Margherita Silengo

Microdeletions in the human H19 DMR result in loss of IGF2 imprinting and Beckwith-Wiedemann syndrome.

The overgrowth- and tumor-associated Beckwith-Wiedemann syndrome results from dysregulation of imprinted genes on chromosome 11p15.5. Here we show that inherited microdeletions in the H19 differentially methylated region (DMR) that abolish two CTCF target sites cause this disease. Maternal transmission of the deletions results in hypermethylation of the H19 DMR, biallelic IGF2 expression, H19 silencing and Beckwith-Wiedemann syndrome, indicative of loss of function of the IGF2-H19 imprinting control element.

Cryptic deletions are a common finding in “balanced” reciprocal and complex chromosome rearrangements: a study of 59 patients

Using array comparative genome hybridisation (CGH) 41 de novo reciprocal translocations and 18 de novo complex chromosome rearrangements (CCRs) were screened. All cases had been interpreted as “balanced” by conventional cytogenetics. In all, 27 cases of reciprocal translocations were detected in patients with an abnormal phenotype, and after array CGH analysis, 11 were found to be unbalanced. Thus 40% (11 of 27) of patients with a “chromosomal phenotype” and an apparently balanced translocation were in fact unbalanced, and 18% (5 of 27) of the reciprocal translocations were instead complex rearrangements with >3 breakpoints. Fourteen fetuses with de novo, apparently balanced translocations, all but two with normal ultrasound findings, were also analysed and all were found to be normal using array CGH. Thirteen CCRs were detected in patients with abnormal phenotypes, two in women who had experienced repeated spontaneous abortions and three in fetuses. Sixteen patients were found to have unbalanced mutations, with up to 4 deletions. These results suggest that genome-wide array CGH may be advisable in all carriers of “balanced” CCRs. The parental origin of the deletions was investigated in 5 reciprocal translocations and 11 CCRs; all were found to be paternal. Using customised platforms in seven cases of CCRs, the deletion breakpoints were narrowed down to regions of a few hundred base pairs in length. No susceptibility motifs were associated with the imbalances. These results show that the phenotypic abnormalities of apparently balanced de novo CCRs are mainly due to cryptic deletions and that spermatogenesis is more prone to generate multiple chaotic chromosome imbalances and reciprocal translocations than oogenesis.

Relative Burden of Large CNVs on a Range of Neurodevelopmental Phenotypes

While numerous studies have implicated copy number variants (CNVs) in a range of neurological phenotypes, the impact relative to disease severity has been difficult to ascertain due to small sample sizes, lack of phenotypic details, and heterogeneity in platforms used for discovery. Using a customized microarray enriched for genomic hotspots, we assayed for large CNVs among 1,227 individuals with various neurological deficits including dyslexia (376), sporadic autism (350), and intellectual disability (ID) (501), as well as 337 controls. We show that the frequency of large CNVs (>1 Mbp) is significantly greater for ID–associated phenotypes compared to autism (p = 9.58×10−11, odds ratio = 4.59), dyslexia (p = 3.81×10−18, odds ratio = 14.45), or controls (p = 2.75×10−17, odds ratio = 13.71). There is a striking difference in the frequency of rare CNVs (>50 kbp) in autism (10%, p = 2.4×10−6, odds ratio = 6) or ID (16%, p = 3.55×10−12, odds ratio = 10) compared to dyslexia (2%) with essentially no difference in large CNV burden among dyslexia patients compared to controls. Rare CNVs were more likely to arise de novo (64%) in ID when compared to autism (40%) or dyslexia (0%). We observed a significantly increased large CNV burden in individuals with ID and multiple congenital anomalies (MCA) compared to ID alone (p = 0.001, odds ratio = 2.54). Our data suggest that large CNV burden positively correlates with the severity of childhood disability: ID with MCA being most severely affected and dyslexics being indistinguishable from controls. When autism without ID was considered separately, the increase in CNV burden was modest compared to controls (p = 0.07, odds ratio = 2.33).

A restricted spectrum of NRAS mutations causes Noonan syndrome

Noonan syndrome, a developmental disorder characterized by congenital heart defects, reduced growth, facial dysmorphism and variable cognitive deficits, is caused by constitutional dysregulation of the RAS-MAPK signaling pathway. Here we report that germline NRAS mutations conferring enhanced stimulus-dependent MAPK activation account for some cases of this disorder. These findings provide evidence for an obligate dependency on proper NRAS function in human development and growth.

