Long read sequencing on its way to the routine diagnostics of genetic diseases.

The clinical application of technological progress in the identification of DNA alterations has always led to improvements of diagnostic yields in genetic medicine. At chromosome side, from cytogenetic techniques evaluating number and gross structural defects to genomic microarrays detecting cryptic copy number variants, and at molecular level, from Sanger method studying the nucleotide sequence of single genes to the high-throughput next-generation sequencing (NGS) technologies, resolution and sensitivity progressively increased expanding considerably the range of detectable DNA anomalies and alongside of Mendelian disorders with known genetic causes. However, particular genomic regions (i.e., repetitive and GC-rich sequences) are inefficiently analyzed by standard genetic tests, still relying on laborious, time-consuming and low-sensitive approaches (i.e., southern-blot for repeat expansion or long-PCR for genes with highly homologous pseudogenes), accounting for at least part of the patients with undiagnosed genetic disorders. Third generation sequencing, generating long reads with improved mappability, is more suitable for the detection of structural alterations and defects in hardly accessible genomic regions. Although recently implemented and not yet clinically available, long read sequencing (LRS) technologies have already shown their potential in genetic medicine research that might greatly impact on diagnostic yield and reporting times, through their translation to clinical settings. The main investigated LRS application concerns the identification of structural variants and repeat expansions, probably because techniques for their detection have not evolved as rapidly as those dedicated to single nucleotide variants (SNV) identification: gold standard analyses are karyotyping and microarrays for balanced and unbalanced chromosome rearrangements, respectively, and southern blot and repeat-primed PCR for the amplification and sizing of expanded alleles, impaired by limited resolution and sensitivity that have not been significantly improved by the advent of NGS. Nevertheless, more recently, with the increased accuracy provided by the latest product releases, LRS has been tested also for SNV detection, especially in genes with highly homologous pseudogenes and for haplotype reconstruction to assess the parental origin of alleles with de novo pathogenic variants. We provide a review of relevant recent scientific papers exploring LRS potential in the diagnosis of genetic diseases and its potential future applications in routine genetic testing.

Characterization of BRCA Deficiency in Ovarian Cancer.

BRCA testing is recommended in all Ovarian Cancer (OC) patients, but the optimal approach is debated. The landscape of BRCA alterations was explored in 30 consecutive OC patients: 6 (20.0%) carried germline pathogenic variants, 1 (3.3%) a somatic mutation of BRCA2, 2 (6.7%) unclassified germline variants in BRCA1, and 5 (16.7%) hypermethylation of the BRCA1 promoter. Overall, 12 patients (40.0%) showed BRCA deficit (BD), due to inactivation of both alleles of either BRCA1 or BRCA2, while 18 (60.0%) had undetected/unclear BRCA deficit (BU). Regarding sequence changes, analysis performed on Formalin-Fixed-Paraffin-Embedded tissue through a validated diagnostic protocol showed 100% accuracy, compared with 96.3% for Snap-Frozen tissue and 77.8% for the pre-diagnostic Formalin-Fixed-Paraffin-Embedded protocol. BD tumors, compared to BU, showed a significantly higher rate of small genomic rearrangements. After a median follow-up of 60.3 months, the mean PFS was 54.9 ± 27.2 months in BD patients and 34.6 ± 26.7 months in BU patients (p = 0.055). The analysis of other cancer genes in BU patients identified a carrier of a pathogenic germline variant in RAD51C. Thus, BRCA sequencing alone may miss tumors potentially responsive to specific treatments (due to BRCA1 promoter methylation or mutations in other genes) while unvalidated FFPE approaches may yield false-positive results.

Exome sequencing in 116 patients with inherited thrombocytopenia that remained of unknown origin after systematic phenotype-driven diagnostic workup.

