Get Your Molar Tooth Right: Joubert Syndrome Misdiagnosis Unmasked by Whole-Exome Sequencing.

Joubert syndrome (JS) is a recessively inherited ciliopathy, characterized by a specific cerebellar and brainstem malformation recognizable on brain imaging as the "molar tooth sign" (MTS). Clinical signs include hypotonia, developmental delay, breathing abnormalities, and ocular motor apraxia. Older patients develop ataxia, intellectual impairment, and variable organ involvement. JS is genetically heterogeneous, with over 40 ciliary genes overall accounting for 65-75% cases. Thus, in recent years, the genetic diagnosis of JS has been based on the analysis of next-generation sequencing targeted gene panels. Since clinical features are unspecific and undistinguishable from other neurodevelopmental syndromes, the recognition of the MTS is crucial to address the patient to the appropriate genetic testing. However, the MTS is not always properly diagnosed, resulting either in false negative diagnoses (patients with the MTS not addressed to JS genetic testing) or in false positive diagnoses (patients with a different brain malformation wrongly addressed to JS genetic testing). Here, we present six cases referred for JS genetic testing based on inappropriate recognition of MTS. While the analysis of JS-related genes was negative, whole-exome sequencing (WES) disclosed pathogenic variants in other genes causative of distinct brain malformative conditions with partial clinical and neuroradiological overlap with JS. Reassessment of brain MRIs from five patients by a panel of expert pediatric neuroradiologists blinded to the genetic diagnosis excluded the MTS in all cases but one, which raised conflicting interpretations. This study highlights that the diagnostic yield of NGS-based targeted panels is strictly related to the accuracy of the diagnostic referral based on clinical and imaging assessment and that WES has an advantage over targeted panel analysis when the diagnostic suspicion is not straightforward.

Small supernumerary marker chromosomes: A legacy of trisomy rescue?

We studied by a whole genomic approach and trios genotyping, 12 de novo, nonrecurrent small supernumerary marker chromosomes (sSMC), detected as mosaics during pre- or postnatal diagnosis and associated with increased maternal age. Four sSMCs contained pericentromeric portions only, whereas eight had additional non-contiguous portions of the same chromosome, assembled together in a disordered fashion by repair-based mechanisms in a chromothriptic event. Maternal hetero/isodisomy was detected with a paternal origin of the sSMC in some cases, whereas in others two maternal alleles in the sSMC region and biparental haplotypes of the homologs were detected. In other cases, the homologs were biparental while the sSMC had the same haplotype of the maternally inherited chromosome. These findings strongly suggest that most sSMCs are the result of a multiple-step mechanism, initiated by maternal meiotic nondisjunction followed by postzygotic anaphase lagging of the supernumerary chromosome and its subsequent chromothripsis.

Role of mycotoxins in the pathobiology of autism: A first evidence.

Objectives: Gene-environment interaction is an emerging hypothesis to expound not only the autism pathogenesis but also the increased incidence of neurodevelopmental disorders (such as autistic spectrum disorder, attention-deficit, hyperactivity disorder). Among xenobiotics, mycotoxins are worldwide contaminants of food that provoke toxicological effects, crucially resembling several symptoms associated with autism such as oxidative stress, intestinal permeability, and inflammation. Here, we focused on a group of mycotoxins to test their role in the manifestation of autism, try to explain their mechanism of action, and discuss possible preventive and therapeutic interventions. Methods: Autistic children (n = 52) and healthy children [n = 58 (31 siblings and 27 unrelated subjects)] were recruited and body fluids and clinical data collected. The diagnosis of autism was made according to DSM V criteria, then with GMDS 0-2, WPPSI, and ADOS. Ochratoxin A (OTA), gliotoxin, zearalenone, and sphingosine/sphinganine ratio were determined by LC analysis in sera and urines. Statistical analysis was performed by the Wilcoxon Rank Sum (Mann-Whitney) test and Spearman test. Results: By comparing the results of autistic patients with those of unrelated controls, a significant association was found for OTA levels in urines (P = 0.0002) and sera (P = 0.0017), and also comparing patients with siblings and unrelated controls together (P = 0.0081). Discussion: Our results are the first describing a possible role of OTA in the pathobiology of autism. Recalling the male prevalence of ASD (male/female = 4-5/1), it is noted that, in animal models, OTA exerts its neurotoxicity especially in males. Moreover, in vitro, OTA increases microRNA-132 that is dysregulated in autistic patients and involved in reciprocal regulation of the autism-related genes MeCP2 and PTEN. A personalized diet coupled with probiotic administration, especially OTA adsorbing Lactobacillus, could ameliorate autistic symptoms in OTA-positive patients.

