Genetic testing for Hennekam syndrome

Abstract Hennekam Syndrome (HS) is a combination of congenital lymphatic malformation, lymphangiectasia and other disorders. It is a very rare disorder with autosomal recessive inheritance. We developed the test protocol "Hennekam Syndrome" on the basis of the latest research findings and diagnostic protocols on lymphatic malformation in HS. The genetic test is useful for confirming diagnosis, as well as for differential diagnosis, couple risk assessment and access to clinical trials

Genetic testing for bicuspid aortic valve

Abstract Bicuspid aortic valve (BAV) is a congenital defect in which the aortic valve has two rather than three leaflets. In many patients valve function may be normal but valve decompensation may occur due to other associated congenital abnormalities and secondary valve and aortic complications. Decompensation manifests as stenosis or regurgitation and thoracic aortic aneurysm and dissection. Cystic medial necrosis plays an important role in the pathogenesis of BAV. Prevalence of BAV is estimated at 0.5-2.0%. In children, 70-85% of stenotic aortic valves are bicuspid, compared to at least 50% in adults. BAV has autosomal dominant inheritance. This Utility Gene Test was developed on the basis of an analysis of the literature and existing diagnostic protocols. It is useful for confirming diagnosis, as well as for differential diagnosis, couple risk assessment and access to clinical trials

Genetic testing for Ebstein anomaly

Abstract Ebstein anomaly (EA) is a rare congenital tricuspid valve malformation, characterized by downward displacement of the septal leaflet and an atrialized right ventricle. About 80% of cases of EA are non-syndromic; in the other 20%, the anomaly is associated with a chromosomal or Mendelian syndrome. The prevalence of EA is estimated at about 1 per 20,000 live births, and accounts for less than 1% of all congenital heart defects. EA has autosomal dominant inheritance. Likely causative genes are: NKX2-5, MYH7 and TPM1. This Utility Gene Test was developed on the basis of an analysis of the literature and existing diagnostic protocols. It is useful for confirming diagnosis, as well as for differential diagnosis, potential risk assessment and access to clinical trials

Genetic testing for vascular Ehlers-Danlos syndrome and other variants with fragility of the middle arteries

Abstract Ehlers-Danlos syndrome (EDS) is an umbrella term for various inherited connective tissue disorders associated with mutations in genes involved in extracellular matrix formation. "The 2017 International Classification of Ehlers-Danlos Syndromes and related disorders" identifies 13 clinical types with mutations in 19 distinct genes. The present module focuses on forms with major vascular involvement: vascular EDS (vEDS) caused by heterozygous mutations in COL3A1, "vascular-like" EDS (vlEDS) caused by recurrent mutations in COL1A1, classical EDS with vascular fragility associated with heterozygous mutations in COL5A1, and kyphoscoliotic EDS associated with recessive variations in PLOD1 and FKBP14. The overall prevalence of EDS is estimated between 1/10,000 and 1/25,000 and vEDS accounts for about 5 to 10% of all EDS cases. This Utility Gene Test was prepared on the basis of an analysis of the literature and existing diagnostic protocols. Molecular testing is useful for diagnosis confirmation, as well as differential diagnosis, appropriate genetic counselling and access to clinical trials

Polymorphisms of the SCN1A gene in children and adolescents with primary headache and idiopathic or cryptogenic epilepsy: is there a linkage?

The purpose of this study was to evaluate the distribution of the polymorphisms of the SCN1A gene in a series of children and adolescents with primary headache and idiopathic or cryptogenic epilepsy compared to controls. Five non-synonymous exonic polymorphisms (1748A > T, 2656T > C, 3199A > G, 5771G > A, 5864T > C) of the SCN1A gene were selected and their genotyping was performed, by high resolution melting (HRM), in 49 cases and 100 controls. We found that among the five polymorphisms, only 3199A > G was a true polymorphism. We did not find a statistically significant difference between distribution of 3199A > G genotypes between cases and controls. We excluded the role of the SCN1A gene in the pathogenesis of comorbidity between headache (especially migraine) and epilepsy. The SCN1A gene is a major gene in different epilepsies and epilepsy syndromes; the HRM could be the new methodology, more rapid and efficacious, for molecular analysis of the SCN1A gene

