetic loci. In this review we focus on Hirschsprung disease (HSCR) as an example of a complex genetic disorder. We describe the genes identified in this congenital malformation and postulate that both common 'low penetrant' variants in combination with rare or private 'high penetrant' variants determine the risk on HSCR, and likely, on other complex diseases. We also discuss how new technological advances can be used to gain further insights in the genetic background of complex diseases. Finally, we outline a few steps to develop functional assays in order to determine the involvement of these variants in disease development.

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.

CLMP is required for intestinal development, and loss-of-function mutations cause congenital short-bowel syndrome.

BACKGROUND & AIMS: Short-bowel syndrome usually results from surgical resection of the small intestine for diseases such as intestinal atresias, volvulus, and necrotizing enterocolitis. Patients with congenital short-bowel syndrome (CSBS) are born with a substantial shortening of the small intestine, to a mean length of 50 cm, compared with a normal length at birth of 190-280 cm. They also are born with intestinal malrotation. Because CSBS occurs in many consanguineous families, it is considered to be an autosomal-recessive disorder. We aimed to identify and characterize the genetic factor causing CSBS. METHODS: We performed homozygosity mapping using 610,000 K single-nucleotide polymorphism arrays to analyze the genomes of 5 patients with CSBS. After identifying a gene causing the disease, we determined its expression pattern in human embryos. We also overexpressed forms of the gene product that were and were not associated with CSBS in Chinese Hamster Ovary and T84 cells and generated a zebrafish model of the disease. RESULTS: We identified loss-of-function mutations in Coxsackie- and adenovirus receptor-like membrane protein (CLMP) in CSBS patients. CLMP is a tight-junction-associated protein that is expressed in the intestine of human embryos throughout development. Mutations in CLMP prevented its normal localization to the cell membrane. Knock-down experiments in zebrafish resulted in general developmental defects, including shortening of the intestine and the absence of goblet cells. Because goblet cells are characteristic for the midintestine in zebrafish, which resembles the small intestine in human beings, the zebrafish model mimics CSBS. CONCLUSIONS: Loss-of-function mutations in CLMP cause CSBS in human beings, likely by interfering with tight-junction formation, which disrupts intestinal development. Furthermore, we developed a zebrafish model of CSBS.

Periconceptional folic acid associated with an increased risk of oral clefts relative to non-folate related malformations in the Northern Netherlands: a population based case-control study.

Periconceptional folic acid has been associated with a reduced risk of neural tube defects, but findings on its effect in oral clefts are largely inconclusive. This case-control study assesses the effects of periconceptional folic acid on cleft risk, using complementary data from the Dutch Oral Cleft Registry and a population-based birth defects registry (Eurocat) of children and foetuses born in the Northern Netherlands between 1997 and 2009. Cases were live-born infants with non-syndromic clefts (n = 367) and controls were infants or foetuses with chromosomal/syndromal (n = 924) or non-folate related anomalies (n = 2,021). We analyzed type/timing/duration of supplement use related to traditional cleft categories as well as to their timing (early/late embryonic periods) and underlying embryological processes (fusion/differentiation defects). Consistent supplement use during the aetiologically relevant period (weeks 0-12 postconception) was associated with an increased risk of clefts (adjusted odds ratio 1.72, 95% confidence interval 1.19-2.49), especially of cleft lip/alveolus (3.16, 1.69-5.91). Further analysis systematically showed twofold to threefold increased risks for late differentiation defects-mainly clefts of the lip/alveolus-with no significant associations for early/late fusion defects. Effects were attributable to folic acid and not to other multivitamin components, and inclusion of partial use (not covering the complete aetiologically relevant period) generally weakened associations. In conclusion, this study presents several lines of evidence indicating that periconceptional folic acid in the Northern Netherlands is associated with an increased risk of clefts, in particular of cleft lip/alveolus. This association is strengthened by the specificity, consistency, systematic pattern, and duration of exposure-response relationship of our findings, underlining the need to evaluate public health strategies regarding folic acid and to further investigate potential adverse effects.

Variant Location Is a Novel Risk Factor for Individuals With Arrhythmogenic Cardiomyopathy Due to a Desmoplakin (DSP) Truncating Variant.

