Cat eye syndrome: Clinical, cytogenetics and familial findings in a large cohort of 43 patients highlighting the importance of congenital heart disease and inherited cases.

Cat Eye Syndrome (CES) is a rare genetic disease caused by the presence of a small supernumerary marker chromosome derived from chromosome 22, which results in a partial tetrasomy of 22p-22q11.21. CES is classically defined by association of iris coloboma, anal atresia, and preauricular tags or pits, with high clinical and genetic heterogeneity. We conducted an international retrospective study of patients carrying genomic gain in the 22q11.21 chromosomal region upstream from LCR22-A identified using FISH, MLPA, and/or array-CGH. We report a cohort of 43 CES cases. We highlight that the clinical triad represents no more than 50% of cases. However, only 16% of CES patients presented with the three signs of the triad and 9% not present any of these three signs. We also highlight the importance of other impairments: cardiac anomalies are one of the major signs of CES (51% of cases), and high frequency of intellectual disability (47%). Ocular motility defects (45%), abdominal malformations (44%), ophthalmologic malformations (35%), and genitourinary tract defects (32%) are other frequent clinical features. We observed that sSMC is the most frequent chromosomal anomaly (91%) and we highlight the high prevalence of mosaic cases (40%) and the unexpectedly high prevalence of parental transmission of sSMC (23%). Most often, the transmitting parent has mild or absent features and carries the mosaic marker at a very low rate (<10%). These data allow us to better delineate the clinical phenotype associated with CES, which must be taken into account in the cytogenetic testing for this syndrome. These findings draw attention to the need for genetic counseling and the risk of recurrence.

Heterozygous deletion of the VEGFC gene in 4q34.3 is associated with Milroy-like lymphedema: First prenatal case report.

Deleterious variants in the vascular endothelial growth factor C (VEGFC) gene have been recently associated with Milroy-like primary lymphedema, an autosomal dominant disorder, characterized mainly by swelling of the lower limbs due to functional impairment of the lymphatic vessels. To date, only 26 patients with congenital lymphedema harboring VEGFC pathogenic variants were documented. Here, we describe the first prenatal case of a fetus with Milroy-like disease. Fetal ultrasound showed bilateral foot swelling. Chromosomal microarray analysis revealed a 137-kb copy number loss in 4q34.3 including only VEGFC gene in the propositus fetus. Segregation analysis showed that the deletion was inherited from the affected mother and grandmother. Taken together, our study highlights the important role of microarray analysis to detect subtle chromosomal imbalances in the prenatal setting and contributes to delineate the fetal phenotype of VEGFC-related primary congenital lymphedema.

Assessment of Multiplex Digital Droplet RT-PCR as a Diagnostic Tool for SARS-CoV-2 Detection in Nasopharyngeal Swabs and Saliva Samples.

BACKGROUND: Reverse transcription-quantitative PCR on nasopharyngeal swabs is currently the reference COVID-19 diagnosis method but exhibits imperfect sensitivity. METHODS: We developed a multiplex reverse transcription-digital droplet PCR (RT-ddPCR) assay, targeting 6 SARS-CoV-2 genomic regions, and evaluated it on nasopharyngeal swabs and saliva samples collected from 130 COVID-19 positive or negative ambulatory individuals, who presented symptoms suggestive of mild or moderate SARS-CoV2 infection. RESULTS: For the nasopharyngeal swab samples, the results obtained using the 6-plex RT-ddPCR and RT-qPCR assays were all concordant. The 6-plex RT-ddPCR assay was more sensitive than RT-qPCR (85% versus 62%) on saliva samples from patients with positive nasopharyngeal swabs. CONCLUSION: Multiplex RT-ddPCR represents an alternative and complementary tool for the diagnosis of COVID-19, in particular to control RT-qPCR ambiguous results. It can also be applied to saliva for repetitive sampling and testing individuals for whom nasopharyngeal swabbing is not possible.

Recurrence of an early postzygotic rescue of an inherited unbalanced translocation resulting in mosaic segmental uniparental isodisomy of chromosome 11q in siblings.