Chromothripsis and ring chromosome 22: a paradigm of genomic complexity in the Phelan-McDermid syndrome (22q13 deletion syndrome).

INTRODUCTION: Phelan-McDermid syndrome (PMS) is caused by SHANK3 haploinsufficiency. Its wide phenotypic variation is attributed partly to the type and size of 22q13 genomic lesion (deletion, unbalanced translocation, ring chromosome), partly to additional undefined factors. We investigated a child with severe global neurodevelopmental delay (NDD) compatible with her distal 22q13 deletion, complicated by bilateral perisylvian polymicrogyria (BPP) and urticarial rashes, unreported in PMS. METHODS: Following the cytogenetic and array-comparative genomic hybridization (CGH) detection of a r(22) with SHANK3 deletion and two upstream duplications, whole-genome sequencing (WGS) in blood and whole-exome sequencing (WES) in blood and saliva were performed to highlight potential chromothripsis/chromoanagenesis events and any possible BPP-associated variants, even in low-level mosaicism. RESULTS: WGS confirmed the deletion and highlighted inversion and displaced order of eight fragments, three of them duplicated. The microhomology-mediated insertion of partial Alu-elements at one breakpoint junction disrupted the topological associating domain joining NFAM1 to the transcriptional coregulator TCF20. WES failed to detect BPP-associated variants. CONCLUSIONS: Although we were unable to highlight the molecular basis of BPP, our data suggest that SHANK3 haploinsufficiency and TCF20 misregulation, both associated with intellectual disability, contributed to the patient's NDD, while NFAM1 interruption likely caused her skin rashes, as previously reported. We provide the first example of chromoanasynthesis in a constitutional ring chromosome and reinforce the growing evidence that chromosomal rearrangements may be more complex than estimated by conventional diagnostic approaches and affect the phenotype by global alteration of the topological chromatin organisation rather than simply by deletion or duplication of dosage-sensitive genes.

De novo unbalanced translocations have a complex history/aetiology.

We investigated 52 cases of de novo unbalanced translocations, consisting in a terminally deleted or inverted-duplicated deleted (inv-dup del) 46th chromosome to which the distal portion of another chromosome or its opposite end was transposed. Array CGH, whole-genome sequencing, qPCR, FISH, and trio genotyping were applied. A biparental origin of the deletion and duplication was detected in 6 cases, whereas in 46, both imbalances have the same parental origin. Moreover, the duplicated region was of maternal origin in more than half of the cases, with 25% of them showing two maternal and one paternal haplotype. In all these cases, maternal age was increased. These findings indicate that the primary driver for the occurrence of the de novo unbalanced translocations is a maternal meiotic non-disjunction, followed by partial trisomy rescue of the supernumerary chromosome present in the trisomic zygote. In contrast, asymmetric breakage of a dicentric chromosome, originated either at the meiosis or postzygotically, in which the two resulting chromosomes, one being deleted and the other one inv-dup del, are repaired by telomere capture, appears at the basis of all inv-dup del translocations. Notably, this mechanism also fits with the origin of some simple translocations in which the duplicated region was of paternal origin. In all cases, the signature at the translocation junctions was that of non-homologous end joining (NHEJ) rather than non-allelic homologous recombination (NAHR). Our data imply that there is no risk of recurrence in the following pregnancies for any of the de novo unbalanced translocations we discuss here.