BACKGROUND: Truncating variants in desmoplakin (DSPtv) are an important cause of arrhythmogenic cardiomyopathy; however the genetic architecture and genotype-specific risk factors are incompletely understood. We evaluated phenotype, risk factors for ventricular arrhythmias, and underlying genetics of DSPtv cardiomyopathy. METHODS: Individuals with DSPtv and any cardiac phenotype, and their gene-positive family members were included from multiple international centers. Clinical data and family history information were collected. Event-free survival from ventricular arrhythmia was assessed. Variant location was compared between cases and controls, and literature review of reported DSPtv performed. RESULTS: There were 98 probands and 72 family members (mean age at diagnosis 43±8 years, 59% women) with a DSPtv, of which 146 were considered clinically affected. Ventricular arrhythmia (sudden cardiac arrest, sustained ventricular tachycardia, appropriate implantable cardioverter defibrillator therapy) occurred in 56 (33%) individuals. DSPtv location and proband status were independent risk factors for ventricular arrhythmia. Further, gene region was important with variants in cases (cohort n=98; Clinvar n=167) more likely to occur in the regions resulting in nonsense mediated decay of both major DSP isoforms, compared with n=124 genome aggregation database control variants (148 [83.6%] versus 29 [16.4%]; P<0.0001). CONCLUSIONS: In the largest series of individuals with DSPtv, we demonstrate that variant location is a novel risk factor for ventricular arrhythmia, can inform variant interpretation, and provide critical insights to allow for precision-based clinical management.

MixupMapper: correcting sample mix-ups in genome-wide datasets increases power to detect small genetic effects.

MOTIVATION: Sample mix-ups can arise during sample collection, handling, genotyping or data management. It is unclear how often sample mix-ups occur in genome-wide studies, as there currently are no post hoc methods that can identify these mix-ups in unrelated samples. We have therefore developed an algorithm (MixupMapper) that can both detect and correct sample mix-ups in genome-wide studies that study gene expression levels. RESULTS: We applied MixupMapper to five publicly available human genetical genomics datasets. On average, 3% of all analyzed samples had been assigned incorrect expression phenotypes: in one of the datasets 23% of the samples had incorrect expression phenotypes. The consequences of sample mix-ups are substantial: when we corrected these sample mix-ups, we identified on average 15% more significant cis-expression quantitative trait loci (cis-eQTLs). In one dataset, we identified three times as many significant cis-eQTLs after correction. Furthermore, we show through simulations that sample mix-ups can lead to an underestimation of the explained heritability of complex traits in genome-wide association datasets. AVAILABILITY AND IMPLEMENTATION: MixupMapper is freely available at http://www.genenetwork.nl/mixupmapper/

A haplotype sharing method for determining the relative age of SNP alleles.

There are two aspects regarding the age of alleles that are relevant as indicators of the timing of mutational events. The first is to know which alleles are species-specific; the second is about the time of origin of species-specific alleles. Both aspects can be analyzed using haplotype-sharing methods, by using the length of shared haplotypes as a measure of the speed of coalescence to common ancestors. The availability of sequence data for closely related species makes it possible to infer the original SNP allele. The allele present in more than one species is the original allele. In general, original alleles are expected to be more frequent, because the cumulative effects of genetic drift determine the maximum frequency a new mutant can reach. The human species is relatively young, and founder effects are still observable as extended linkage disequilibrium. Coalescence to a single founder takes place in human populations over a time frame that is so small that original haplotypes spanning several markers are still observable in current high-density SNP genotyping arrays. We show here that the length of shared haplotypes surrounding alleles is an indicator of the relative ages of alleles, and it is applicable to original and species-specific alleles.

Current smoking-specific gene expression signature in normal bronchial epithelium is enhanced in squamous cell lung cancer.