Inherited thrombocytopenias (IT) are genetic diseases characterized by low platelet count, sometimes associated with congenital defects or a predisposition to develop additional conditions. Next-generation sequencing has substantially improved our knowledge of IT, with more than 40 genes identified so far, but obtaining a molecular diagnosis remains a challenge especially for patients with non-syndromic forms, having no clinical or functional phenotypes that raise suspicion about specific genes. We performed exome sequencing (ES) in a cohort of 116 IT patients (89 families), still undiagnosed after a previously validated phenotype-driven diagnostic algorithm including a targeted analysis of suspected genes. ES achieved a diagnostic yield of 36%, with a gain of 16% over the diagnostic algorithm. This can be explained by genetic heterogeneity and unspecific genotype-phenotype relationships that make the simultaneous analysis of all the genes, enabled by ES, the most reasonable strategy. Furthermore, ES disentangled situations that had been puzzling because of atypical inheritance, sex-related effects or false negative laboratory results. Finally, ES-based copy number variant analysis disclosed an unexpectedly high prevalence of RUNX1 deletions, predisposing to hematologic malignancies. Our findings demonstrate that ES, including copy number variant analysis, can substantially contribute to the diagnosis of IT and can solve diagnostic problems that would otherwise remain a challenge.

Partial trisomy 21 with or without highly restricted Down syndrome critical region (HR-DSCR): report of two new cases and reanalysis of the genotype-phenotype association.

BACKGROUND: Down syndrome (DS) is caused by the presence of an extra copy of full or partial human chromosome 21 (Hsa21). Partial (segmental) trisomy 21 (PT21) is the duplication of only a delimited region of Hsa21 and can be associated or not to DS: the study of PT21 cases is an invaluable model for addressing genotype-phenotype correlation in DS. Previous works reported systematic reanalyses of 132 subjects with PT21 and allowed the identification of a 34-kb highly restricted DS critical region (HR-DSCR) as the minimal region whose duplication is shared by all PT21 subjects diagnosed with DS. METHODS: We report clinical data and cytogenetic analysis of two children with PT21, one with DS and the other without DS. Moreover, we performed a systematic bibliographic search for any new PT21 report. RESULTS: Clinical and cytogenetic analyses of the two PT21 children have been reported: in Case 1 the duplication involves the whole long arm of Hsa21, except for the last 2.7 Mb, which are deleted as a consequence of an isodicentric 21: the HR-DSCR is within the duplicated regions and the child is diagnosed with DS. In Case 2 the duplication involves 7.1 Mb of distal 21q22, with a deletion of 2.1 Mb of proximal 20p, as a consequence of an unbalanced translocation: the HR-DSCR is not duplicated and the child presents with psychomotor development delay but no clinical signs of DS. Furthermore, two PT21 reports recently published (named Case 3 and 4) have been discussed: Case 3 has DS diagnosis, nearly full trisomy for Hsa21 and a monosomy for the 21q22.3 region. Case 4 is a baby without DS and a 0.56-Mb duplication of 21q22.3. Genotype-phenotype correlation confirmed the presence of three copies of the HR-DSCR in all DS subjects and two copies in all non-DS individuals. CONCLUSIONS: The results presented here are fully consistent with the hypothesis that the HR-DSCR is critically associated with DS diagnosis. No exception to this pathogenetic model was found. Further studies are needed to detect genetic determinants likely located in the HR-DSCR and possibly responsible for core DS features, in particular intellectual disability.

Defective lipid signalling caused by mutations in PIK3C2B underlies focal epilepsy.