Molecular mechanisms generating and stabilizing terminal 22q13 deletions in 44 subjects with Phelan/McDermid syndrome.

In this study, we used deletions at 22q13, which represent a substantial source of human pathology (Phelan/McDermid syndrome), as a model for investigating the molecular mechanisms of terminal deletions that are currently poorly understood. We characterized at the molecular level the genomic rearrangement in 44 unrelated patients with 22q13 monosomy resulting from simple terminal deletions (72%), ring chromosomes (14%), and unbalanced translocations (7%). We also discovered interstitial deletions between 17-74 kb in 9% of the patients. Haploinsufficiency of the SHANK3 gene, confirmed in all rearrangements, is very likely the cause of the major neurological features associated with PMS. SHANK3 mutations can also result in language and/or social interaction disabilities. We determined the breakpoint junctions in 29 cases, providing a realistic snapshot of the variety of mechanisms driving non-recurrent deletion and repair at chromosome ends. De novo telomere synthesis and telomere capture are used to repair terminal deletions; non-homologous end-joining or microhomology-mediated break-induced replication is probably involved in ring 22 formation and translocations; non-homologous end-joining and fork stalling and template switching prevail in cases with interstitial 22q13.3. For the first time, we also demonstrated that distinct stabilizing events of the same terminal deletion can occur in different early embryonic cells, proving that terminal deletions can be repaired by multistep healing events and supporting the recent hypothesis that rare pathogenic germline rearrangements may have mitotic origin. Finally, the progressive clinical deterioration observed throughout the longitudinal medical history of three subjects over forty years supports the hypothesis of a role for SHANK3 haploinsufficiency in neurological deterioration, in addition to its involvement in the neurobehavioral phenotype of PMS.

Further evidence supporting the role of GTDC1 in glycine metabolism and neurodevelopmental disorders.

Copy number variants (CNVs) represent the genetic cause of about 15-20% of neurodevelopmental disorders (NDDs). We identified a ~67 kb de novo intragenic deletion on chromosome 2q22.3 in a female individual showing a developmental encephalopathy characterised by epilepsy, severe intellectual disability, speech delay, microcephaly, and thin corpus callosum with facial dysmorphisms. The microdeletion involved exons 5-6 of GTDC1, encoding a putative glycosyltransferase, whose expression is particularly enriched in the nervous system. In a previous study, a balanced de novo translocation encompassing GTDC1 was reported in a male child with global developmental delay and delayed speech and language development. Based on these premises, we explored the transcriptomic profile of our proband to evaluate the functional consequences of the novel GTDC1 de novo intragenic deletion in relation to the observed neurodevelopmental phenotype. RNA-seq on the proband's lymphoblastoid cell line (LCL) showed expression changes of glycine/serine and cytokine/chemokine signalling pathways, which are related to neurodevelopment and epileptogenesis. Subsequent analysis by ELISA (enzyme-linked immunosorbent assay) and HPLC (high-performance liquid chromatography) revealed increased levels of glycine in the proband's LCL and serum compared to matched controls. Given that an increased level of glycine has been observed in the plasma samples of individuals with Rett syndrome, a condition sharing epilepsy, microcephaly, and intellectual disability with our proband, we proposed that the GTDC1 downregulation is implicated in neurodevelopmental impairment by altering glycine metabolism. Furthermore, our findings expanded the phenotypic spectrum of the novel GTDC1-related condition, including microcephaly and epilepsy among relevant clinical features.

Case Report: Decrypting an interchromosomal insertion associated with Marfan's syndrome: how optical genome mapping emphasizes the morbid burden of copy-neutral variants.