Genotype-phenotype characterization of novel variants in six Italian patients with familial exudative vitreoretinopathy

Familial exudative vitreoretinopathy (FEVR) is a complex disorder characterized by incomplete development of the retinal vasculature. Here, we report the results obtained on the spectrum of genetic variations and correlated phenotypes found in a cohort of Italian FEVR patients. Eight probands (age range 7-19 years) were assessed by genetic analysis and comprehensive age-appropriate ophthalmic examination. Genetic testing investigated the genes most widely associated in literature with FEVR: FZD4, LRP5, TSPAN12, and NDP. Clinical and genetic evaluations were extended to relatives of probands positive to genetic testing. Six out of eight probands (75%) showed a genetic variation probably related to the phenotype. We identified four novel genetic variants, one variant already described in association with Norrie disease and one previously described linked to autosomal dominant FEVR. Pedigree analysis of patients led to the classification of four autosomal dominant cases of FEVR (caused by FZD4 and TSPAN12 variants) and two X-linked FEVR probands (NDP variants). None of the patients showed variants in the LRP5 gene. This study represents the largest cohort study in Italian FEVR patients. Our findings are in agreement with the previous literature confirming that FEVR is a clinically and genetically heterogeneous retinal disorder, even when it manifests in the same family

A genome-wide association study of anorexia nervosa

Anorexia nervosa (AN) is a complex and heritable eating disorder characterized by dangerously low body weight. Neither candidate gene studies nor an initial genome wide association study (GWAS) have yielded significant and replicated results. We performed a GWAS in 2,907 cases with AN from 14 countries (15 sites) and 14,860 ancestrally matched controls as part of the Genetic Consortium for AN (GCAN) and the Wellcome Trust Case Control Consortium 3 (WTCCC3). Individual association analyses were conducted in each stratum and meta-analyzed across all 15 discovery datasets. Seventy-six (72 independent) SNPs were taken forward for in silico (two datasets) or de novo (13 datasets) replication genotyping in 2,677 independent AN cases and 8,629 European ancestry controls along with 458 AN cases and 421 controls from Japan. The final global meta-analysis across discovery and replication datasets comprised 5,551 AN cases and 21,080 controls. AN subtype analyses (1,606 AN restricting; 1,445 AN binge-purge) were performed. No findings reached genome-wide significance. Two intronic variants were suggestively associated: rs9839776 (P=3.01×10−7) in SOX2OT and rs17030795 (P=5.84×10−6) in PPP3CA. Two additional signals were specific to Europeans: rs1523921 (P=5.76×10−6) between CUL3 and FAM124B and rs1886797 (P=8.05×10−6) near SPATA13. Comparing discovery to replication results, 76% of the effects were in the same direction, an observation highly unlikely to be due to chance (P= 4×10−6), strongly suggesting that true findings exist but that our sample, the largest yet reported, was underpowered for their detection. The accrual of large genotyped AN case-control samples should be an immediate priority for the field

A genome-wide association study of anorexia nervosa suggests a risk locus implicated in dysregulated leptin signaling

J. Kaprio, A. Palotie, A. Raevuori-Helkamaa ja S. Ripatti ovat työryhmän Eating Disorders Working Group of the Psychiatric Genomics Consortium jäseniä. Erratum in: Sci Rep. 2017 Aug 21;7(1):8379, doi: 10.1038/s41598-017-06409-3We conducted a genome-wide association study (GWAS) of anorexia nervosa (AN) using a stringently defined phenotype. Analysis of phenotypic variability led to the identification of a specific genetic risk factor that approached genome-wide significance (rs929626 in EBF1 (Early B-Cell Factor 1); P = 2.04 x 10(-7); OR = 0.7; 95% confidence interval (CI) = 0.61-0.8) with independent replication (P = 0.04), suggesting a variant-mediated dysregulation of leptin signaling may play a role in AN. Multiple SNPs in LD with the variant support the nominal association. This demonstrates that although the clinical and etiologic heterogeneity of AN is universally recognized, further careful sub-typing of cases may provide more precise genomic signals. In this study, through a refinement of the phenotype spectrum of AN, we present a replicable GWAS signal that is nominally associated with AN, highlighting a potentially important candidate locus for further investigation.Peer reviewe