Epilepsy is one of the most frequent neurological diseases, with focal epilepsy accounting for the largest number of cases. The genetic alterations involved in focal epilepsy are far from being fully elucidated. Here, we show that defective lipid signalling caused by heterozygous ultra-rare variants in PIK3C2B, encoding for the class II phosphatidylinositol 3-kinase PI3K-C2ß, underlie focal epilepsy in humans. We demonstrate that patients' variants act as loss-of-function alleles, leading to impaired synthesis of the rare signalling lipid phosphatidylinositol 3,4-bisphosphate, resulting in mTORC1 hyperactivation. In vivo, mutant Pik3c2b alleles caused dose-dependent neuronal hyperexcitability and increased seizure susceptibility, indicating haploinsufficiency as a key driver of disease. Moreover, acute mTORC1 inhibition in mutant mice prevented experimentally induced seizures, providing a potential therapeutic option for a selective group of patients with focal epilepsy. Our findings reveal an unexpected role for class II PI3K-mediated lipid signalling in regulating mTORC1-dependent neuronal excitability in mice and humans.

HDAC9 structural variants disrupting TWIST1 transcriptional regulation lead to craniofacial and limb malformations.

Structural variants (SVs) can affect protein-coding sequences as well as gene regulatory elements. However, SVs disrupting protein-coding sequences that also function as cis-regulatory elements remain largely uncharacterized. Here, we show that craniosynostosis patients with SVs containing the histone deacetylase 9 (HDAC9) protein-coding sequence are associated with disruption of TWIST1 regulatory elements that reside within the HDAC9 sequence. Based on SVs within the HDAC9-TWIST1 locus, we defined the 3'-HDAC9 sequence as a critical TWIST1 regulatory region, encompassing craniofacial TWIST1 enhancers and CTCF sites. Deletions of either Twist1 enhancers (eTw5-7Δ/Δ) or CTCF site (CTCF-5Δ/Δ) within the Hdac9 protein-coding sequence led to decreased Twist1 expression and altered anterior/posterior limb expression patterns of SHH pathway genes. This decreased Twist1 expression results in a smaller sized and asymmetric skull and polydactyly that resembles Twist1+/- mouse phenotype. Chromatin conformation analysis revealed that the Twist1 promoter interacts with Hdac9 sequences that encompass Twist1 enhancers and a CTCF site, and that interactions depended on the presence of both regulatory regions. Finally, a large inversion of the entire Hdac9 sequence (Hdac9 INV/+) in mice that does not disrupt Hdac9 expression but repositions Twist1 regulatory elements showed decreased Twist1 expression and led to a craniosynostosis-like phenotype and polydactyly. Thus, our study elucidates essential components of TWIST1 transcriptional machinery that reside within the HDAC9 sequence. It suggests that SVs encompassing protein-coding sequences could lead to a phenotype that is not attributed to its protein function but rather to a disruption of the transcriptional regulation of a nearby gene.

Third-Generation Cytogenetic Analysis: Diagnostic Application of Long-Read Sequencing.

Copy number variants (CNVs) play important roles in the pathogenesis of several genetic syndromes. Traditional and molecular karyotyping are considered the first-tier diagnostic tests to detect macroscopic and cryptic deletions/duplications. However, their time-consuming and laborious experimental protocols protract diagnostic times from 3 to 15 days. Nanopore sequencing has the ability to reduce time to results for the detection of CNVs with the same resolution of current state-of-the-art diagnostic tests. Nanopore sequencing was compared to molecular karyotyping for the detection of pathogenic CNVs of seven patients with previously diagnosed causative CNVs of different sizes and cellular fractions. Larger chromosomal anomalies included trisomy 21 and mosaic tetrasomy 12p. Among smaller CNVs, two genomic imbalances of 1.3 Mb, a small deletion of 170 kb, and two mosaic deletions (1.2 Mb and 408 kb) were tested. DNA was sequenced and data generated during runs were analyzed in online mode. All pathogenic CNVs were identified with detection time inversely proportional to size and cellular fraction. Aneuploidies were called after only 30 minutes of sequencing, whereas 30 hours were needed to call small CNVs. These results demonstrate the clinical utility of our approach that allows the molecular diagnosis of genomic disorders within a 30-minute to 30-hour time frame and its easy implementation as a routinary diagnostic tool.

Case Report: Hereditary Alpha Tryptasemia in Children: A Pediatric Case Series and a Brief Overview of Literature.