Balanced translocations are associated with a risk of transmission of unbalanced chromosomal rearrangements in the offspring. Such inherited chromosomal abnormalities are typically non-mosaic as they are present in the germline. We report the recurrence in two siblings of a mosaicism for a chromosomal rearrangement inherited from their asymptomatic father who carried a balanced t(2;11)(q35;q25) translocation. Both siblings exhibited a similar phenotype including intellectual disability, dysmorphic features, kyphoscoliosis, and cervical spinal stenosis. Karyotyping, fluorescence in situ hybridization and SNP array analysis of blood lymphocytes of both siblings identified two cell lines: one carrying a 2q35q37.3 duplication and a 11q25qter deletion (~90% cells), and one carrying an 11q uniparental isodisomy of maternal origin (~10% cells). We hypothesize that these mosaics were related to a postzygotic rescue mechanism which unexpectedly recurred in both siblings.

YY1 Haploinsufficiency Causes an Intellectual Disability Syndrome Featuring Transcriptional and Chromatin Dysfunction.

Yin and yang 1 (YY1) is a well-known zinc-finger transcription factor with crucial roles in normal development and malignancy. YY1 acts both as a repressor and as an activator of gene expression. We have identified 23 individuals with de novo mutations or deletions of YY1 and phenotypic features that define a syndrome of cognitive impairment, behavioral alterations, intrauterine growth restriction, feeding problems, and various congenital malformations. Our combined clinical and molecular data define "YY1 syndrome" as a haploinsufficiency syndrome. Through immunoprecipitation of YY1-bound chromatin from affected individuals' cells with antibodies recognizing both ends of the protein, we show that YY1 deletions and missense mutations lead to a global loss of YY1 binding with a preferential retention at high-occupancy sites. Finally, we uncover a widespread loss of H3K27 acetylation in particular on the YY1-bound enhancers, underscoring a crucial role for YY1 in enhancer regulation. Collectively, these results define a clinical syndrome caused by haploinsufficiency of YY1 through dysregulation of key transcriptional regulators.

Haploinsufficiency of the Primary Familial Brain Calcification Gene SLC20A2 Mediated by Disruption of a Regulatory Element.

OBJECTIVE: Primary familial brain calcification (PFBC) is a rare cerebral microvascular calcifying disorder with diverse neuropsychiatric expression. Five genes were reported as PFBC causative when carrying pathogenic variants. Haploinsufficiency of SLC20A2, which encodes an inorganic phosphate importer, is a major cause of autosomal-dominant PFBC. However, PFBC remains genetically unexplained in a proportion of patients, suggesting the existence of additional genes or cryptic mutations. We analyzed exome sequencing data of 71 unrelated, genetically unexplained PFBC patients with the aim to detect copy number variations that may disrupt the expression of core PFBC-causing genes. METHODS: After the identification of a deletion upstream of SLC20A2, we assessed its consequences on gene function by reverse transcriptase droplet digital polymerase chain reaction (RT-ddPCR), an ex vivo inorganic phosphate uptake assay, and introduced the deletion of a putative SLC20A2 enhancer mapping to this region in human embryonic kidney 293 (HEK293) cells by clustered regularly interspaced short palindromic repeats (CRISPR) - CRISPR-associated protein 9 (Cas9). RESULTS: The 8p11.21 deletion, segregating with PFBC in a family, mapped 35 kb upstream of SLC20A2. The deletion carriers/normal controls ratio of relative SLC20A2 mRNA levels was 60.2% (P < 0.001). This was comparable with that of patients carrying an SLC20A2 premature stop codon (63.4%; P < 0.001). The proband exhibited a 39.3% decrease of inorganic phosphate uptake in blood (P = 0.015). In HEK293 cells, we observed a 39.8% decrease in relative SLC20A2 mRNA levels after normalization on DNA copy number (P < 0.001). DISCUSSION: We identified a deletion of an enhancer of SLC20A2 expression, with carriers showing haploinsufficiency in similar ranges to loss-of-function alleles, and we observed reduced mRNA levels after deleting this element in a cellular model. We propose a 3-step strategy to identify and easily assess the effect of such events. © 2020 International Parkinson and Movement Disorder Society.