Germline BRAF mutations in noonan, LEOPARD, and cardiofaciocutaneous Syndromes: Molecular diversity and associated phenotypic spectrum

Noonan, LEOPARD, and cardiofaciocutaneous syndromes (NS, LS, and CFCS) are developmental disorders with overlapping features including distinctive facial dysmorphia, reduced growth, cardiac defects, skeletal and ectodermal anomalies, and variable cognitive deficits. Dysregulated RAS–mitogen‐activated protein kinase (MAPK) signal traffic has been established to represent the molecular pathogenic cause underlying these conditions. To investigate the phenotypic spectrum and molecular diversity of germline mutations affecting BRAF, which encodes a serine/threonine kinase functioning as a RAS effector frequently mutated in CFCS, subjects with a diagnosis of NS (N=270), LS (N=6), and CFCS (N=33), and no mutation in PTPN11, SOS1, KRAS, RAF1, MEK1, or MEK2, were screened for the entire coding sequence of the gene. Besides the expected high prevalence of mutations observed among CFCS patients (52%), a de novo heterozygous missense change was identified in one subject with LS (17%) and five individuals with NS (1.9%). Mutations mapped to multiple protein domains and largely did not overlap with cancer‐associated defects. NS‐causing mutations had not been documented in CFCS, suggesting that the phenotypes arising from germline BRAF defects might be allele specific. Selected mutant BRAF proteins promoted variable gain of function of the kinase, but appeared less activating compared to the recurrent cancer‐associated p.Val600Glu mutant. Our findings provide evidence for a wide phenotypic diversity associated with mutations affecting BRAF, and occurrence of a clinical continuum associated with these molecular lesions. Hum Mutat 0:1–8, 2009.

First Reported Patient with Human ERCC1 Deficiency Has Cerebro-Oculo-Facio-Skeletal Syndrome with a Mild Defect in Nucleotide Excision Repair and Severe Developmental Failure

Nucleotide excision repair (NER) is a genome caretaker mechanism responsible for removing helix-distorting DNA lesions, most notably ultraviolet photodimers. Inherited defects in NER result in profound photosensitivity and the cancer-prone syndrome xeroderma pigmentosum (XP) or two progeroid syndromes: Cockayne and trichothiodystrophy syndromes. The heterodimer ERCC1-XPF is one of two endonucleases required for NER. Mutations in XPF are associated with mild XP and rarely with progeria. Mutations in ERCC1 have not been reported. Here, we describe the first case of human inherited ERCC1 deficiency. Patient cells showed moderate hypersensitivity to ultraviolet rays and mitomycin C, yet the clinical features were very severe and, unexpectedly, were compatible with a diagnosis of cerebro-oculo-facio-skeletal syndrome. This discovery represents a novel complementation group of patients with defective NER. Further, the clinical severity, coupled with a relatively mild repair defect, suggests novel functions for ERCC1.

Frequency of Ret mutations in long- and short-segment Hirschsprung disease

Hirschsprung disease, or congenital aganglionic megacolon, is a genetic disorder of neural crest development affecting 1:5,000 newborns. Mutations in the RET proto‐oncogene, repeatedly identified in the heterozygous state in both long‐ and short‐segment Hirschsprung patients, lead to loss of both transforming and differentiating capacities of the activated RET through a dominant negative effect when expressed in appropriate cellular systems. The approach of single‐strand conformational polymorphism analysis established for all the 20 exons of the RET proto‐oncogene, and previously used to screen for point mutations in Hirschsprung patients allowed us to identify seven additional mutations among 39 sporadic and familial cases of Hirschsprung disease (detection rate 18%). This relatively low efficiency in detecting mutations of RET in Hirschsprung patients cannot be accounted by the hypothesis of genetic heterogeneity, which is not supported by the results of linkage analysis in the pedigrees analyzed so far. Almost 74% of the point mutations in our series, as well as in other patient series, were identified among long segment patients, who represented only 25% of our patient population. The finding of a C620R substitution in a patient affected with total colonic aganglionosis confirms the involvement of this mutation in the pathogenesis of different phenotypes (i.e., medullary thyroid carcinoma and Hirschsprung). Finally the R313Q mutation identified for the first time in homozygosity in a child born of consanguineous parents is associated with the most severe Hirschsprung phenotype (total colonic aganglionosis with small bowel involvement). Hum Mutat 9:243–249, 1997.