Hereditary alpha tryptasemia (HαT) is a recently described autosomal dominant genetic trait caused by an increased copy number of the TPSAB1 gene. It commonly leads to elevated basal serum tryptase levels, and it is associated with heterogeneous clinical manifestations. Some individuals report few to no symptoms, while others present with a spectrum of debilitating features. Most symptoms related to HαT may be explained by mast cell activation and mediator release, namely multiple allergies, anaphylaxis, and skin rash. However, the genotype-phenotype correlation has not yet been clearly understood. In particular, the characterization of the clinical spectrum lacks in children, where differential diagnosis could be challenging. Systemic mastocytosis, HαT, and mast cell activation syndrome are all associated with overlapping pathophysiology and symptoms, making the distinction between these conditions a difficult task. We herein describe two pediatric cases of HαT and their respective families at our tertiary care teaching hospital, highlighting the diagnostic workup and differential diagnosis. We also provide a brief review of the literature to underline the peculiar features of this condition in children.

De novo and bi-allelic variants in AP1G1 cause neurodevelopmental disorder with developmental delay, intellectual disability, and epilepsy.

Adaptor protein (AP) complexes mediate selective intracellular vesicular trafficking and polarized localization of somatodendritic proteins in neurons. Disease-causing alleles of various subunits of AP complexes have been implicated in several heritable human disorders, including intellectual disabilities (IDs). Here, we report two bi-allelic (c.737C>A [p.Pro246His] and c.1105A>G [p.Met369Val]) and eight de novo heterozygous variants (c.44G>A [p.Arg15Gln], c.103C>T [p.Arg35Trp], c.104G>A [p.Arg35Gln], c.229delC [p.Gln77Lys∗11], c.399_400del [p.Glu133Aspfs∗37], c.747G>T [p.Gln249His], c.928-2A>C [p.?], and c.2459C>G [p.Pro820Arg]) in AP1G1, encoding gamma-1 subunit of adaptor-related protein complex 1 (AP1γ1), associated with a neurodevelopmental disorder (NDD) characterized by mild to severe ID, epilepsy, and developmental delay in eleven families from different ethnicities. The AP1γ1-mediated adaptor complex is essential for the formation of clathrin-coated intracellular vesicles. In silico analysis and 3D protein modeling simulation predicted alteration of AP1γ1 protein folding for missense variants, which was consistent with the observed altered AP1γ1 levels in heterologous cells. Functional studies of the recessively inherited missense variants revealed no apparent impact on the interaction of AP1γ1 with other subunits of the AP-1 complex but rather showed to affect the endosome recycling pathway. Knocking out ap1g1 in zebrafish leads to severe morphological defect and lethality, which was significantly rescued by injection of wild-type AP1G1 mRNA and not by transcripts encoding the missense variants. Furthermore, microinjection of mRNAs with de novo missense variants in wild-type zebrafish resulted in severe developmental abnormalities and increased lethality. We conclude that de novo and bi-allelic variants in AP1G1 are associated with neurodevelopmental disorder in diverse populations.

An increased burden of rare exonic variants in NRXN1 microdeletion carriers is likely to enhance the penetrance for autism spectrum disorder.