A Simple, Universal, and Cost-Efficient Digital PCR Method for the Targeted Analysis of Copy Number Variations.

BACKGROUND: Rare copy number variations (CNVs) are a major cause of genetic diseases. Simple targeted methods are required for their confirmation and segregation analysis. We developed a simple and universal CNV assay based on digital PCR (dPCR) and universal locked nucleic acid (LNA) hydrolysis probes. METHODS: We analyzed the mapping of the 90 LNA hydrolysis probes from the Roche Universal ProbeLibrary (UPL). For each CNV, selection of the optimal primers and LNA probe was almost automated; probes were reused across assays and each dPCR assay included the CNV amplicon and a reference amplicon. We assessed the assay performance on 93 small and large CNVs and performed a comparative cost-efficiency analysis. RESULTS: UPL-LNA probes presented nearly 20000000 occurrences on the human genome and were homogeneously distributed with a mean interval of 156 bp. The assay accurately detected all the 93 CNVs, except one (<200 bp), with coefficient of variation <10%. The assay was more cost-efficient than all the other methods. CONCLUSIONS: The universal dPCR CNV assay is simple, robust, and cost-efficient because it combines a straightforward design allowed by universal probes and end point PCR, the advantages of a relative quantification of the target to the reference within the same reaction, and the high flexibility of the LNA hydrolysis probes. This method should be a useful tool for genomic medicine, which requires simple methods for the interpretation and segregation analysis of genomic variations.

Autologous Myoblasts for the Treatment of Fecal Incontinence: Results of a Phase 2 Randomized Placebo-controlled Study (MIAS).

OBJECTIVE: The aim of this study was to evaluate the efficacy of intrasphincteric injections of autologous myoblasts (AMs) in fecal incontinence (FI) in a controlled study. SUMMARY OF BACKGROUND DATA: Adult stem cell therapy is expected to definitively cure FI by regenerating damaged sphincter. Preclinical data and results of open-label trials suggest that myoblast therapy may represent a noninvasive treatment option. METHODS: We conducted a phase 2 randomized, double-blind, placebo-controlled study of intrasphincteric injections of AM in 24 patients. The study compared outcome after AM (n = 12) or placebo (n = 12) injection using Cleveland Clinic Incontinence (CCI), score at 6 and 12 months. Patients in the placebo group were eligible to receive frozen AM after 1 year. RESULTS: At 6 months, the median CCI score significantly decreased from baseline in both the AM (9 vs 15, P = 0.02) and placebo (10 vs 15, P = 0.01) groups. Hence, no significant difference was found between the 2 groups (primary endpoint) at 6 months. At 12 months, the median CCI score continued to ameliorate in the AM group (6.5 vs 15, P = 0.006), while effect was lost in the placebo group (14 vs 15, P = 0.35). Consequently, there was a higher response rate at 12 months in the treated than the placebo arm (58% vs 8%, P = 0.03). After delayed frozen AM injection in the placebo group, the response rate was 60% (6/10) at 12 months. CONCLUSIONS: Intrasphincteric AM injections in FI patients have shown tolerance, safety, and clinical benefit at 12 months despite a transient placebo effect at 6 months.

Pregnancy outcomes in prenatally diagnosed 47, XXX and 47, XYY syndromes: a 30-year French, retrospective, multicentre study.

OBJECTIVE: Sex chromosome aneuploidies are frequently detected fortuitously in a prenatal diagnosis. Most cases of 47, XXX and 47, XYY syndromes are diagnosed in this context, and parents are thus faced with an unexpected situation. The objective of the present study was to characterize a French cohort of prenatally diagnosed cases of 47, XXX and 47, XYY and to evaluate the termination of pregnancy (TOP) rate before and after France's implementation of multidisciplinary centres for prenatal diagnosis in 1997. METHODS: This retrospective study identified respectively 291 and 175 cases of prenatally diagnosed 47, XXX and 47, XYY between 1976 and 2012. For each case, the indication, maternal age, karyotype and outcome were recorded. RESULTS: Most diagnoses of the two conditions were fortuitous. The occurrence of 47, XXX was associated with advanced maternal age. The overall TOP rate was higher for 47, XXX (22.9%) than for 47, XYY (14.6%), although this difference was not statistically significant. However, the TOP rates fell significantly after 1997 (from 41.1% to 11.8% for 47, XXX and from 25.8% to 6.7% for 47, XYY). CONCLUSION: The TOP rates after prenatal diagnoses of 47, XXX and 47, XYY fell significantly after 1997, following France's implementation of multidisciplinary centres for prenatal diagnosis. © 2016 John Wiley & Sons, Ltd.

