Sandra Monfort

Detection of known and novel genomic rearrangements by array based comparative genomic hybridisation: deletion of ZNF533 and duplication of CHARGE syndrome genes

Background: Mental retardation can be caused by copy number variations (deletions, insertions, duplications), ranging in size from 1 kb to several megabases. Array based comparative genomic hybridisation (array-CGH) allows detection of an increasing number of genomic alterations. Methods: A series of 46 patients with mental retardation and congenital abnormalities (previously screened for subtelomeric rearrangements) were evaluated for cryptic chromosomal imbalances by array-CGH. This array contains 6465 large-insert BAC/PAC clones, representing sequences uniformly distributed throughout the human genome. The results were confirmed by alternative techniques. Results: Four pathogenic rearrangements were detected: two of them were novel, a deletion at 2q31.2 and a duplication at 8q12 band; the other two have been previously reported—a duplication of the Williams–Beuren region and a deletion of 3q29. By adding the subtelomeric alterations previously identified, a total rate of 18% of pathogenic rearrangements was found in the series. Conclusion: Based on our results, ZNF533 is the only gene contained in the overlapping region with other deletions at 2q31.2, and it is most probably the fourth zinc-finger gene implied in mental retardation. On the other hand, we propose that the CHD7 gene, associated with CHARGE syndrome by haploinsufficiency, causes a different phenotype by gain-of-dosage.

TAF1 Variants Are Associated with Dysmorphic Features, Intellectual Disability, and Neurological Manifestations.

We describe an X-linked genetic syndrome associated with mutations in TAF1 and manifesting with global developmental delay, intellectual disability (ID), characteristic facial dysmorphology, generalized hypotonia, and variable neurologic features, all in male individuals. Simultaneous studies using diverse strategies led to the identification of nine families with overlapping clinical presentations and affected by de novo or maternally inherited single-nucleotide changes. Two additional families harboring large duplications involving TAF1 were also found to share phenotypic overlap with the probands harboring single-nucleotide changes, but they also demonstrated a severe neurodegeneration phenotype. Functional analysis with RNA-seq for one of the families suggested that the phenotype is associated with downregulation of a set of genes notably enriched with genes regulated by E-box proteins. In addition, knockdown and mutant studies of this gene in zebrafish have shown a quantifiable, albeit small, effect on a neuronal phenotype. Our results suggest that mutations in TAF1 play a critical role in the development of this X-linked ID syndrome.

Corpus Callosum Abnormalities and the Controversy about the Candidate Genes Located in 1q44

Submicroscopic deletions of 1q44–qter cause severe mental retardation, profound growth retardation, microcephaly and corpus callosum hypo/agenesis in most patients. At least 3 intervals in 1q44 have been described as critical regions containing genes leading to corpus callosum abnormalities. In this report we describe a patient with a de novo small interstitial 1q44 deletion of 1,152 kb detected with 44K oligonucleotide array-CGH (44K Agilent Technologies) and a mild phenotype lacking corpus callosum abnormalities. The first deleted oligonucleotide was located at 242.638 Mb (within the ADSS gene), and the last deleted oligonucleotide at 243.791 Mb (within the KIF26B gene). The clinical and molecular findings of the patient here reported remain consistent with a role for the AKT3 or ZNF238 genes in corpus callosum development.

High diagnostic yield of syndromic intellectual disability by targeted next-generation sequencing

Background Intellectual disability is a very complex condition where more than 600 genes have been reported. Due to this extraordinary heterogeneity, a large proportion of patients remain without a specific diagnosis and genetic counselling. The need for new methodological strategies in order to detect a greater number of mutations in multiple genes is therefore crucial. Methods In this work, we screened a large panel of 1256 genes (646 pathogenic, 610 candidate) by next-generation sequencing to determine the molecular aetiology of syndromic intellectual disability. A total of 92 patients, negative for previous genetic analyses, were studied together with their parents. Clinically relevant variants were validated by conventional sequencing. Results A definitive diagnosis was achieved in 29 families by testing the 646 known pathogenic genes. Mutations were found in 25 different genes, where only the genes KMT2D, KMT2A and MED13L were found mutated in more than one patient. A preponderance of de novo mutations was noted even among the X linked conditions. Additionally, seven de novo probably pathogenic mutations were found in the candidate genes AGO1, JARID2, SIN3B, FBXO11, MAP3K7, HDAC2 and SMARCC2. Altogether, this means a diagnostic yield of 39% of the cases (95% CI 30% to 49%). Conclusions The developed panel proved to be efficient and suitable for the genetic diagnosis of syndromic intellectual disability in a clinical setting. Next-generation sequencing has the potential for high-throughput identification of genetic variations, although the challenges of an adequate clinical interpretation of these variants and the knowledge on further unknown genes causing intellectual disability remain to be solved.