Distinct methylation changes at the IGF2-H19 locus in congenital growth disorders and cancer.

Background Differentially methylated regions (DMRs) are associated with many imprinted genes. In mice methylation at a DMR upstream of the H19 gene known as the Imprint Control region (IC1) is acquired in the male germline and influences the methylation status of DMRs 100 kb away in the adjacent Insulin-like growth factor 2 (Igf2) gene through long-range interactions. In humans, germline-derived or post-zygotically acquired imprinting defects at IC1 are associated with aberrant activation or repression of IGF2, resulting in the congenital growth disorders Beckwith-Wiedemann (BWS) and Silver-Russell (SRS) syndromes, respectively. In Wilms tumour and colorectal cancer, biallelic expression of IGF2 has been observed in association with loss of methylation at a DMR in IGF2. This DMR, known as DMR0, has been shown to be methylated on the silent maternal IGF2 allele presumably with a role in repression. The effect of IGF2 DMR0 methylation changes in the aetiology of BWS or SRS is unknown. Methodology/Principal Findings We analysed the methylation status of the DMR0 in BWS, SRS and Wilms tumour patients by conventional bisulphite sequencing and pyrosequencing. We show here that, contrary to previous reports, the IGF2 DMR0 is actually methylated on the active paternal allele in peripheral blood and kidney. This is similar to the IC1 methylation status and is inconsistent with the proposed silencing function of the maternal IGF2 allele. Beckwith-Wiedemann and Silver-Russell patients with IC1 methylation defects have similar methylation defects at the IGF2 DMR0, consistent with IC1 regulating methylation at IGF2 in cis. In Wilms tumour, however, methylation profiles of IC1 and IGF2 DMR0 are indicative of methylation changes occurring on both parental alleles rather than in cis. Conclusions/Significance These results support a model in which DMR0 and IC1 have opposite susceptibilities to global hyper and hypomethylation during tumorigenesis independent of the parent of origin imprint. In contrast, during embryogenesis DMR0 is methylated or demethylated according to the germline methylation imprint at the IC1, indicating different mechanisms of imprinting loss in neoplastic and non-neoplastic cells.

Ectodermal dysplasias: not only ‘skin’ deep

The ectodermal dysplasias (EDs) are a large and complex nosologic group of diseases; more than 170 different pathologic clinical conditions have been identified. Despite the great number of EDs described so far, few causative genes have been identified. We review EDs in the light of the most recent molecular findings and propose a new classification of EDs integrating both molecular‐genetic data and corresponding clinical findings of related diseases.

The KCNQ1OT1 Imprinting Control Region and non-coding RNA: new properties derived from the study of Beckwith-Wiedemann syndrome and Silver-Russell syndrome cases

A cluster of imprinted genes at chromosome 11p15.5 is associated with the growth disorders, Silver–Russell syndrome (SRS) and Beckwith–Wiedemann syndrome (BWS). The cluster is divided into two domains with independent imprinting control regions (ICRs). We describe two maternal 11p15.5 microduplications with contrasting phenotypes. The first is an inverted and in cis duplication of the entire 11p15.5 cluster associated with the maintenance of genomic imprinting and with the SRS phenotype. The second is a 160 kb duplication also inverted and in cis, but resulting in the imprinting alteration of the centromeric domain. It includes the centromeric ICR (ICR2) and the most 5′ 20 kb of the non-coding KCNQ1OT1 gene. Its maternal transmission is associated with ICR2 hypomethylation and the BWS phenotype. By excluding epigenetic mosaicism, cell clones analysis indicated that the two closely located ICR2 sequences resulting from the 160 kb duplication carried discordant DNA methylation on the maternal chromosome and supported the hypothesis that the ICR2 sequence is not sufficient for establishing imprinted methylation and some other property, possibly orientation-dependent, is needed. Furthermore, the 1.2 Mb duplication demonstrated that all features are present for correct imprinting at ICR2 when this is duplicated and inverted within the entire cluster. In the individuals maternally inheriting the 160 kb duplication, ICR2 hypomethylation led to the expression of a truncated KCNQ1OT1 transcript and to down-regulation of CDKN1C. We demonstrated by chromatin RNA immunopurification that the KCNQ1OT1 RNA interacts with chromatin through its most 5′ 20 kb sequence, providing a mechanism likely mediating the silencing activity of this long non-coding RNA.