[Malignant meningioma with adenocarcinoma-like metaplasia: a rare entity to be not misdiagnosed]. / Le méningiome malin avec métaplasie adénocarcinomateuse: une entité rare à ne pas méconnaître.

We report on a 51-year-old woman who presented with a cervical spinal cord tumor clinically suspected to be a metastasis. Histological examination revealed an anaplastic meningioma containing epithelial nests arranged in a gland-like pattern suggestive of adenocarcinoma. This component strongly expressed cytokeratins whereas the meningothelial component was vimentin--epithelial membrane antigen--and progesterone receptor-immunoreactive, suggesting either anaplastic meningioma with adenocarcinoma-like metaplasia, or adenocarcinoma metastasis in a meningioma, but the search for a primitive neoplasia including thoracic-abdominal-pelvic computed tomography and mammography was negative. Anaplastic meningiomas with adenocarcinoma-like metaplasia are uncommon lesions, 4 cases having been reported in the literature so far. Their immunohistochemical and chromosomal characteristics are similar to those observed in secretory meningiomas. When available, fluorescence in situ hybridization detects the same chromosomal alterations in the two components, confirming a common clonal origin. This observation demonstrates the necessity to perform the correct diagnosis of malignant meningioma with adenocarcinomatous metaplasia, whose prognosis and treatment radically differ from those of metastatic adenocarcinoma located in a meningioma.

Generation of 17q21.31 duplication iPSC-derived neurons as a model for primary tauopathies.

Tau proteins belong to the microtubule associated protein family and are mainly expressed in neurons. Tau accumulates in patients' brain in several neurodegenerative diseases, including Fronto-temporal dementia and Alzheimer's disease. Recently, we described a 17q21.31 duplication in patients presenting different cognitive or motor symptoms and characterized by the accumulation of different Tau isoforms. This duplication involves four genes, including the MAPT gene that encodes the Tau protein. The main pathophysiological consequence associated with this duplication was a 1.6-1.9-fold increase in the MAPT messenger RNA as measured in blood samples of duplication carriers. However, the pathophysiological consequences of this duplication in a cell type relevant for neurodegenerative diseases have never been explored so far. In this study, we developed the first model of primary tauopathy linked to a 17q21.31 duplication in iPSC-induced neurons from 2 unrelated carriers. As in patients' blood, we demonstrated that this duplication was associated with an increase in MAPT mRNA resulting in elevated Tau protein levels in iPSC-derived cortical neurons. We believe that these iPSC lines will be a pertinent tool to elucidate how a same genetic cause could lead to distinct types of tauopathies and the pathophysiological mechanisms associated with Tau-mediated neurodegeneration in the 17q21.31 duplication context.

Evidence for high breakpoint variability in 46, XX, SRY-positive testicular disorder and frequent ARSE deletion that may be associated with short stature.