De novo Interstitial Triplication of MECP2 in a Girl with Neurodevelopmental Disorder and Random X Chromosome Inactivation

Loss-of-function mutations of the MECP2 gene are the cause of most cases of Rett syndrome in females, a progressive neurodevelopmental disorder characterized by severe mental retardation, global regression, hand stereotypies, and microcephaly. On the other hand, gain of dosage of this gene causes the MECP2 duplication syndrome in males characterized by severe mental retardation, absence of speech development, infantile hypotonia, progressive spasticity, recurrent infections, and facial dysmorphism. Female carriers of a heterozygous duplication show a skewed X-inactivation pattern which is the most probable cause of the lack of clinical symptoms. In this paper, we describe a girl with a complex de novo copy number gain at Xq28 and non-skewed X-inactivation pattern that causes mental retardation and motor and language delay. This rearrangement implies triplication of the MECP2 and IRAK1 genes, but it does not span other proximal genes located in the common minimal region of patients affected by the MECP2 duplication syndrome. We conclude that the triplication leads to a severe phenotype due to random X-inactivation, while the preferential X chromosome inactivation in healthy carriers may be caused by a negative selection effect of the duplication on some proximal genes like ARD1A or HCFC1.

Duplication of 14q11.2 associates with short stature and mild mental retardation: A putative relation with quantitative trait loci†

Submicroscopic chromosomal rearrangements are a frequent cause of mental retardation in patients with additional phenotypic features. The clinical consequences of these genomic copy-number abnormalities are determined by the kind of alteration (deletion or duplication) and by the number and function of those genes affected by the dosage alteration [Biesecker, 2002; de Vries et al., 2005]. However, the identification of the specific genes directly implied in the phenotypic consequences is a question far from being resolved. Here we report our findings on a patient with short stature and mild mental retardation with a de novo duplication of 14q11.2, a region where significant linkage of quantitative trait loci (QTL) for stature or for a component of the intelligencehavebeen reported [Beck et al., 2003; Buyske et al., 2006]. The patient is a 14-year-old male referred for short stature (<3rd centile), hypogenitalism and mild mental retardation. He is the second child of healthy non-consanguineous parents. After pre-term delivery (38 weeks), the following signs or conditions were recorded: birth weight 2,350 g (<3rd centile), length 45 cm (<3rd centile), occipitofrontal circumference33 cm(3rd–10th centile), iris colobomaat the left eye and retrognathia. Psychomotor development was subsequently delayed: he started to walk at 16 months of age, and he spoke his first words at 2 years old. After 11 years pubertal development stopped and growth development declined dramatically. Upon clinical examination at 13 years, he showed short stature (144 cm; <3rd centile), mild hypogenitalism, and dysmorphic features including micrognathia, a bulbous nose, short philtrum, thin lips, clinodactyly and bilateral partial syndactyly between toes 2–3 (see Fig. 1). Hormone determinations, including FSH, LH, testosterone and GH, were reported at normal levels. No relevant family history was reported, except for short stature of the paternal grandmother and one half-sibling of the father. Blood samples of the patient, and the parents were obtained after informed consent. Routine chromosome analysis was normal. However, one 14q11 copy-number gain was detected by quantitative analysis of the subtelomeric regionswith the use of MLPA (SALSA P036B and P019 from MRCHolland,Amsterdam, TheNetherlands), as described elsewhere [Monfort et al., 2006], where this case was referred as case 90. The duplication was validated by microsatellite segregation analysis with markers D14S72, D14S1023, and D14S990. In addition, MLPA testing on the parental samples allowed to establish a de novo condition in the patient. On the other hand, FISH studies with BAC DNA clone RP11-52401 ruled out the possibility of a cryptic chromosomal translocation with another acrocentric chromosome. In order to determine the size of the duplication, the patient’s DNA sample was tested against a pool of normal DNA samples by array-based comparative genomic hybridization (human genome CGH microarray G4410B from Agilent Technologies, Palo Alto,

Enrichment of ultraconserved elements among genomic imbalances causing mental delay and congenital anomalies

