Positive predictive values and outcomes for uninformative cell-free DNA tests: An Italian multicentric Cytogenetic and cytogenomic Audit of diagnOstic testing (ICARO study).

OBJECTIVES: To establish the positive predictive values (PPV) of cfDNA testing based on data from a nationwide survey of independent clinical cytogenetics laboratories. METHODS: Prenatal diagnostic test results obtained by Italian laboratories between 2013 and March 2020 were compiled for women with positive non-invasive prenatal tests (NIPT), without an NIPT result, and cases where there was sex discordancy between the NIPT and ultrasound. PPV and other summary data were reviewed. RESULTS: Diagnostic test results were collected for 1327 women with a positive NIPT. The highest PPVs were for Trisomy (T) 21 (624/671, 93%) and XYY (26/27, 96.3%), while rare autosomal trisomies (9/47, 19.1%) and recurrent microdeletions (8/55, 14.5%) had the lowest PPVs. PPVs for T21, T18, and T13 were significantly higher when diagnostic confirmation was carried out on chorionic villi (97.5%) compared to amniotic fluid (89.5%) (p < 0.001). In 19/139 (13.9%), of no result cases, a cytogenetic abnormality was detected. Follow-up genetic testing provided explanations for 3/6 cases with a fetal sex discordancy between NIPT and ultrasound. CONCLUSIONS: NIPT PPVs differ across the conditions screened and the tissues studied in diagnostic testing. This variability, issues associated with fetal sex discordancy, and no results, illustrate the importance of pre- and post-test counselling.

Discovering chimeric transcripts in paired-end RNA-seq data by using EricScript.

MOTIVATION: The discovery of novel gene fusions can lead to a better comprehension of cancer progression and development. The emergence of deep sequencing of trancriptome, known as RNA-seq, has opened many opportunities for the identification of this class of genomic alterations, leading to the discovery of novel chimeric transcripts in melanomas, breast cancers and lymphomas. Nowadays, few computational approaches have been developed for the detection of chimeric transcripts. Although all of these computational methods show good sensitivity, much work remains to reduce the huge number of false-positive calls that arises from this analysis. RESULTS: We proposed a novel computational framework, named chimEric tranScript detection algorithm (EricScript), for the identification of gene fusion products in paired-end RNA-seq data. Our simulation study on synthetic data demonstrates that EricScript enables to achieve higher sensitivity and specificity than existing methods with noticeably lower running times. We also applied our method to publicly available RNA-seq tumour datasets, and we showed its capability in rediscovering known gene fusions.

Identification and characterization of novel ETV4 splice variants in prostate cancer.

ETV4, one of ETS proteins overexpressed in prostate cancer, promotes migration, invasion, and proliferation in prostate cells. This study identifies a series of previously unknown ETV4 alternatively spliced transcripts in human prostate cell lines. Their expression has been validated using several unbiased techniques, including Nanopore sequencing. Most of these transcripts originate from an in-frame exon skipping and, thus, are expected to be translated into ETV4 protein isoforms. Functional analysis of the most abundant among these isoforms shows that they still bear an activity, namely a reduced ability to promote proliferation and a residual ability to regulate the transcription of ETV4 target genes. Alternatively spliced genes are common in cancer cells: an analysis of the TCGA dataset confirms the abundance of these novel ETV4 transcripts in prostate tumors, in contrast to peritumoral tissues. Since none of their translated isoforms have acquired a higher oncogenic potential, such abundance is likely to reflect the tumor deranged splicing machinery. However, it is also possible that their interaction with the canonical variants may contribute to the biology and the clinics of prostate cancer. Further investigations are needed to elucidate the biological role of these ETV4 transcripts and of their putative isoforms.

Easy genotyping of complement C3 'slow' and 'fast' allotypes by tetra-primer amplification refractory mutation system PCR.

Complement C3 'slow' and 'fast' allotypes are associated with immune-mediated disorders and may affect the outcome of renal transplantation. We report a tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) that provides a rapid, reproducible and cost-effective method to genotype both complement C3 'slow' and 'fast' alleles by a single tube reaction.

Erratum to: Clinical and molecular characterization of Italian patients affected by Cohen syndrome.

The complete affiliations of two authors appeared incorrectly. The affiliations of A. Selicorni and D. Milani should be given as.

Italian Rett database and biobank.