BACKGROUND: The translocation of SRY onto one of the two X chromosomes results in a 46,XX testicular disorder of sex development; this is supposedly because of non-allelic homologous recombination between the protein kinase X gene (PRKX) and the inverted protein kinase Y pseudogene (PRKY). Although 46,XX SRY-positive men are infertile, the literature data indicate that some of these individuals are of short stature (relative to the general population). We sought to determine whether short stature was linked to additional, more complex chromosomal rearrangements. METHODS: Twelve laboratories gathered detailed clinical, anthropomorphic, cytogenetic and genetic data (including chromosome microarray data) on patients with 46,XX SRY-positive male syndrome. RESULTS: SRY was present (suggesting a der(X)t(X;Y)) in 34 of the 38 cases (89.5%). When considering only the 20 patients with chromosome microarray data, we identified several chromosomal rearrangements and breakpoints, especially on the X chromosome. In the five cases for whom the X chromosome breakpoint was located in the pseudoautosomal region, there was partial duplication of the derivate X chromosome. In contrast, in the 15 cases for whom the breakpoint was located downstream of the pseudoautosomal region, part of the derivate X chromosome had been deleted (included the arylsulfatase E [ARSE] gene in 11 patients). For patients with versus without ARSE deletion, the mean height was, respectively, 167.7 ± 4.5 and 173.1 ± 4.0 cm; this difference was not statistically significant (p = 0.1005). CONCLUSION: Although 46,XX SRY-positive male syndromes were mainly because of imbalanced crossover between the X and Y chromosome during meiosis, the breakpoints differed markedly from one patient to another (especially on the X chromosome); this suggests the presence of a replication-based mechanism for recombination between non-homologous sequences. In some patients, the translocation of SRY to the X chromosome was associated with ARSE gene deletion, which might have led to short stature. With a view to explaining this disorder of sex development, whole exome sequencing could be suggested for SRY-negative patients.

Neuropathological hallmarks of fetal hydrocephalus linked to CCDC88C pathogenic variants.

The prevalence of congenital hydrocephalus has been estimated at 1.1 per 1000 infants when including cases diagnosed before 1 year of age after exclusion of neural tube defects. Classification criteria are based either on CSF dynamics, pathophysiological mechanisms or associated lesions. Whereas inherited syndromic hydrocephalus has been associated with more than 100 disease-causing genes, only four genes are currently known to be linked to congenital hydrocephalus either isolated or as a major clinical feature: L1CAM, AP1S2, MPDZ and CCDC88C. In the past 10 years, pathogenic variants in CCDC88C have been documented but the neuropathology remains virtually unknown. We report the neuropathology of two foetuses from one family harbouring two novel compound heterozygous pathogenic variants in the CCDC88C gene: a maternally inherited indel in exon 22, c.3807_3809delinsACCT;p.(Gly1270Profs*53) and a paternally inherited deletion of exon 23, c.3967-?_c.4112-?;p.(Leu1323Argfs*10). Medical termination of pregnancy was performed at 18 and 23 weeks of gestation for severe bilateral ventriculomegaly. In both fetuses, brain lesions consisted of multifocal atresia-forking along the aqueduct of Sylvius and the central canal of the medulla, periventricular neuronal heterotopias and choroid plexus hydrops. The second fetus also presented lumbar myelomeningocele, left diaphragmatic hernia and bilateral renal agenesis. CCDC88C encodes the protein DAPLE which contributes to ependymal cell planar polarity by inhibiting the non-canonical Wnt signaling pathway and interacts with MPDZ and PARD3. Interestingly, heterozygous variants in PARD3 result in neural tube defects by defective tight junction formation and polarization process of the neuroepithelium. Besides, during organ formation Wnt signalling is a prerequisite for planar cell polarity pathway activation, and mutations in planar cell polarity genes lead to heart, lung and kidney malformations. Hence, candidate variants in CCDC88C should be carefully considered whether brain lesions are isolated or associated with malformations suspected to result from disorders of planar cell polarity.

Detection of copy-number variations from NGS data using read depth information: a diagnostic performance evaluation.