Optical genome mapping (OGM), which allows analysis of ultra-high molecular weight (UHMW) DNA molecules, represents a response to the restriction created by short-read next-generation-sequencing, even in cases where the causative variant is a neutral copy-number-variant insensitive to quantitative investigations. This study aimed to provide a molecular diagnosis to a boy with Marfan syndrome (MFS) and intellectual disability (ID) carrying a de novo translocation involving chromosomes 3, 4, and 13 and a 1.7 Mb deletion at the breakpoint of chromosome 3. No FBN1 alteration explaining his Marfan phenotype was highlighted. UHMW gDNA was isolated from both the patient and his parents and processed using OGM. Genome assembly was followed by variant calling and annotation. Multiple strategies confirmed the results. The 3p deletion, which disrupted ROBO2, (MIM*602431) included three copy-neutral insertions. Two came from chromosome 13; the third contained 15q21.1, including the FBN1 from intron-45 onwards, thus explaining the MFS phenotype. We could not attribute the ID to a specific gene variant nor to the reshuffling of topologically associating domains (TADs). Our patient did not have vesicular reflux-2, as reported by missense alterations of ROBO2 (VUR2, MIM#610878), implying that reduced expression of all or some isoforms has a different effect than some of the point mutations. Indeed, the ROBO2 expression pattern and its role as an axon-guide suggests that its partial deletion is responsible for the patient's neurological phenotype. Conclusion: OGM testing 1) highlights copy-neutral variants that could remain invisible if no loss of heterozygosity is observed and 2) is mandatory before other molecular studies in the presence of any chromosomal rearrangement for an accurate genotype-phenotype relationship.

Trait - driven analysis of the 2p15p16.1 microdeletion syndrome suggests a complex pattern of interactions between candidate genes.

BACKGROUND: Individuals with the 2p15p16.1 microdeletion syndrome share a complex phenotype including neurodevelopmental delay, brain malformations, microcephaly, and autistic behavior. The analysis of the shortest region of overlap (SRO) between deletions in ~ 40 patients has led to the identification of two critical regions and four strongly candidate genes (BCL11A, REL, USP34 and XPO1). However, the delineation of their role in the occurrence of specific traits is hampered by their incomplete penetrance. OBJECTIVE: To better delineate the role of hemizygosity of specific regions in selected traits by leveraging information both from penetrant and non - penetrant deletions. METHODS: Deletions in patients that do not present a specific trait cannot contribute to delineate the SROs. We recently developed a probabilistic model that, by considering also the non - penetrant deletions, allows a more reliable assignment of peculiar traits to specific genomic segments. We apply this method adding two new patients to the published cases. RESULTS: Our results delineate an intricate pattern of genotype - phenotype correlation where BCL11A emerges as the main gene for autistic behavior while USP34 and/or XPO1 haploinsufficiency are mainly associated with microcephaly, hearing loss and IUGR. BCL11A, USP34 and XPO1 genes are broadly related with brain malformations albeit with distinct patterns of brain damage. CONCLUSIONS: The observed penetrance of deletions encompassing different SROs and that predicted when considering each single SRO as acting independently, may reflect a more complex model than the additive one. Our approach may improve the genotype/phenotype correlation and may help to identify specific pathogenic mechanisms in contiguous gene syndromes.

Rare familial 16q21 microdeletions under a linkage peak implicate cadherin 8 (CDH8) in susceptibility to autism and learning disability.

BACKGROUND: Autism spectrum disorder (ASD) is characterised by impairments in social communication and by a pattern of repetitive behaviours, with learning disability (LD) typically seen in up to 70% of cases. A recent study using the PPL statistical framework identified a novel region of genetic linkage on chromosome 16q21 that is limited to ASD families with LD. METHODS: In this study, two families with autism and/or LD are described which harbour rare >1.6 Mb microdeletions located within this linkage region. The deletion breakpoints are mapped at base-pair resolution and segregation analysis is performed using a combination of 1M single nucleotide polymorphism (SNP) technology, array comparative genomic hybridisation (CGH), long-range PCR, and Sanger sequencing. The frequency of similar genomic variants in control subjects is determined through analysis of published SNP array data. Expression of CDH8, the only gene disrupted by these microdeletions, is assessed using reverse transcriptase PCR and in situ hybridisation analysis of 9 week human embryos. RESULTS: The deletion of chr16: 60 025 584-61 667 839 was transmitted to three of three boys with autism and LD and none of four unaffected siblings, from their unaffected mother. In a second family, an overlapping deletion of chr16: 58 724 527-60 547 472 was transmitted to an individual with severe LD from his father with moderate LD. No copy number variations (CNVs) disrupting CDH8 were observed in 5023 controls. Expression analysis indicates that the two CDH8 isoforms are present in the developing human cortex. CONCLUSION: Rare familial 16q21 microdeletions and expression analysis implicate CDH8 in susceptibility to autism and LD.