BackgroundThe ultraconserved elements (UCEs) are defined as stretches of at least 200 base pairs of human DNA that match identically with corresponding regions in the mouse and rat genomes, albeit their real significance remains an intriguing issue. These elements are most often located either overlapping exons in genes involved in RNA processing or in introns or nearby genes involved in the regulation of transcription and development. Interestingly, human UCEs have been reported to be strongly depleted among segmental duplications and benign copy number variants (CNVs). However no comprehensive survey of a putative enrichment of these elements among pathogenic dose variants has yet been reported.ResultsA survey for UCEs was performed among the 26 cryptic genomic rearrangements detected in our series of 200 patients with idiopathic neurodevelopmental disorders associated to congenital anomalies. A total of 29 elements, out of the 481 described UCEs, were contained in 13 of the 26 pathogenic gains or losses detected in our series, what represents a highly significant enrichment of ultraconserved elements. In addition, here we show that these elements are preferentially found in pathogenic deletions (enrichment ratio 3.6 vs. 0.5 in duplications), and that this association is not related with a higher content of genes. In contrast, pathogenic CNVs lacking UCEs showed almost a threefold higher content in genes.ConclusionsWe propose that these elements may be interpreted as hallmarks for dose-sensitive genes, particularly for those genes whose gain or loss may be directly implied in neurodevelopmental disorders. Therefore, their presence in genomic imbalances of unknown effect might be suggestive of a clinically relevant condition.

Novel UBE3A mutations causing Angelman syndrome: Different parental origin for single nucleotide changes and multiple nucleotide deletions or insertions†

Angelman syndrome (AS) is a genetic disorder caused by a deficiency of UBE3A imprinted gene expression from the maternal chromosome 15. In 10% of AS cases the genetic cause is a mutation affecting the maternal copy of the UBE3A gene. In two large Spanish series of clinically stringently selected and nonstringently selected patients, we have identified 11 pathological mutations—eight of them novel mutations—and 14 sequence changes considered polymorphic variants. Remarkably, single nucleotide substitutions are more likely to be inherited, while multiple nucleotide deletions or insertions are less frequently inherited, thus indicating that single nucleotide substitutions are more likely to originate from the paternal germline. Additionally, there seems to be a different distribution of nucleotide changes and multiple nucleotide deletions or insertions along the UBE3A gene sequence.

Intronic mutations affecting splicing of MBTPS2 cause ichthyosis follicularis, alopecia and photophobia (IFAP) syndrome

Abstract:  Ichthyosis follicularis, alopecia and photophobia (IFAP) syndrome is an X‐linked genodermatosis with congenital atrichia being the most prominent feature. Recently, we have shown that functional deficiency of MBTPS2 (membrane‐bound transcription factor protease site 2) – a zinc metalloprotease essential for cholesterol homeostasis and endoplasmic reticulum stress response – causes the disease. Here, we present results obtained by analysing two intronic MBTPS2 mutations, c.671‐9T>G and c.225‐6T>A, using in silico and cell‐based splicing assays. Accordingly, the c.225‐6T>A transversion generated a new splice acceptor site, which caused extension of exon 3 by four bases and subsequently introduced a premature stop codon. Both, minigene experiments and RT‐PCR analysis with patient‐derived mRNA, demonstrated that the c.671‐9T>G mutation resulted in skipping of exon 6, most likely because of disruption of the polypyrimidin tract or a putative intronic splicing enhancer (ISE). Our combined biocomputational and experimental analysis strongly suggested that both intronic alterations are disease‐causing mutations.

Phenotype profiling of patients with intellectual disability and copy number variations

BACKGROUND Nowadays the microarray technology allows whole-genome analysis with a high resolution and performance for the genetic diagnosis in any patient with intellectual disability or autism spectrum disorder. However in the immediate future, with the development of massive sequencing systems for application at clinical diagnosis, it will be necessary to have clinical criteria to guide studies. AIM To perform an exhaustive clinical definition of patients with pathogenic copy number variations in order to establish the clinical criteria most suggestive of this kind of genomic rearrangements. METHOD We designed and implemented a database to collect 190 different clinical variables (pregnancy, neonatal, facial dysmorphism, congenital anomalies, neurological features and family history) in a series of 246 patients, with developmental delay/intellectual disability. All cases were studied with array comparative genomic hybridization. RESULTS We have found a pathogenic genomic imbalance in 73 patients. Frequency analysis of all clinical variables showed that growth disorder, abnormalities of hands, low-set ears and hypertelorism are the more frequent features among patients with genomic rearrangements. However other clinical features, such as genital abnormalities and aggressiveness, are more specifically associated with pathogenic copy number variations in spite of their low frequencies in the overall series, yielding higher statistical significance values than other traits. CONCLUSIONS The genotype-phenotype comparison may be useful to set in the future the main clinical manifestations associated with deletions, duplications and unbalanced translocations. Theses analyses will improve the clinical indications and protocols to implement genomic arrays in the genetic study of patients with neurodevelopment disorders.