The detection of copy-number variations (CNVs) from NGS data is underexploited as chip-based or targeted techniques are still commonly used. We assessed the performances of a workflow centered on CANOES, a bioinformatics tool based on read depth information. We applied our workflow to gene panel (GP) and whole-exome sequencing (WES) data, and compared CNV calls to quantitative multiplex PCR of short fluorescent fragments (QMSPF) or array comparative genomic hybridization (aCGH) results. From GP data of 3776 samples, we reached an overall positive predictive value (PPV) of 87.8%. This dataset included a complete comprehensive QMPSF comparison of four genes (60 exons) on which we obtained 100% sensitivity and specificity. From WES data, we first compared 137 samples with aCGH and filtered comparable events (exonic CNVs encompassing enough aCGH probes) and obtained an 87.25% sensitivity. The overall PPV was 86.4% following the targeted confirmation of candidate CNVs from 1056 additional WES. In addition, our CANOES-centered workflow on WES data allowed the detection of CNVs with a resolution of single exons, allowing the detection of CNVs that were missed by aCGH. Overall, switching to an NGS-only approach should be cost-effective as it allows a reduction in overall costs together with likely stable diagnostic yields. Our bioinformatics pipeline is available at: https://gitlab.bioinfo-diag.fr/nc4gpm/canoes-centered-workflow .

Patients with 10q22.3q23.1 recurrent deletion syndrome are at risk for juvenile polyposis.

Juvenile polyposis syndrome (JPS) is a rare autosomal dominant predisposition to hamartomatous polyps within the gastrointestinal tract, at high risk for malignant transformation. BMPR1A and SMAD4 loss-of-function variants account for 50% of the cases. More specifically, point mutations and structural abnormalities in BMPR1A lead to a highly penetrant yet variable phenotype of JPS. Intriguingly, in the developmental disorder caused by a recurrent 10q22.3q23.1 7 Mb deletion which includes BMPR1A, juvenile polyps have never been reported. We present the case of a young adult harboring this recurrent deletion, in a context of intellectual disability, ventricular septal defect and severe juvenile polyposis syndrome diagnosed at the age of 25 years, requiring a surgical preventive colectomy. She developed a gastric adenocarcinoma from which she died at the age of 32. We hypothesize that with the current available pangenomic CNV arrays, the diagnosis of 10q22.3q23.1 deletion is often made several years before the onset of the digestive phenotype, which could explain the absence of reports for juvenile polyps. This observation highlights the importance of an active digestive surveillance of patients with 10q22.3q23.1 deletion.

Simultaneous detection of EGFR amplification and EGFRvIII variant using digital PCR-based method in glioblastoma.

Epidermal growth factor receptor (EGFR) amplification and EGFR variant III (EGFRvIII, deletion of exons 2-7) are of clinical interest for glioblastoma. The aim was to develop a digital PCR (dPCR)-based method using locked nucleic acid (LNA)-based hydrolysis probes, allowing the simultaneous detection of the EGFR amplification and EGFRvIII variant. Sixty-two patients were included. An exploratory cohort (n = 19) was used to develop the dPCR assay using three selected amplicons within the EGFR gene, targeting intron 1 (EGFR1), junction of exon 3 and intron 3 (EGFR2) and intron 22 (EGFR3). The copy number of EGFR was estimated by the relative quantification of EGFR1, EGFR2 and EGFR3 amplicon droplets compared to the droplets of a reference gene. EGFRvIII was identified by comparing the copy number of the EGFR2 amplicon to either the EGFR1 or EGFR3 amplicon. dPCR results were compared to fluorescence in situ hybridization (FISH) and next-generation sequencing for amplification; and to RT-PCR-based method for EGFRvIII. The dPCR assay was then tested in a validation cohort (n = 43). A total of 8/19 EGFR-amplified and 5/19 EGFRvIII-positive tumors were identified in the exploratory cohort. Compared to FISH, the EGFR3 dPCR assay detected all EGFR-amplified tumors (8/8, 100%) and had the highest concordance with the copy number estimation by NGS. The concordance between RT-PCR and dPCR was also 100% for detecting EGFRvIII using an absolute difference of 10.8 for the copy number between EGFR2 and EGFR3 probes. In the validation cohort, the sensitivity and specificity of dPCR using EGFR3 probes were 100% for the EGFR amplification detection compared to FISH (19/19). EGFRvIII was detected by dPCR in 8 EGFR-amplified patients and confirmed by RT-PCR. Compared to FISH, the EGFR2/EGFR3 dPCR assay was estimated with a one-half cost value. These results highlight that dPCR allowed the simultaneous detection of EGFR amplification and EGFRvIII for glioblastoma.