Sviluppo di metodi per l'identificazione e l'analisi di sequenze geniche fetali nella circolazione materna per una diagnosi prenatale non invasiva

During last decade, molecular biology evolution has lead to a deep knowledge of human genome allowing the in-utero diagnosis of a large number of genetic diseases. Now adays performing a prenatal diagnosis of chromosomal abnormalities and of genetic diseases, requires genetic material from the fetus and this is possible only by invasive procedures such as amniocentesis and chorionic villus sampling. The risk of miscarriage of these technics is between 0.5% and 1% and considering these limits, new alternative and non-invasive screening methods, based on ultrasound and/or biochemical parameters of maternal blood, were developed. The disavantages of these methods are the probabilistic aspect of the results. There was an increasing interest in a new approach that could keep to zero the risk of miscarriage, with an extension of fetal genetic materials screening to a large number of women at the beginning of pregnancy, leading a decreased physic and psychological impact. In 1997 it was demonstrated the presence of cell-free fetal DNA (cffDNA) in the maternal blood and this has opened up new possibilities for a non-invasive prenatal diagnosis (NIPD). The cell-free fetal DNA originates from apoptotic trophoblasts on placenta’s surface and comprises around 3-6% of total cell-free DNA in maternal circulation. The cffDNA consists of short DNA fragments (<200bp). Fetal DNA can be detected from 32 days of gestation (though only reliably from 7 weeks) and it’s concentration increases with gestational age (21% per week during the first three months) with a sharp peak during last 8 weeks of pregnancy. The cffDNA is completely removed after delivery. The main problem related to the application of this NIPD is the high maternal background, so researchers have initially focused their attention on the detection of sequences that are absent in the maternal genome, such as DNA sequences from chromosome Y of male fetuses, the determination of fetal RhD genotype in women RhD-negative, and the detection of paternal mutations in maternal plasma. Although, the Real Time qPCR amplification of fetal sequences is the widespread method for cffDNA study, there are still a lot of studies focused on the development different protocols for detection of minimal differences between maternal and fetal DNA. The aim of this PhD thesis is the study and setup of different methods to identify fetal genomic sequences in maternal circulation. The research project was divided in three steps: the first one was focused on the validation of Real-Time qPCR protocol, applied to the detection and quantification of total free DNA and of cffDNA in pregnancies with a male fetus; the second was focused on the develop of a reliable protocol for the identification of fetal genomic sequences useful to recognize cffDNA in pregnancies with a female fetus, by the mini-sequencing technics; finally, the last step was focused on the validation of an analytical protocol to verify the presence/absence of paternal mutations in maternal plasma using nested PCR allele-specific. The results demonstrated here for each technic, show a high sensibility and resolution capacity, together with an easy and rapid handling. Furthermore, the combination of the two technics (Real-Time qPCR and mini-sequencing reaction) was essential in order to exclude the presence of a paternal mutation in the fetal genome in every case where a mutation wasn’t identified. The ability to exclude the presence of the paternal mutation in the fetus by analysis of maternal plasma avoid the utilization of invasive prenatal tests and, at the same time, reassures the couple early in the course of the pregnancy about the recurrence of the disease, thus avoiding significant anxiety.Negli ultimi anni l'evoluzione della biologia molecolare ha permesso una conoscenza sempre maggiore del genoma umano e consente ormai la diagnosi in utero di un numero crescente di patologie genetiche. Al momento attuale la diagnostica prenatale di anomalie cromosomiche o di malattie genetiche richiede il prelievo di cellule fetali ottenibili con metodiche invasive, l'amniocentesi e la villocentesi. Queste tecniche comportano un rischio di provocare un aborto compreso fra 0.5% e 1%. Di fronte a questi limiti delle metodiche invasive sono stati messi a punto alcuni metodi di screening alternativi non invasivi, basati su parametri ecografici fetali e/o biochimici su sangue materno, che però hanno il limite di fornire risposte solo di tipo