Autism spectrum disorder (ASD) is characterized by a complex polygenic background, but with the unique feature of a subset of cases (~15%-30%) presenting a rare large-effect variant. However, clinical interpretation in these cases is often complicated by incomplete penetrance, variable expressivity and different neurodevelopmental trajectories. NRXN1 intragenic deletions represent the prototype of such ASD-associated susceptibility variants. From chromosomal microarrays analysis of 104 ASD individuals, we identified an inherited NRXN1 deletion in a trio family. We carried out whole-exome sequencing and deep sequencing of mitochondrial DNA (mtDNA) in this family, to evaluate the burden of rare variants which may contribute to the phenotypic outcome in NRXN1 deletion carriers. We identified an increased burden of exonic rare variants in the ASD child compared to the unaffected NRXN1 deletion-transmitting mother, which remains significant if we restrict the analysis to potentially deleterious rare variants only (P = 6.07 × 10-5 ). We also detected significant interaction enrichment among genes with damaging variants in the proband, suggesting that additional rare variants in interacting genes collectively contribute to cross the liability threshold for ASD. Finally, the proband's mtDNA presented five low-level heteroplasmic mtDNA variants that were absent in the mother, and two maternally inherited variants with increased heteroplasmic load. This study underlines the importance of a comprehensive assessment of the genomic background in carriers of large-effect variants, as penetrance modulation by additional interacting rare variants to might represent a widespread mechanism in neurodevelopmental disorders.

Expanding Phenotype of Schimke Immuno-Osseous Dysplasia: Congenital Anomalies of the Kidneys and of the Urinary Tract and Alteration of NK Cells.

Schimke immuno-osseous dysplasia (SIOD) is a rare multisystemic disorder with a variable clinical expressivity caused by biallelic variants in SMARCAL1. A phenotype-genotype correlation has been attempted and variable expressivity of biallelic SMARCAL1 variants may be associated with environmental and genetic disturbances of gene expression. We describe two siblings born from consanguineous parents with a diagnosis of SIOD revealed by whole exome sequencing (WES). Results: A homozygous missense variant in the SMARCAL1 gene (c.1682G>A; p.Arg561His) was identified in both patients. Despite carrying the same variant, the two patients showed substantial renal and immunological phenotypic differences. We describe features not previously associated with SIOD-both patients had congenital anomalies of the kidneys and of the urinary tract and one of them succumbed to a classical type congenital mesoblastic nephroma. We performed an extensive characterization of the immunophenotype showing combined immunodeficiency characterized by a profound lymphopenia, lack of thymic output, defective IL-7Rα expression, and disturbed B plasma cells differentiation and immunoglobulin production in addition to an altered NK-cell phenotype and function. Conclusions: Overall, our results contribute to extending the phenotypic spectrum of features associated with SMARCAL1 mutations and to better characterizing the underlying immunologic disorder with critical implications for therapeutic and management strategies.

Accurate Detection of Hot-Spot MTOR Somatic Mutations in Archival Surgical Specimens of Focal Cortical Dysplasia by Molecular Inversion Probes.

BACKGROUND: Formalin-fixed, paraffin-embedded brain specimens are a potentially rich resource to identify somatic variants, but their DNA is characterised by low yield and extensive degradation, and matched peripheral samples are usually unavailable for analysis. METHODS: We designed single-molecule molecular inversion probes to target 18 MTOR somatic mutational hot-spots in unmatched, histologically proven focal cortical dysplasias from formalin-fixed, paraffin-embedded tissues of 50 patients. RESULTS: We achieved adequate DNA and sequencing quality in 28 focal cortical dysplasias, mostly extracted within 2 years from fixation, showing a statistically significant effect of time from fixation as a major determinant for successful genetic analysis. We identified and validated seven encompassing hot-spot residues (found in 14% of all patients and in 25% of those sequenced and analysed). The allele fraction had a range of 2-5% and variants were absent in available neighbouring non-focal cortical dysplasia specimens. We computed an alternate allele threshold for calling true variants, based on an experiment-wise mismatch count distribution, well predicting call reliability. CONCLUSIONS: Single-molecule molecular inversion probes are experimentally simple, cost effective and scalable, accurately detecting clinically relevant somatic variants in challenging brain formalin-fixed, paraffin-embedded tissues.

Autozygosity-driven genetic diagnosis in consanguineous families from Italy and the Greater Middle East.