Rare genetic susceptibility variants assessment in autism spectrum disorder: detection rate and practical use.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder with a strong genetic component whose knowledge evolves quickly. Next-generation sequencing is the only effective technology to deal with the high genetic heterogeneity of ASD in a clinical setting. However, rigorous criteria to classify rare genetic variants conferring ASD susceptibility are currently lacking. We have performed whole-exome sequencing to identify both nucleotide variants and copy number variants (CNVs) in 253 ASD patients, including 68 patients with intellectual disability (ID) and 90 diagnosed as Asperger syndrome. Using explicit criteria to classify both susceptibility genes and susceptibility variants we prioritized 217 genes belonging to the following categories: syndromic genes, genes with an excess of de novo protein truncating variants and genes targeted by rare CNVs. We obtained a susceptibility variant detection rate of 19.7% (95% CI: [15-25.2%]). The rate for CNVs was 7.1% (95% CI: [4.3-11%]) and 12.6% (95% CI: [8.8-17.4%]) for nucleotide variants. The highest rate (30.1%, 95% CI: [20.2-43.2%]) was obtained in the ASD + ID subgroup. A strong contributor for at risk nucleotide variants was the recently identified set of genes (n = 81) harboring an excess of de novo protein truncating variants. Since there is currently no evidence that the genes targeted here are necessary and sufficient to cause ASD, we recommend to avoid the term "causative of ASD" when delivering the information about a variant to a family and to use instead the term "genetic susceptibility factor contributing to ASD".

Clinical phenotype of germline RUNX1 haploinsufficiency: from point mutations to large genomic deletions.

Germline RUNX1 mutations result in a rare autosomal dominant condition characterized by qualitative and quantitative platelet defects and predisposition to the development of myeloid malignancies (familial platelet disorder with propensity to acute myeloid leukaemia, FPD/AML). Only 13 pedigrees have previously been described so far. We report on two novel germline RUNX1 mutations: (1) an out-of-frame 8 bp heterozygous deletion (c.442_449del) in an FPD/AML pedigree and (2) a de novo 3.5 Mb deletion in the 21q22.11.21q22.12 region encompassing the RUNX1 gene in a mentally retarded female patient with short stature and thrombocytopenia. Interestingly, a similar de novo submicroscopic deletion has been recently reported in the literature in a mentally retarded patient. Mental retardation is one of the most common disorders and primary causes of thrombocytopenia are rare. When occurring together, these features should prompt to test for 21q22 deletion for comprehensive genetic counselling and clinical management.

Metabolic causes of nonimmune hydrops fetalis: A next-generation sequencing panel as a first-line investigation.

PURPOSES: Hydrops fetalis is a life-threatening fetal condition, and 85% of all cases are classified as nonimmune hydrops fetalis (NIHF). Up to 15% of NIHF cases may be due to inborn errors of metabolism (IEM), but a large proportion of cases linked to metabolic disorders remains undiagnosed. This lack of diagnosis may be related to the limitations of conventional biological procedures, which involve sequential investigations and require multiple samples and steps. In addition, this approach is time consuming. We have developed a next-generation sequencing (NGS) panel to investigate metabolic causes of NIHF, ascites, and polyhydramnios associated to another fetal abnormality. METHODS: The hydrops fetalis (HydFet) panel was designed to cover the coding regions and flanking intronic sequences of 41 genes. A retrospective study of amniotic fluid samples from 40 subjects was conducted. A prospective study was subsequently initiated, and six samples were analyzed using the NGS panel. RESULTS: Five IEM diagnoses were made using the HydFet panel (Niemann-Pick type C (NPC), Barth syndrome, HNF1Β deficiency, GM1 gangliosidosis, and Gaucher disease). This analysis also allowed the identification of 8p sequence triplication in an additional case. CONCLUSION: NGS combined with robust bioinformatics analyses is a useful tool for identifying the causative variants of NIHF. Subsequent functional characterization of the protein encoded by the altered gene and morphological studies may confirm the diagnosis. This paradigm shift allows a significant improvement of IEM diagnosis in NIHF.