Rett syndrome is the second most common cause of severe mental retardation in females, with an incidence of approximately 1 out of 10,000 live female births. In addition to the classic form, a number of Rett variants have been described. MECP2 gene mutations are responsible for about 90% of classic cases and for a lower percentage of variant cases. Recently, CDKL5 mutations have been identified in the early onset seizures variant and other atypical Rett patients. While the high percentage of MECP2 mutations in classic patients supports the hypothesis of a single disease gene, the low frequency of mutated variant cases suggests genetic heterogeneity. Since 1998, we have performed clinical evaluation and molecular analysis of a large number of Italian Rett patients. The Italian Rett Syndrome (RTT) database has been developed to share data and samples of our RTT collection with the scientific community (http://www.biobank.unisi.it). This is the first RTT database that has been connected with a biobank. It allows the user to immediately visualize the list of available RTT samples and, using the "Search by" tool, to rapidly select those with specific clinical and molecular features. By contacting bank curators, users can request the samples of interest for their studies. This database encourages collaboration projects with clinicians and researchers from around the world and provides important resources that will help to better define the pathogenic mechanisms underlying Rett syndrome.

Validation of a method for noninvasive prenatal testing for fetal aneuploidies risk and considerations for its introduction in the Public Health System.

OBJECTIVE: The aim of this study was to validate noninvasive prenatal testing (NIPT) for fetal aneuploidies by whole-genome massively parallel sequencing (MPS). METHODS: MPS was performed on cell-free DNA (cfDNA) isolated from maternal plasma in two groups: a first set of 186 euploid samples and a second set of 195 samples enriched of aneuploid cases (n = 69); digital PCR for fetal fraction (FF) assessment was performed on 178/381 samples. Cases with <10 × 106 reads (n = 54) were excluded for downstream data analysis. Follow-up data (invasive testing results or neonatal information) were available for all samples. Performances in terms of specificity/sensitivity and Z-score distributions were evaluated. RESULTS: All positive samples for trisomy 21 (T21) (n = 43), trisomy 18 (T18) (n = 6) and trisomy 13 (T13) (n = 7) were correctly identified (sensitivity: 99.9%); 5 false positive results were reported: 3 for T21 (specificity = 98.9%) and 2 for T13 (specificity = 99.4%). Besides FF, total cfDNA concentration seems another important parameter for MPS, since it influences the number of reads. CONCLUSIONS: The overall test accuracy allowed us introducing NIPT for T21, T18 and T13 as a clinical service for pregnant women after 10 + 4 weeks of gestation. Sex chromosome aneuploidy assessment needs further validation due to the limited number of aneuploid cases in this study.

Molecular Dissection Using Array Comparative Genomic Hybridization and Clinical Evaluation of An Infertile Male Carrier of An Unbalanced Y;21 Translocation: A Case Report and Review of The Literature.

Chromosomal defects are relatively frequent in infertile men however, translocations between the Y chromosome and autosomes are rare and less than 40 cases of Y-autosome translocation have been reported. In particular, only three individuals has been described with a Y;21 translocation, up to now. We report on an additional case of an infertile man in whom a Y;21 translocation was associated with the deletion of a large part of the Y chromosome long arm. Applying various techniques, including conventional cytogenetic procedures, fluorescence in situ hybridisation (FISH) analysis and array comparative genomic hybridization (array-CGH) studies, we identified a derivative chromosome originating from a fragment of the short arm of the chromosome Y translocated on the short arm of the 21 chromosome. The Y chromosome structural rearrangement resulted in the intactness of the entire short arm, including the sex-determining region Y (SRY) and the short stature homeobox (SHOX) loci, although translocated on the 21 chromosome, and the loss of a large part of the long arm of the Y chromosome, including azoospermia factor-a (AZFa), AZFb, AZFc and Yq heterochromatin regions. This is the first case in which a (Yp;21p) translocation has been ascertained using an array-CGH approach, thus reporting details of such a rearrangement at higher resolution.

Thin and thick primary cutaneous melanomas reveal distinct patterns of somatic copy number alterations.