Cigarette smoking is the main risk factor for the development of squamous cell lung carcinoma (SCC). However, the smoking-related molecular changes in SCC have not been studied. Gene expression studies in both histologically normal bronchial epithelium and SCC epithelial samples identified genes differentially expressed between current and ex-smokers. Subsequently, expression levels of the smoking-related genes in normal bronchial epithelium were compared with those in SCC cells, since we hypothesized that the smoking-induced changes would be also deregulated in SCC. Gene expression profiles were generated using Agilent whole human genome microarrays on laser-microdissected normal bronchial epithelium and SCC samples. Expression levels of 246 genes, mainly related to oxidative stress response, were significantly different between normal bronchial epithelium of current and ex-smokers. Such a differential gene expression profile did not exist in SCC cells of smokers and ex-smokers. Interestingly, when comparing SCC and normal bronchial epithelium from ex-smokers, the vast majority of these 246 genes were also deregulated in SCC. When comparing SCC with normal epithelium from smokers, 22% of the up-regulated genes showed a similar high expression in SCC whereas 79% of the down-regulated genes were even further reduced in SCC as compared to current smokers. The down-regulated genes included several tumour suppressor genes, such as C9orf9, INHBB, LRIG1, SCGB3A1, SERPINI2, STEAP3 and ZMYND10. Thus, our study shows that the majority of genes up-regulated in normal bronchial epithelium of current smokers show similar high expression levels in SCC, while down-regulated genes are even further repressed in SCC. Our data indicate that smoking-related changes in normal bronchial epithelial cells persist in malignant transformed squamous cells.

Survival-related profile, pathways, and transcription factors in ovarian cancer.

BACKGROUND: Ovarian cancer has a poor prognosis due to advanced stage at presentation and either intrinsic or acquired resistance to classic cytotoxic drugs such as platinum and taxoids. Recent large clinical trials with different combinations and sequences of classic cytotoxic drugs indicate that further significant improvement in prognosis by this type of drugs is not to be expected. Currently a large number of drugs, targeting dysregulated molecular pathways in cancer cells have been developed and are introduced in the clinic. A major challenge is to identify those patients who will benefit from drugs targeting these specific dysregulated pathways.The aims of our study were (1) to develop a gene expression profile associated with overall survival in advanced stage serous ovarian cancer, (2) to assess the association of pathways and transcription factors with overall survival, and (3) to validate our identified profile and pathways/transcription factors in an independent set of ovarian cancers. METHODS AND FINDINGS: According to a randomized design, profiling of 157 advanced stage serous ovarian cancers was performed in duplicate using approximately 35,000 70-mer oligonucleotide microarrays. A continuous predictor of overall survival was built taking into account well-known issues in microarray analysis, such as multiple testing and overfitting. A functional class scoring analysis was utilized to assess pathways/transcription factors for their association with overall survival. The prognostic value of genes that constitute our overall survival profile was validated on a fully independent, publicly available dataset of 118 well-defined primary serous ovarian cancers. Furthermore, functional class scoring analysis was also performed on this independent dataset to assess the similarities with results from our own dataset. An 86-gene overall survival profile discriminated between patients with unfavorable and favorable prognosis (median survival, 19 versus 41 mo, respectively; permutation p-value of log-rank statistic = 0.015) and maintained its independent prognostic value in multivariate analysis. Genes that composed the overall survival profile were also able to discriminate between the two risk groups in the independent dataset. In our dataset 17/167 pathways and 13/111 transcription factors were associated with overall survival, of which 16 and 12, respectively, were confirmed in the independent dataset. CONCLUSIONS: Our study provides new clues to genes, pathways, and transcription factors that contribute to the clinical outcome of serous ovarian cancer and might be exploited in designing new treatment strategies.

Serum chemokine levels in Hodgkin lymphoma patients: highly increased levels of CCL17 and CCL22.

