Sonia Mayo

Detection of Hypomethylation Syndrome among Patients with Epigenetic Alterations at the GNAS Locus

CONTEXT Genomic imprinting is the modification of the genome so that genes from only one (rather than two) of the parental alleles are expressed. The mechanism underlying imprinting is epigenetic, occurring via changes in DNA methylation and histone modifications rather than through alterations in the DNA sequence. To date, nine different imprinting disorders have been clinically and genetically identified and a considerable research effort has been focused on determining the cause of the corresponding methylation defects. OBJECTIVE Our objective was to identify multilocus imprinting defects and characterize any mutations in trans-acting genes in patients with pseudohypoparathyroidism (PHP) caused by epigenetic alterations at GNAS locus. DESIGN We have investigated multilocus imprinting defects in 22 PHP patients with aberrant methylation at the GNAS locus not due to previously described deletions or to paternal uniparental disomy (UPD) of chromosome 20. RESULTS We found that, in contrast to what has been described in growth disorders, multilocus hypomethylation is an uncommon event in PHP patients. We were also unable to identify any genetic alteration causative of the epigenetic defects in the currently known methylation regulatory genes. CONCLUSION Our work suggests that a trans-acting gene regulating the establishment or maintenance of imprinting at GNAS locus, if it exists, should be specific to PHP cases caused by epigenetic defects at GNAS.

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.

Identification of Intellectual Disability Genes in Female Patients with a Skewed X-Inactivation Pattern.

Intellectual disability (ID) is a heterogeneous disorder with an unknown molecular etiology in many cases. Previously, X‐linked ID (XLID) studies focused on males because of the hemizygous state of their X chromosome. Carrier females are generally unaffected because of the presence of a second normal allele, or inactivation of the mutant X chromosome in most of their cells (skewing). However, in female ID patients, we hypothesized that the presence of skewing of X‐inactivation would be an indicator for an X chromosomal ID cause. We analyzed the X‐inactivation patterns of 288 females with ID, and found that 22 (7.6%) had extreme skewing (>90%), which is significantly higher than observed in the general population (3.6%; P = 0.029). Whole‐exome sequencing of 19 females with extreme skewing revealed causal variants in six females in the XLID genes DDX3X, NHS, WDR45, MECP2, and SMC1A. Interestingly, variants in genes escaping X‐inactivation presumably cause both XLID and skewing of X‐inactivation in three of these patients. Moreover, variants likely accounting for skewing only were detected in MED12, HDAC8, and TAF9B. All tested candidate causative variants were de novo events. Hence, extreme skewing is a good indicator for the presence of X‐linked variants in female patients.

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.

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.

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.

De novo mutations in genes of mediator complex causing syndromic intellectual disability: mediatorpathy or transcriptomopathy?

Background:Mutations in the X-linked gene MED12 cause at least three different, but closely related, entities of syndromic intellectual disability. Recently, a new syndrome caused by MED13L deleterious variants has been described, which shows similar clinical manifestations including intellectual disability, hypotonia, and other congenital anomalies.Methods:Genotyping of 1,256 genes related with neurodevelopment was performed by next-generation sequencing in three unrelated patients and their healthy parents. Clinically relevant findings were confirmed by conventional sequencing.Results:Each patient showed one de novo variant not previously reported in the literature or databases. Two different missense variants were found in the MED12 or MED13L genes and one nonsense mutation was found in the MED13L gene.Conclusion:The phenotypic consequences of these mutations are closely related and/or have been previously reported in one or other gene. Additionally, MED12 and MED13L code for two closely related partners of the mediator kinase module. Consequently, we propose the concept of a common MED12/MED13L clinical spectrum, encompassing Opitz-Kaveggia syndrome, Lujan-Fryns syndrome, Ohdo syndrome, MED13L haploinsufficiency syndrome, and others.

Novel mutations of NFIX gene causing Marshall-Smith syndrome or Sotos-like syndrome: one gene, two phenotypes

Background:Only 15 point mutations in NFIX gene have been reported so far, nine of them cause the Marshall-Smith syndrome (MSS) and the remaining mutations lead to an overgrowth disorder with a less severe phenotype, defined as Sotos-like.Methods:The clinical findings in three patients with MSS and two patients with a Sotos-like phenotype are presented. Analysis of the NFIX gene was performed both by conventional or next-generation sequencing.Results:Five de novo mutations in NFIX gene were identified, four of them not previously reported. Two frameshift mutations and a donor-splice one caused MSS, while two missense mutations in the DNA binding/dimerisation domain entailed an overgrowth syndrome with some clinical features resembling Sotos syndrome, accompanied by a marfanoid habitus, very low BMI, long narrow face, or arachnodactyly.Conclusion:Marshall-Smith mutations are scattered through exons 6–10 of NFIX gene, while most point mutations causing an overgrowth syndrome are clustered in exon 2. Clinical features of this overgrowth syndrome may well be considered an intermediate phenotype between Sotos and Marfan syndromes.