The embryo battle against adverse genomes: Are de novo terminal deletions the rescue of unfavorable zygotic imbalances?

De novo distal deletions are structural variants considered to be already present in the zygote. However, investigations especially in the prenatal setting have documented that they are often in mosaic with cell lines in which the same deleted chromosome shows different types of aberrations such as: 1) neutral copy variants with loss of heterozygosity that replace the deleted region with equivalent portions of the homologous chromosome and create distal uniparental disomy (UPD); 2) derivative chromosomes where the deleted one ends with the distal region of another chromosome or has the shape of a ring; 3) U-type mirror dicentric or inv-dup del rearrangements. Unstable dicentrics had already been entailed as causative of terminal deletions even when no trace of the reciprocal inv-dup del had been detected. To clarify the mechanism of origin of distal deletions, we examined PubMed using as keywords: complex/mosaic chromosomal deletions, distal UPD, U-type dicentrics, inv-dup del chromosomes, excluding the recurrent inv-dup del(8p)s which are known to originate by NAHR at the maternal meiosis. The literature has shown that U-type dicentrics leading to nearly complete trisomy and therefore incompatible with zygotic survival underlie many types of de novo unbalanced rearrangements, including terminal deletions. In the early embryo, the position of the postzygotic breaks of the dicentric, the different ways of acquiring telomeres by the broken portions and the selection of the most favorable cell lines in the different tissues determine the prevalence of one or the other rearrangement. Multiple lines with simple terminal deletions, inv-dup dels, unbalanced translocations and segmental UPDs can coexist in various mosaic combinations although it is rare to identify them all in the blood. Regarding the origin of the dicentric, among the 30 cases of non-recurrent inv-dup del with sufficient genotyping information, paternal origin was markedly prevalent with consistently identical polymorphisms within the duplication region, regardless of parental origin. The non-random parental origin made any postzygotic origin unlikely and suggested the occurrence of these dicentrics mainly in spermatogenesis. This study strengthens the evidence that non-recurrent de novo structural rearrangements are often secondary to the rescue of a zygotic genome incompatible with embryo survival.

Low-level complex mosaic with multiple cell lines affecting the 18q21.31q21.32 region in a patient with de novo 18q terminal deletion.

We describe a 5-year-old girl who was diagnosed at birth with 18q de novo homogeneous deletion at G-banding karyotype. Her clinical condition, characterized by hypotonia, psychomotor retardation, short stature, deafness secondary to bilateral atresia of the external auditory canals, was in agreement with the 18q deletion syndrome though presence of coloboma of a single eye only suggested a mosaic condition as an unusual sign. By combining multiple technologies including array-CGH, FISH, and WGS, we found that the terminal deletion 18q21.32q23 (21 Mb) was in segmental mosaicism of the proximal region 18q21.31q21.32 (2.7 Mb), which showed a variable number of copies: one, two, or three, in 7, 41 and 55% of the cells respectively. Breakpoint junction analysis demonstrated the presence of an inv-dup del (18q) with a disomic segment of 4.7 kb between the inverted and non-inverted copies of the duplicated region 18q21.31q21.32. From these results, we propose that all three types of abnormal chr18 (the inv-dup del and the two 18q terminal deletions of different sizes) arisen from breaks in a dicentric mirror chromosome 18q, either in more than one embryo cell or from subsequent breaking-fusion-bridge cycles. The duplication region was with identical polymorphisms as in all non-recurrent inv-dup del rearrangements though, in contrast with most of them, the 18q abnormality was of maternal origin. Taking into account that distal 18q deletions are not rarely associated with inv-dup del(18q) cell lines, and that the non-disjunction of chromosome 18 takes place especially at maternal meiosis II rather than meiosis I, multiple rescue events starting from trisomic zygotes could be considered alternative to the postmitotic ones. From the clinical point of view, our case, as well as those of del(18q) in mosaic with the dic(18q), shows that the final phenotype is the sum of the different cell lines that acted on embryonic development with signs typical of both the 18q deletion syndrome and trisomy 18. Asymmetrical malformations, such as coloboma of the iris only in the right eye, confirm the underlying mosaicism regardless of whether it is still detectable in the blood.