Hodgkin lymphoma (HL) is characterized by a minority of neoplastic Hodgkin-Reed Sternberg (HRS) cells surrounded by a non-neoplastic reactive infiltrate. As immunological mechanisms appear to be crucial in classical HL pathogenesis, altered serum chemokine levels might be related to disease activity. Serum levels of nine chemokines were examined in 163 untreated HL patients and 334 controls. We investigated single nucleotide polymorphisms (SNPs) for association with serum CCL17 (thymus and activation-regulated chemokine, TARC) levels and HL susceptibility. Serum CCL17 and CCL22 (macrophage-derived chemokine, MDC) levels were significantly increased in 82% and 57% of the HL patients. Nodular sclerosis cases showed increased serum CCL17 and CCL22 levels (P < 0.001) and serum levels were correlated with Ann Arbor stage. Of nine patients with pre- and post-treatment serum samples, the majority showed decreased CCL17 and CCL22 levels after treatment. HRS cells expressed CCL17 and CCL22 in 77% and 75% of 74 cases. Three SNPs showed a trend of increased serum CCL17 levels with minor alleles in controls, but were not associated with HL susceptibility. CCL17 and CCL22 were the only chemokines with increased serum levels in the vast majority of HL patients, which provides further insight into the molecular mechanism(s) leading to infiltrations of reactive lymphocytes in HL.

A "late-but-fitter revertant cell" explains the high frequency of revertant mosaicism in epidermolysis bullosa.

Revertant mosaicism, or "natural gene therapy", is the phenomenon in which germline mutations are corrected by somatic events. In recent years, revertant mosaicism has been identified in all major types of epidermolysis bullosa, the group of heritable blistering disorders caused by mutations in the genes encoding epidermal adhesion proteins. Moreover, revertant mosaicism appears to be present in all patients with a specific subtype of recessive epidermolysis bullosa. We therefore hypothesized that revertant mosaicism should be expected at least in all patients with recessive forms of epidermolysis bullosa. Naturally corrected, patient-own cells are of extreme interest for their promising therapeutic potential, and their presence in all patients would open exciting, new treatment perspectives to those patients. To test our hypothesis, we determined the probability that single nucleotide reversions occur in patients' skin using a mathematical developmental model. According to our model, reverse mutations are expected to occur frequently (estimated 216x) in each patient's skin. Reverse mutations should, however, occur early in embryogenesis to be able to drive the emergence of recognizable revertant patches, which is expected to occur in only one per ~10,000 patients. This underestimate, compared to our clinical observations, can be explained by the "late-but-fitter revertant cell" hypothesis: reverse mutations arise at later stages of development, but provide revertant cells with a selective growth advantage in vivo that drives the development of recognizable healthy skin patches. Our results can be extrapolated to any other organ with stem cell division numbers comparable to skin, which may offer novel future therapeutic options for other genetic conditions if these revertant cells can be identified and isolated.

Microarray amplification bias: loss of 30% differentially expressed genes due to long probe - poly(A)-tail distances.

BACKGROUND: Laser microdissection microscopy has become a rising tool to assess gene expression profiles of pure cell populations. Given the low yield of RNA, a second round of amplification is usually mandatory to yield sufficient amplified-RNA for microarray approaches. Since amplification induces truncation of RNA molecules, we studied the impact of a second round of amplification on identification of differentially expressed genes in relation to the probe - poly(A)-tail distances. RESULTS: Disagreement was observed between gene expression profiles acquired after a second round of amplification compared to a single round. Thirty percent of the differentially expressed genes identified after one round of amplification were not detected after two rounds. These inconsistent genes have a significant longer probe - poly(A)-tail distance. qRT-PCR on unamplified RNA confirmed differential expression of genes with a probe - poly(A)-tail distance >500 nucleotides appearing only after one round of amplification. CONCLUSION: Our data demonstrate a marked loss of 30% of truly differentially expressed genes after a second round of amplification. Therefore, we strongly recommend improvement of amplification procedures and importance of microarray probe design to allow detection of all differentially expressed genes in case of limited amounts of RNA.

Catechol-o-methyltransferase polymorphism and susceptibility to major depressive disorder modulates psychological stress response.