Cutaneous melanoma is one of the most aggressive type of skin tumor. Early stage melanoma can be often cured by surgery; therefore current management guidelines dictate a different approach for thin (<1mm) versus thick (>4mm) melanomas. We have carried out whole-exome sequencing in 5 thin and 5 thick fresh-frozen primary cutaneous melanomas. Unsupervised hierarchical clustering analysis of somatic copy number alterations (SCNAs) identified two groups corresponding to thin and thick melanomas. The most striking difference between them was the much greater abundance of SCNAs in thick melanomas, whereas mutation frequency did not significantly change between the two groups. We found novel mutations and focal SCNAs in genes that are embryonic regulators of axon guidance, predominantly in thick melanomas. Analysis of publicly available microarray datasets provided further support for a potential role of Ephrin receptors in melanoma progression. In addition, we have identified a set of SCNAs, including amplification of BRAF and ofthe epigenetic modifier EZH2, that are specific for the group of thick melanomas that developed metastasis during the follow-up. Our data suggest that mutations occur early during melanoma development, whereas SCNAs might be involved in melanoma progression.

Chromosomal alterations detected by comparative genomic hybridization in nonfunctioning endocrine pancreatic tumors.

We have used comparative genomic hybridization to investigate changes in DNA copy number in 20 nonfunctioning endocrine pancreatic tumors. The total number of regional DNA imbalances per tumor showed variation from case to case and high genetic heterogeneity was observed. From 1 to 22 chromosomal anomalies were detected in 13 of the 20 cases evaluated. Overall gains predominated over losses with a ratio of about 3.9:1 (58 gains/15 losses). The mean total number of regions displaying imbalances increased from 1.25 per tumor for benign tumors to 5.25 for malignant tumors, although statistical significance was not reached (P=0.074).

Rett syndrome: the complex nature of a monogenic disease.

Rett syndrome (RTT) is a severe neurodevelopmental disorder affecting almost exclusively girls. It is currently considered a monogenic X-linked dominant disorder due to mutations in MECP2 gene, encoding the methyl-CpG binding protein 2. A few RTT male cases, resulting from mosaicism for MECP2 mutations, have been reported. Male germline MECP2 mutations cause either severe encephalopathy with death at birth (usually in brothers of classical RTT females) or X-linked recessive mental retardation (XLMR). To date the wide phenotypic heterogeneity associated with MECP2 mutations in females (from classical RTT to healthy carriers) has been explained by differences in X chromosome inactivation. However, conflicting results have been obtained in different studies, with both random and highly skewed X-inactivation reported in healthy carrier females. Consequently it is possible that mechanisms other than X-inactivation play a role in the expressivity of MECP2 mutations. To explain the phenotypic heterogeneity associated with MECP2 mutations we propose a digenic model in which the presence of a "mutated" allele in a second gene, leading to a less functional protein, determines the clinical severity of the MECP2 mutation. The model is supported by the identification of the same mutation in XLMR and RTT cases. The carrier mothers of XLMR families are clinically asymptomatic and present balanced X chromosome inactivation. Therefore the same mutation arising in different genetic backgrounds can cause XLMR in males, remain silent in the carrier females and cause classic RTT in females. MECP2 mutations account for approximately 70-80% of classic RTT cases. MECP2 negative cases might result from mutations in noncoding regions of MECP2 gene. Alternatively, these cases might be due to mutations in other genes (locus heterogeneity). This hypothesis is supported by the identification of several chromosomal rearrangements in MECP2 negative patients with RTT and RTT-like phenotypes. MeCP2 is considered a general transcriptional repressor. However, conditional mouse mutants with selective loss of Mecp2 in the brain develop clinical manifestations similar to RTT, indicating that MECP2 is exclusively required for central nervous system function. The involvement of MeCP2 in methylation-specific transcriptional repression suggests that MECP2 related disorders result from dysregulated gene expression. Studies on gene expression have been performed in mouse and human brains. A relatively small number of gene expression changes were identified. It is possible that MeCP2 causes dysregulation of a very small subset of genes that are not detected with this method of analysis, or that very subtle changes in many genes cause the neuronal phenotype.

Real-time quantitative PCR as a routine method for screening large rearrangements in Rett syndrome: Report of one case of MECP2 deletion and one case of MECP2 duplication.