probabilistico. L'introduzione di un test realmente alternativo di diagnosi prenatale non invasiva che permetta di eliminare il rischio di aborto legato all’amniocentesi e alla villocentesi è fortemente attesa. Infatti un simile test permetterebbe una estensione dell'analisi del materiale genetico fetale a molte più donne all'inizio della gravidanza e diminuirebbe l'impatto fisico e psicologico di questo tipo di indagine. Nel 1997 uno studio ha dimostrato la presenza di DNA libero fetale (cell-free fetal DNA, cffDNA) nel plasma materno e ha evidenziato la sua potenzialità per una diagnosi prenatale non invasiva (NIPD). Durante i 15 anni successivi, si è sviluppata la ricerca di base sulle caratteristiche del DNA libero fetale e molti ricercatori hanno studiato la biologia e il significato clinico del trasporto di cffDNA nel circolo materno. Il DNA libero fetale origina dall’apoptosi dei trofoblasti costantemente rimpiazzati sulla superficie della placenta e comprende circa il 3-6% del DNA libero totale nella circolazione materna. E’ stato inoltre osservato che è costituito da corti frammenti di DNA (<200bp) piuttosto che interi cromosomi e che la sua concentrazione aumenta gradualmente durante la gravidanza: è rilevabile nella circolazione materna già a 32 giorni circa di gestazione (in quantità affidabile solo dopo le 7 settimane) e aumenta del 21% ogni settimana nel primo trimestre, con un forte picco durante le ultime 8 settimane di gravidanza per poi venire rapidamente rimosso dalla circolazione materna subito dopo il parto. L’utilizzo del DNA fetale libero come mezzo per la diagnosi prenatale non invasiva di malattie genetiche fino ad ora è fortemente limitato dall’elevato background di DNA materno pertanto le prime strategie messe a punto per l’identificazione del DNA libero fetale si sono basate sul rilevamento di sequenze assenti nel genoma materno, come le sequenze Y-specifiche, la determinazione del genotipo RhD fetale in donne RhD-negative e l’identificazione di sequenze fetali con mutazioni ereditate dal padre. L’amplificazione tramite Real Time qPCR di sequenze unicamente fetali nel plasma di donne gravide rappresenta ad oggi il metodo di elezione per lo studio del DNA libero fetale, ma numerosi sforzi sono stati realizzati per la messa a punto di sistemi sempre più sensibili ed universali per rilevare le più piccole differenze tra il DNA fetale libero e quello materno, al fine di consentire un utilizzo su larga scala del cffDNA per la diagnosi prenatale non invasiva. Lo scopo di questa ricerca è stato quindi, la messa a punto di protocolli validi per l’identificazione e l’analisi di sequenze geniche fetali nella circolazione materna. Il progetto è stato quindi suddiviso in tre fasi: la prima fase ha comportato la messa a punto di opportuni protocolli di Real-Time qPCR per il rilevamento e la quantificazione sia del DNA libero totale estratto sia del cffDNA estratto in gravidanze con feto di sesso maschile; la seconda fase del progetto si è occupata di testare e sviluppare un protocollo affidabile per l’identificazione di sequenze geniche fetali utili al riconoscimento di cffDNA nelle gravidanze con feto di sesso femminile, mediante l’utilizzo della tecnica di mini-sequencing; l’ultima fase del progetto si è concentrata infine sulla messa a punto di un protocollo di verifica della presenza/assenza di mutazioni paterne in plasma materno mediante nested PCR allele-specifica. L’analisi dei risultati ha dimostrato, per ogni tecnica sviluppata, elevate sensibilità e capacità di risoluzione accoppiate a velocità e facilità di esecuzione, confermandone quindi l’applicabilità per una diagnosi prenatale non invasiva. Inoltre l’utilizzo in combinazione delle due tecniche (Real-Time qPCR e mini-sequencing) per la conferma della presenza del cffDNA nel campione in analisi, si è rilevata indispensabile per poter escludere che il feto abbia ereditato la mutazione paterna, in tutti i casi in cui non viene identificata la mutazione. L’esclusione della mutazione paterna nel feto attraverso l’analisi su plasma materno permetterebbe quindi di evitare l’utilizzo di procedure invasive per il prelievo di cellule fetali e avere questa informazione alla 9-10 settimana di gravidanza consentirebbe di rassicurare la coppia prima di qualsiasi altro test diagnostico di routine

Genetic testing for Mendelian stroke due to cerebrovascular anomalies and other syndromes

Stroke is defined as a focal or at times global neurological impairment of sudden onset and presumed vascular origin. 85% of strokes are due to cerebral ischemia and the other 15% to primary intracerebral hemorrhage