OBJECTIVES: The stress response is related to both physiological and psychological factors and is strongly marked by a neuroendocrine component. Genetic factors are believed to underlie individual differences in the degree of stress resilience and thereby contribute in determining susceptibility to stress-related pathologies like major depressive disorder (MDD). Little, however, is known about the genetic influence on the endocrine and behavioural stress response in relation to MDD. METHODS: Here, we sought to examine the effects of the catechol-o-methyltransferase polymorphism on psychological stress in three groups of individuals with different degrees of susceptibility to MDD (i.e. healthy controls, healthy high risk probands to MDD and those suffering from MDD). This genotype is involved in the metabolism of catecholamines (dopamine, norepinephrine and epinephrine). RESULTS: Allelic variations of this polymorphism were found to influence the degree of subjective stress experience and plasma epinephrine stress response. Interactions between catechol-o-methyltransferase polymorphism and diagnostic group in measures of plasma epinephrine, cortisol and subjective responses to psychological stress were also found, with the influence of the different alleles on these measures differing between healthy controls relative to MDD patients and high risk probands. CONCLUSION: These observations support a possible role for catechol-o-methyltransferase polymorphism in the endocrine and subjective response to psychological stress and thus may qualify as a possible candidate gene involved in the pathogenesis of MDD.

Lamin A/C-Related Cardiac Disease: Late Onset With a Variable and Mild Phenotype in a Large Cohort of Patients With the Lamin A/C p.(Arg331Gln) Founder Mutation.

BACKGROUND: Interpretation of missense variants can be especially difficult when the variant is also found in control populations. This is what we encountered for the LMNA c.992G>A (p.(Arg331Gln)) variant. Therefore, to evaluate the effect of this variant, we combined an evaluation of clinical data with functional experiments and morphological studies. METHODS AND RESULTS: Clinical data of 23 probands and 35 family members carrying this variant were retrospectively collected. A time-to-event analysis was performed to compare the course of the disease with carriers of other LMNA mutations. Myocardial biopsies were studied with electron microscopy and by measuring force development of the sarcomeres. Morphology of the nuclear envelope was assessed with immunofluorescence on cultured fibroblasts. The phenotype in probands and family members was characterized by atrioventricular conduction disturbances (61% and 44%, respectively), supraventricular arrhythmias (69% and 52%, respectively), and dilated cardiomyopathy (74% and 14%, respectively). LMNA p.(Arg331Gln) carriers had a significantly better outcome regarding the composite end point (malignant ventricular arrhythmias, end-stage heart failure, or death) compared with carriers of other pathogenic LMNA mutations. A shared haplotype of 1 Mb around LMNA suggested a common founder. The combined logarithm of the odds score was 3.46. Force development in membrane-permeabilized cardiomyocytes was reduced because of decreased myofibril density. Structural nuclear LMNA-associated envelope abnormalities, that is, blebs, were confirmed by electron microscopy and immunofluorescence microscopy. CONCLUSIONS: Clinical, morphological, functional, haplotype, and segregation data all indicate that LMNA p.(Arg331Gln) is a pathogenic founder mutation with a phenotype reminiscent of other LMNA mutations but with a more benign course.

A biological question and a balanced (orthogonal) design: the ingredients to efficiently analyze two-color microarrays with Confirmatory Factor Analysis.

BACKGROUND: Factor analysis (FA) has been widely applied in microarray studies as a data-reduction-tool without any a-priori assumption regarding associations between observed data and latent structure (Exploratory Factor Analysis).A disadvantage is that the representation of data in a reduced set of dimensions can be difficult to interpret, as biological contrasts do not necessarily coincide with single dimensions. However, FA can also be applied as an instrument to confirm what is expected on the basis of pre-established hypotheses (Confirmatory Factor Analysis, CFA). We show that with a hypothesis incorporated in a balanced (orthogonal) design, including 'SelfSelf' hybridizations, dye swaps and independent replications, FA can be used to identify the latent factors underlying the correlation structure among the observed two-color microarray data. An orthogonal design will reflect the principal components associated with each experimental factor. We applied CFA to a microarray study performed to investigate cisplatin resistance in four ovarian cancer cell lines, which only differ in their degree of cisplatin resistance. RESULTS: Two latent factors, coinciding with principal components, representing the differences in cisplatin resistance between the four ovarian cancer cell lines were easily identified. From these two factors 315 genes associated with cisplatin resistance were selected, 199 genes from the first factor (False Discovery Rate (FDR): 19%) and 152 (FDR: 24%) from the second factor, while both gene sets shared 36. The differential expression of 16 genes was validated with reverse transcription-polymerase chain reaction. CONCLUSION: Our results show that FA is an efficient method to analyze two-color microarray data provided that there is a pre-defined hypothesis reflected in an orthogonal design.