Mutations in the X-linked methyl-CpG-binding protein 2 gene (MECP2) are found in 70-80% of cases of classical Rett syndrome (RTT) and in about 50% of cases of preserved speech variant (PSV). This high percentage of MECP2 mutations, especially in classical RTT cases, suggests that another major RTT locus is unlikely. Missed mutations may be due to the limited sensitivity of the methodology used for mutation scanning and/or the presence of intronic mutations. In a double-copy gene, such as MECP2 in females, current methodologies (e.g., DGGE, SSCP, DHPLC, direct sequencing) are prone to miss gross rearrangements. Three previous reports during 2001-2003 have shown the presence of large deletions in a fraction of MECP2-negative classical RTT patients. We developed a reliable, single tube, quantitative PCR assay for rapid determination of MECP2 gene dosage. This method involves a multiplex reaction using a FAM labeled TaqMan probe with a TAMRA quencher derived from MECP2 exon 4 and two primers derived from the same exon and RNAaseP as an internal reference. The copy number of the MECP2 gene was determined by the comparative threshold cycle method (ddCt). Each sample was run in quadruplicate. We validated this assay through the analysis of 30 healthy controls (15 female and 15 male) and we then applied this method to eight classical RTT and six PSV patients, all negative for MECP2 mutations. We identified gross rearrangements in two patients: a deletion in a classical RTT patient and a duplication in a PSV patient. Our results confirm that a fraction of MECP2-negative RTT cases have MECP2 gross rearrangements and we propose real-time quantitative PCR as a simple and reliable method for routine screening of MECP2 in addition to DHPLC analysis.

Three Rett patients with both MECP2 mutation and 15q11-13 rearrangements.

Autism and Rett syndrome, a severe neurological disorder with autistic behavior, are classified as separate disorders on clinical and etiological ground. Rett syndrome is a monogenic X-linked dominant condition due to de novo mutations in the MECP2 gene, whereas autism is a neurodevelopmental and behavioral disorder with complex genetic basis. Maternally inherited duplications on 15q11-q13 are found in a fraction of autistic children suggesting that an abnormal dosage of gene(s) within this region might cause susceptibility to autism. Now we show that three Rett patients are carriers of both a MECP2 mutation and a 15q11-q13 rearrangement, suggesting that there might be a relationship between autism-related genes and the MECP2 gene.

Type-IV collagen related diseases.

Alport syndrome (ATS) is a progressive inherited glomerulonephritis accounting for 1-2% of all patients who start renal replacement therapy, with an estimated gene frequency of approximately 1 in 5000. ATS is a genetically heterogeneous disease, commonly inherited as an X-linked semi-dominant trait, caused by mutations in COL4A5, on the X-chromosome, and only rarely (less than 10% of cases) caused by the COL4A3 or the COL4A4 gene on chromosome 2q. In the X-linked form females are generally less affected than males, microhematuria being the only sign present throughout life, although approximately 30% can progress to end-stage renal disease. It became evident in recent years that mutations in the COL4A3 or the COL4A4 gene can give rise not only to autosomal recessive ATS syndrome, in which males and females are severely affected, but also to an autosomal dominant form, where the clinical progression towards impaired renal function can be very slow and also to benign familial hematuria (BFH) in which renal function is preserved.

16p11.2 de novo microdeletion encompassing SRCAP gene in a patient with speech impairment, global developmental delay and behavioural problems.

We describe a patient with speech impairment, global developmental delay, behavioural problems and a 186 kb de novo microdeletion on 16p11.2. There are four OMIM Phenotypes entries partially overlapping with the deleted region and related to recurrent microdeletions/microduplications in 16p11.2. A detailed review of published data shows that microdeletions/microduplications' boundaries do not include genes that are deleted in the case here reported. The deletion encompasses 9 RefSeq genes and includes SRCAP (Snf2-related CREBBP activator protein, OMIM*611421), a disease causing gene. Recently, truncating mutations in the SRCAP gene have been shown to cause Floating-Harbor syndrome (FHS, OMIM#136140), a rare disorder characterized by peculiar facial features, short stature with delayed osseous maturation and speech impairment. The patient reported here shows few subtle phenotypic features resembling that of FHS, but she does not have sufficient signs and symptoms for the clinical diagnosis and a clinical classification based on facial gestalt is not possible. This is the first report of a 16p11.2 deletion completely removing one copy of SRCAP, suggesting that haploinsufficiency of this gene could be associated to speech impairment, global developmental delay, behavioural problems and few subtle phenotypic features resembling FHS. However, further evidence for the putative causative role of SRCAP isolated deletion is needed.

Novel α-actinin 2 variant associated with familial hypertrophic cardiomyopathy and juvenile atrial arrhythmias: a massively parallel sequencing study.