Autozygosity-driven exome analysis has been shown effective for identification of genes underlying recessive diseases especially in countries of the so-called Greater Middle East (GME), where high consanguinity unravels the phenotypic effects of recessive alleles and large family sizes facilitate homozygosity mapping. In Italy, as in most European countries, consanguinity is estimated low. Nonetheless, consanguineous Italian families are not uncommon in publications of genetic findings and are often key to new associations of genes with rare diseases. We collected 52 patients from 47 consanguineous families with suspected recessive diseases, 29 originated in GME countries and 18 of Italian descent. We performed autozygosity-driven exome analysis by detecting long runs of homozygosity (ROHs > 1.5 Mb) and by prioritizing candidate clinical variants within. We identified a pathogenic synonymous variant that had been previously missed in NARS2 and we increased an initial high diagnostic rate (47%) to 55% by matchmaking our candidate genes and including in the analysis shorter ROHs that may also happen to be autozygous. GME and Italian families contributed to diagnostic yield comparably. We found no significant difference either in the extension of the autozygous genome, or in the distribution of candidate clinical variants between GME and Italian families, while we showed that the average autozygous genome was larger and the mean number of candidate clinical variants was significantly higher (p = 0.003) in mutation-positive than in mutation-negative individuals, suggesting that these features influence the likelihood that the disease is autozygosity-related. We highlight the utility of autozygosity-driven genomic analysis also in countries and/or communities, where consanguinity is not widespread cultural tradition.

SSBP1 mutations cause mtDNA depletion underlying a complex optic atrophy disorder.

Inherited optic neuropathies include complex phenotypes, mostly driven by mitochondrial dysfunction. We report an optic atrophy spectrum disorder, including retinal macular dystrophy and kidney insufficiency leading to transplantation, associated with mitochondrial DNA (mtDNA) depletion without accumulation of multiple deletions. By whole-exome sequencing, we identified mutations affecting the mitochondrial single-strand binding protein (SSBP1) in 4 families with dominant and 1 with recessive inheritance. We show that SSBP1 mutations in patient-derived fibroblasts variably affect the amount of SSBP1 protein and alter multimer formation, but not the binding to ssDNA. SSBP1 mutations impaired mtDNA, nucleoids, and 7S-DNA amounts as well as mtDNA replication, affecting replisome machinery. The variable mtDNA depletion in cells was reflected in severity of mitochondrial dysfunction, including respiratory efficiency, OXPHOS subunits, and complex amount and assembly. mtDNA depletion and cytochrome c oxidase-negative cells were found ex vivo in biopsies of affected tissues, such as kidney and skeletal muscle. Reduced efficiency of mtDNA replication was also reproduced in vitro, confirming the pathogenic mechanism. Furthermore, ssbp1 suppression in zebrafish induced signs of nephropathy and reduced optic nerve size, the latter phenotype complemented by WT mRNA but not by SSBP1 mutant transcripts. This previously unrecognized disease of mtDNA maintenance implicates SSBP1 mutations as a cause of human pathology.

Loss of SMPD4 Causes a Developmental Disorder Characterized by Microcephaly and Congenital Arthrogryposis.

Sphingomyelinases generate ceramide from sphingomyelin as a second messenger in intracellular signaling pathways involved in cell proliferation, differentiation, or apoptosis. Children from 12 unrelated families presented with microcephaly, simplified gyral pattern of the cortex, hypomyelination, cerebellar hypoplasia, congenital arthrogryposis, and early fetal/postnatal demise. Genomic analysis revealed bi-allelic loss-of-function variants in SMPD4, coding for the neutral sphingomyelinase-3 (nSMase-3/SMPD4). Overexpression of human Myc-tagged SMPD4 showed localization both to the outer nuclear envelope and the ER and additionally revealed interactions with several nuclear pore complex proteins by proteomics analysis. Fibroblasts from affected individuals showed ER cisternae abnormalities, suspected for increased autophagy, and were more susceptible to apoptosis under stress conditions, while treatment with siSMPD4 caused delayed cell cycle progression. Our data show that SMPD4 links homeostasis of membrane sphingolipids to cell fate by regulating the cross-talk between the ER and the outer nuclear envelope, while its loss reveals a pathogenic mechanism in microcephaly.