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.

Definition of the neurological phenotype associated with dup (X)(p11.22-p11.23).

The aim of this study was to describe in detail the neurological features of nine patients carrying the recently reported microduplication at Xp11.22-11.23. Clinical and neurological examination, brain magnetic resonance imaging (except for two patients), electroencephalography and a neuropsychological assessment specific for language disturbances were performed in nine patients with microduplication at Xp11.22-11.23, disclosed by comparative genomic hybridisation array. Six patients were familial cases belonging to three unrelated pedigrees and three were sporadic cases. The patients had the following characteristics: mild dysmorphic facial features (except for two patients), mental retardation with moderate to severe global language deterioration, electroencephalographic epileptiform discharges during wakefulness and especially during sleep or electrical status epilepticus during slow sleep in younger cases, and negative brain magnetic resonance imaging. The main clinical features of this new microduplication syndrome were mild facial dysmorphisms, from increased electroencephalogram abnormalities during sleep to electrical status epilepticus during slow sleep, and mental retardation mainly involving language function in the absence of detectable brain lesions. In the absence of detectable brain lesions, speech delay may be associated with electrical status epilepticus during slow sleep or, alternatively, related to abnormal brain expression of a dosage-sensitive gene contained within the duplication region.

Long-term follow-up in a cohort of children with isolated corpus callosum agenesis at fetal MRI.

OBJECTIVE: This long-term retrospective follow-up study aimed to address the knowledge gap between prenatal diagnosis of complete isolated Agenesis of Corpus Callosum (cACC) at fetal MRI and postnatal neurodevelopmental outcome to improve prenatal counseling for parents. METHODS: Data on fetuses with isolated cACC from a single-center MRI database built up in two decades were considered. Detailed postnatal clinical, neuropsychological evaluations were performed and descriptions of available neuroradiological and genetic data were provided. RESULTS: Following a detailed neuropsychological evaluation and a long-term follow-up, the subsequent results emerged: 38 school-aged children (older than 6 years) of 50 (aged 2.5-15 years) showed normal intellectual functions (50%), intellectual disability (21%), and borderline intelligence quotient (29%). Deficits in motor functions (58%), executive functions (37%), language (61%), memory abilities (58%), and academic performances (53%) were found. Twenty-one percent of participants showed behavioral difficulties. Almost half of the participants underwent rehabilitation. Additional findings (21%) were detected at postnatal brain MRI, and a significant association between additional findings at postnatal imaging and abnormal neurodevelopmental outcome was observed. INTERPRETATIONS: This study supports the view that children with prenatal diagnosis of isolated cACC may present with several degrees of neurologic and neuropsychological impairment which become more evident only in their second decade of life. Postnatal MRI and detailed genetic analysis may add crucial information to prenatal data and substantially influence final judgment on the outcome and orient clinical management and counseling.

Targeted next-generation sequencing identifies the disruption of the SHANK3 and RYR2 genes in a patient carrying a de novo t(1;22)(q43;q13.3) associated with signs of Phelan-McDermid syndrome.