BACKGROUND: Next-generation sequencing might be particularly advantageous in genetically heterogeneous conditions, such as hypertrophic cardiomyopathy (HCM), in which a considerable proportion of patients remain undiagnosed after Sanger. In this study, we present an Italian family with atypical HCM in which a novel disease-causing variant in α-actinin 2 (ACTN2) was identified by next-generation sequencing. METHODS AND RESULTS: A large family spanning 4 generations was examined, exhibiting an autosomal dominant cardiomyopathic trait comprising a variable spectrum of (1) midapical HCM with restrictive evolution with marked biatrial dilatation, (2) early-onset atrial fibrillation and atrioventricular block, and (3) left ventricular noncompaction. In the proband, 48 disease genes for HCM, selected on the basis of published reports, were analyzed by targeted resequencing with a customized enrichment system. After bioinformatics analysis, 4 likely pathogenic variants were identified: TTN c.21977G>A (p.Arg7326Gln); TTN c.8749A>C (p.Thr2917Pro); ACTN2 c.683T>C (p.Met228Thr); and OBSCN c.13475T>G (p.Leu4492Arg). The novel variant ACTN2 c.683T>C (p.Met228Thr), located in the actin-binding domain, proved to be the only mutation fully cosegregating with the cardiomyopathic trait in 18 additional family members (of whom 11 clinically affected). ACTN2 c.683T>C (p.Met228Thr) was absent in 570 alleles of healthy controls and in 1000 Genomes Project and was labeled as Damaging by in silico analysis using polymorphism phenotyping v2, as Deleterious by sorts intolerant from tolerant, and as Disease-Causing by Mutation Taster. CONCLUSIONS: A targeted next-generation sequencing approach allowed the identification of a novel ACTN2 variant associated with midapical HCM and juvenile onset of atrial fibrillation, emphasizing the potential of such approach in HCM diagnostic screening.

372 kb microdeletion in 18q12.3 causing SETBP1 haploinsufficiency associated with mild mental retardation and expressive speech impairment.

Several cases of interstitial deletion encompassing band 18q12.3 are described in patients with mild dysmorphic features, mental retardation and impairment of expressive language. The critical deleted region contains SETBP1 gene (SET binding protein 1). Missense heterozygous mutations in this gene cause Schinzel-Giedion syndrome (SGS, MIM#269150), characterized by profound mental retardation and multiple congenital malformations. Recently, a 18q12.3 microdeletion causing SETBP1 haploinsufficiency has been described in two patients that show expressive speech impairment, moderate developmental delay and peculiar facial features. The phenotype of individual with partial chromosome 18q deletions does not resemble SGS. The deletion defines a critical region in which SETBP1 is the major candidate gene for expressive speech defect. We describe an additional patient with the smallest 18q12.3 microdeletion never reported that causes the disruption of SETBP1. The patient shows mild mental retardation and expressive speech impairment with striking discrepancy between expressive and receptive language skills. He is able to communicate using gestures and mimic expression of face and body with surprising efficacy. The significant phenotypic overlap between this patient and the cases previously reported enforce the hypothesis that SETBP1 haploinsufficiency may have a role in expressive language development.

Association between primary open-angle glaucoma (POAG) and WDR36 sequence variance in Italian families affected by POAG.

BACKGROUND/AIMS: To assess the involvement of WDR36 sequence variance in primary open-angle glaucoma (POAG) in Italian patients. METHODS: A cohort of 34 Italian families affected by POAG was analysed by denaturing high-performance liquid chromatography for mutation in the WDR36 gene. Among the 34 families enrolled, 25 were affected by high-tension glaucoma (HTG), four by juvenile open-angle glaucoma and one by normal tension glaucoma. In addition, four families presented both juvenile open-angle glaucoma and HTG-POAG patients within the same pedigree. RESULTS: Four previously identified intronic polymorphisms (IVS5+30C→T; IVS12+90 G→T; IVS13+89G→A; IVS16-30A→G) and a novel one (IVS21-75G→A) have been identified. In addition, one proband was found to carry the p.D658G mutation reported as the more recurrent disease-causing allele. CONCLUSIONS: The findings suggest that WDR36 sequence variance is only a rare cause of glaucoma in Italian families. Clearly, investigation of additional families with extensive studies is needed to clarify the role of WDR36 in the pathophysiology of glaucoma.