Large deletion in the Factor VIII gene (F8) involving segmental duplications in int22h shows no haematological phenotype in female carriers, but may be embryonic lethal in males

A common inversion of the Factor VIII gene (F8) causes half of all severe haemophilia A cases. The initial hypothesis proposed a homologous recombination between repeat int22h-1, in intron 22 of F8, and one of the two duplicons, int22h-2 or int22h-3, in the reverse direction. At the present time, these duplicons are known to be inversely oriented in relation to each other, and that they form part of an imperfect palindrome with a central unique loop of 67 3 kb and arms of 50 5 kb. Bagnall et al (2006) considered that intra-chromosomal recombinations between these arms generate polymorphic inversions of the internal segment of the palindrome, so that int22h-2 changes place with int22h-3 (Fig 1A). They described a new long-polymerase chain reaction (PCR) discriminatory test for detecting type 1 and type 2 inversions or putative deletions. During the genetic studies carried out on a woman with mental retardation, with no relevant family history, a deletion of approximately 440 kb was detected in Xq28 by an arraycomparative genomic hybridization (CGH) analysis (Martı́nez et al, 2010). In addition, a completely skewed X-inactivation of the paternal X chromosome was observed by analysis of the polymorphic CAG repeat in the androgen receptor gene. The Multiplex Ligation-dependent Probe Amplification (MLPA) analysis confirmed the loss of one F8 copy from exon 1 to exon 22, although the patient did not present a haemophilic phenotype. To confirm these findings, long-PCR was carried out as previously described (Bagnall et al, 2006), with minor modifications (TaKaRa LA TaqTM with GC Buffer, Takara Bio Inc., Japan). Amplification of patient DNA with H1F, H1R, H2F and H3F primers (Fig 1A) showed two bands, of 9 and 12 7 kb, corresponding to the normal allele and to the inversion type 2, respectively, which is the pattern usually seen in heterozygous carrier females (Fig 1B, lanes 2 and 5). In contrast, amplification with primers H1R and H2F yielded a segment of approximately 12.7 kb, while no band was generated with primers H1F and H2/3R (Fig 1B, lanes 3 and 4), or with the H1R-H3F pair. These results indicated a 497 kb deletion from int22h-1 to int22h-2, in accordance with the array-CGH analysis. The complete inactivation of the X chromosome bearing the deletion is most probably due to the loss of any gene contained between both segmental duplications. Analogous X-inactivation patterns have been documented in other X chromosome rearrangements with lethality in males, such as those causing incontinentia pigmenti, focal dermal hypoplasia or MIDAS syndrome (microphthalmia, dermal aplasia, sclerocornea) among others, as well as in various X-linked non-lethal phenotypes (reviewed in Van den Veyver, 2001). Considering that the paternal X chromosome was completely inactivated, we deduced that this deletion arose de novo by non-allelic homologous intra-chromosomal recombination between the two segmental duplications int22h-1 and int22h2 in one paternal gamete (Fig 1B). Similarly, the inversion of F8 causing severe haemophilia A usually also occurs in the paternal germline, rather than in maternal germline. Pegoraro et al (1997) reported a deletion of <800 kb covering at least part of F8 in Xq28. They studied a family with 50 female members and demonstrated the segregation of the preferential X chromosome inactivation associated with the deletion. Furthermore, the deletion in this family was not associated with either mental delay or any other clinical finding except for a statistically significant increase in spontaneous abortion rate in the females carrying the deletion. They hypothesized that this deletion could cause a male-lethal phenotype. Recurrent spontaneous abortion is defined as loss of three or more consecutive pregnancies prior to gestation weeks 20 –28, which affects up to 5% of fertile couples (Pandey et al, 2005). In the majority of cases, aetiology is unknown, but the knowledge of a possible cause of recurrent miscarriages is an important issue to avoid unnecessary treatments. Further studies could identify if similar deletions as those described herein could be involved in recurrent spontaneous abortion, especially in families where X-linked lethal transmission is suspected; i.e., affecting about 50% of male pregnancies and an excess of female members. In agreement with Bagnall et al (2006), the mutation rate causing deletions among int22h repeats should be at least as frequent as those causing inversions, as the latter only result from recombinations in the same chromosome, while deletions could arise through both intraand inter-chromosomal recombinations. However such recurrent deletion has never been detected in haemophilia A patients. Very recently, during the preparation of this letter, this same deletion has been reported in a girl and her mother, who also had two spontaneous abortions (El-Hattab et al, 2011). There are nine genes in the region comprising the deletion: H2AFB2, F8A1, FUNDC2, MTCP1NB, MTCP1, BRCC3,

Localización cromosómica de duplicaciones submicroscópicas en pacientes con trastornos del neurodesarrollo para identificar casos con alto riesgo de recurrencia familiar

BACKGROUND AND OBJECTIVE An important proportion of neurodevelopmental disorders (NDDs) results from unbalanced genomic alterations (duplication or deletion). These chromosomal rearrangements may be considered as de novo, despite they arise as a result of a balanced rearrangement not detected in a phenotypically normal parent. Therefore, if the rearrangements are inherited, the recurrence risk and the genetic counseling of these cases change radically. Fluorescence in situ hybridization (FISH) is a technique that allows detecting both balanced and unbalanced rearrangements, identifying also the location of duplicated segments. We tried to locate in the genome the duplicated segments detected in patients with NDDs in order to identify those cases due to inherited rearrangements. PATIENTS AND METHOD The study was conducted in 13 patients with NDDs and genomic duplications detected by compared genomic hybridization-array (CGH-array). Two approaches of FISH technique were taken: hybridization with painting chromosome probes and with specific probes for each duplication. RESULTS In the studied series of 13 patients with duplication, 11 patients were found to carry tandem duplications, one with an intrachromosomal insertional translocation, and another with an interchromosomal insertional translocation. Therefore, 2 of the duplications considered de novo were actually an unbalanced rearrangement inherited from a parent who is a balanced carrier. CONCLUSION The results illustrate the need to characterize by FISH technique the rearrangements that are detected by CGH-array to identify those cases with a high risk of recurrence.