BACKGROUND: It has been known for more than 30 years that balanced translocations, especially if de novo, can associate with congenital malformations and / or neurodevelopmental disorders, following the disruption of a disease gene or its cis-regulatory elements at one or both breakpoints. CASE PRESENTATION: We describe a 10-year-old girl with a non-specific neurodevelopmental disorder characterized by moderate intellectual disability (ID), gross motor clumsiness, social and communication deficits. She carries a de novo reciprocal translocation between chromosomes 1q43 and 22q13.3, the latter suggesting the involvement of SHANK3. Indeed, its haploinsufficiency associates with Phelan-McDermid Syndrome, whose main symptoms are characterized by global developmental delay and absent or severely delayed expressive speech. A deep molecular approach, including next-generation sequencing of SHANK3 locus, allowed demonstrating the breakage of RYR2 and SHANK3 on the derivative chromosomes 1 and 22 respectively, and the formation of two fusion genes SHANK3-RYR2 and RYR2-SHANK3 with concomitant cryptic deletion of 3.6 and 4.1 kilobases at translocation junction of both derivatives chromosomes 22 and 1, respectively. CONCLUSIONS: Although the interruption of SHANK3 accounts for the patient's psychomotor retardation and autism-like behavior, we do not exclude that the interruption of RYR2 may also have a role on her disorder, or result in further pathogenicity in the future. Indeed, RYR2 that has a well-established role in the etiology of two autosomal dominant adulthood cardiac disorders (#600996 and #604772) is also expressed in the brain (cerebellum, hippocampus, and cerebral cortex) and about half of RYR2 mutation carriers present late onset primary generalized epilepsy without cardiac arrhythmogenic disorders. Moreover, RYR2 variants have also been sporadically reported in individuals with early onset schizophrenia or ID, and its constraint values suggest intolerance to loss-of-function. This study not only confirms the usefulness of the molecular mapping of de novo balanced rearrangements in symptomatic individuals, but also underscores the need for long-term clinical evaluation of the patients, for better evaluating the pathogenicity of the chromosomal breakpoints.

Detailed phenotype-genotype study in five patients with chromosome 6q16 deletion: narrowing the critical region for Prader-Willi-like phenotype.

Most patients with an interstitial deletion of 6q16 have Prader-Willi-like phenotype, featuring obesity, hypotonia, short hands and feet, and developmental delay. In all reported studies, the chromosome rearrangement was detected by karyotype analysis, which provides an overview of the entire genome but has limited resolution. Here we describe a detailed clinical presentation of five patients, two of whom were previously reported, with overlapping interstitial 6q16 deletions and Prader-Willi-like phenotype. Our patients share the following main features with previously reported cases: global developmental delay, hypotonia, obesity, hyperphagia, and eye/vision anomalies. All rearrangement breakpoints have been accurately defined through array-CGH at about 100 Kb resolution. We were able to narrow the shortest region of deletion overlap for the presumed gene(s) involved in the Prader-Willi-like syndrome to 4.1 Mb located at 6q16.1q16.2. Our results support the evidence that haploinsufficiency of the SIM1 gene is responsible for obesity in these patients. A possible involvement of the GRIK2 gene in autistic-like behaviour, of POPDC3 in heart development, and of MCHR2 in the control of feeding behaviour and energy metabolism is also hypothesized.

Concurrent transposition of distal 6p and 20q to the 22q telomere: a recurrent benign chromosomal variant.