P5CS expression study in a new family with ALDH18A1-associated hereditary spastic paraplegia SPG9.

In 2015-2016, we and others reported ALDH18A1 mutations causing dominant (SPG9A) or recessive (SPG9B) spastic paraplegia. In vitro production of the ALDH18A1 product, Δ1 -pyrroline-5-carboxylate synthetase (P5CS), appeared necessary for cracking SPG9 disease-causing mechanisms. We now describe a baculovirus-insect cell system that yields mgs of pure human P5CS and that has proven highly valuable with two novel P5CS mutations reported here in new SPG9B patients. We conclude that both mutations are disease-causing, that SPG9B associates with partial P5CS deficiency and that it is clinically more severe than SPG9A, as reflected in onset age, disability, cognitive status, growth, and dysmorphic traits.

Partial trisomy 21 map: Ten cases further supporting the highly restricted Down syndrome critical region (HR-DSCR) on human chromosome 21.

BACKGROUND: Down syndrome (DS) is characterized by the presence of an extra full or partial human chromosome 21 (Hsa21). An invaluable model to define genotype-phenotype correlations in DS is the study of the extremely rare cases of partial (segmental) trisomy 21 (PT21), the duplication of only a delimited region of Hsa21 associated or not to DS. A systematic retrospective reanalysis of 125 PT21 cases described up to 2015 allowed the creation of the most comprehensive PT21 map and the identification of a 34-kb highly restricted DS critical region (HR-DSCR) as the minimal region whose duplication is shared by all PT21 subjects diagnosed with DS. We reanalyzed at higher resolution three cases previously published and we accurately searched for any new PT21 reports in order to verify whether HR-DSCR limits could prospectively be confirmed and possibly refined. METHODS: Hsa21 partial duplications of three PT21 subjects were refined by adding array-based comparative genomic hybridization data. Seven newly described PT21 cases fulfilling stringent cytogenetic and clinical criteria have been incorporated into the PT21 integrated map. RESULTS: The PT21 map now integrates fine structure of Hsa21 sequence intervals of 132 subjects onto a common framework fully consistent with the presence of a duplicated HR-DSCR, on distal 21q22.13 sub-band, only in DS subjects and not in non-DS individuals. No documented exception to the HR-DSCR model was found. CONCLUSIONS: The findings presented here further support the association of the HR-DSCR with the diagnosis of DS, representing an unbiased validation of the original model. Further studies are needed to identify and characterize genetic determinants presumably located in the HR-DSCR and functionally associated to the critical manifestations of DS.

Challenges in the clinical interpretation of small de novo copy number variants in neurodevelopmental disorders.

In clinical genetics, the need to discriminate between benign and pathogenic variants identified in patients with neurodevelopmental disorders is an absolute necessity. Copy number variants (CNVs) of small size can enable the identification of genes that are critical for neurologic development. However, assigning a definite association with a specific disorder is a difficult task. Among 328 trios analyzed over seven years of activity in a single laboratory, we identified 19 unrelated patients (5.8%) who carried a small (<500 kb) de novo CNV. Four patients had an additional independent de novo CNV. Nine had a variant that could be assigned as definitely pathogenic, whereas the remaining CNVs were considered as variants of unknown significance (VUS). We report clinical and molecular findings of patients harboring VUS. We reviewed the medical literature available for genes impacted by CNVs, obtained the probability of truncating loss-of-function intolerance, and compared overlapping CNVs reported in databases. The classification of small non-recurrent CNVs remains difficult but, among our findings, we provide support for a role of SND1 in the susceptibility of autism, describe a new case of the rare 17p13.1 microduplication syndrome, and report an X-linked duplication involving KIF4A and DLG3 as a likely cause of epilepsy.