We report the second instance of a complex unbalanced rearrangement consisting of distal trisomy 6p and 20q due to the concurrent transposition of distal 6p and 20q to the 22q telomere, previously described as a benign familial chromosomal variant. In the previous case, the nonpathogenicity of the rearrangement was based on the absence of genotypic differences between the affected proband and his normal father, and on the absence of imprinted genes in the unbalanced region. We now describe the same variant in an unrelated affected subject, in whom testing confirmed the diagnosis of Angelman syndrome, and in his healthy father. Molecular investigations confirmed that the two families have an identical subtelomeric rearrangement. However, genotyping of the flanking sequences on 22q showed a completely different pattern in the two families, demonstrating that they are indeed unrelated. Array-CGH analysis with a resolution of approximately 20 kb (Kit 244A, Agilent) defined a deletion size of 5.9 Mb on 15q11.2. No other imbalances were visible at subtelomeric regions. Further Array-CGH analysis using DNA of the proband (as test) and his mother (as reference) did not detect any duplication at the 6p and 20q subtelomeric regions. The proband and his father appear to have a copy number of the transposed regions equal to that of individuals with a normal repartition of the subtelomeric regions. This is not suggestive of a trisomy but rather of CNV regions. This type of rearrangement could define a new class of polymorphic variants, i.e. positional variants, as observed for pericentromeric heterochromatin.

Refinement of the critical 7p22.1 deletion region: Haploinsufficiency of ACTB is the cause of the 7p22.1 microdeletion-related developmental disorders.

Non-recurrent microdeletion (≤2 Mb in size) in 7p22.1 is a rarely described cytogenetic aberration, only recently reported in patients with developmental delay/intellectual disability, short stature and microcephaly. The size of the deletions ranged from 0.37 to 1.5 Mb, and reported genotype-phenotype correlations identified a minimum deleted region of 0.37 Mb involving the FBLX18, ACTB, FSCN1, RNF216 and ZNF815P genes. The authors suggested that deletion of ACTB, which encodes ß-actin, an essential component of the cytoskeleton, could be responsible for the clinical features observed in the patients with a 7p22.1 microdeletion. Here, we describe a 23-month-old child displaying developmental delay, short stature, microcephaly and distinctive facial features. Chromosomal microarray analysis performed using high-resolution SNP-array platform revealed a de novo interstitial 60 Kb microdeletion in the 7p22.1 region (from 5,509,127 bp to 5,569,096 bp, hg19) encompassing only two genes: FBXL18 and ACTB. To the best of our knowledge, this is the smallest deletion at 7p22.1 to date reported in medical literature (Pubmed). Combining our data with phenotypic and genotypic data of cases from literature, we were able to narrow the minimal critical region, which contained only two genes, i.e., FBXL18 and ACTB. Our finding is useful to perform a more accurate genotype-phenotype correlation and strongly strengthen the hypothesis that haploinsufficiency of ACTB is the main cause of the clinical phenotype observed in the patients with 7p22.1 microdeletions, facilitating genetic diagnosis and counseling.

Two classes of low-copy repeats comediate a new recurrent rearrangement consisting of duplication at 8p23.1 and triplication at 8p23.2.

We describe a new type of rearrangement consisting of the duplication of 8p23.1 and the triplication of 8p23.2 [dup trp(8p)] in two patients affected by mental retardation and minor facial dysmorphisms. Array-comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH), and genotyping of polymorphic loci allowed us to demonstrate that this rearrangement is mediated by the combined effects of two unrelated low-copy repeats (LCRs). The first set of LCRs consists of the two clusters of olfactory receptor genes (OR-REPs) lying at 8p23.1. The second type of LCRs consists of a 15-kb segmental duplication, lying in inverted orientation at 8p23.2 and enclosing a nonrepeated sequence of approximately 130 kb, named MYOM2-REP because of its proximity to the MYOM2 gene. The molecular characterization of a third case with a dicentric chromosome 8 demonstrated that the rearrangement had been generated by nonallelic homologous recombination between the two MYOM2-REPs. Based on our findings, we propose a model showing that a second recombination event at the level of the OR-REPs leads to the formation of the dup trp(8p) chromosome. This rearrangement can only arise during meiosis in heterozygous carriers of the polymorphic 8p23.1 inversion, whereas in subjects with noninverted chromosomes 8 or homozygous for the inversion only the dicentric chromosome can be formed. Our study demonstrates that nonallelic homologous recombination involving multiple LCRs can generate more complex rearrangements and cause a greater variety of genomic diseases.