Complete Sequence of the 22q11.2 Allele in 1,053 Subjects with 22q11.2 Deletion Syndrome Reveals Modifiers of Conotruncal Heart Defects.

The 22q11.2 deletion syndrome (22q11.2DS) results from non-allelic homologous recombination between low-copy repeats termed LCR22. About 60%-70% of individuals with the typical 3 megabase (Mb) deletion from LCR22A-D have congenital heart disease, mostly of the conotruncal type (CTD), whereas others have normal cardiac anatomy. In this study, we tested whether variants in the hemizygous LCR22A-D region are associated with risk for CTDs on the basis of the sequence of the 22q11.2 region from 1,053 22q11.2DS individuals. We found a significant association (FDR p < 0.05) of the CTD subset with 62 common variants in a single linkage disequilibrium (LD) block in a 350 kb interval harboring CRKL. A total of 45 of the 62 variants were associated with increased risk for CTDs (odds ratio [OR) ranges: 1.64-4.75). Associations of four variants were replicated in a meta-analysis of three genome-wide association studies of CTDs in affected individuals without 22q11.2DS. One of the replicated variants, rs178252, is located in an open chromatin region and resides in the double-elite enhancer, GH22J020947, that is predicted to regulate CRKL (CRK-like proto-oncogene, cytoplasmic adaptor) expression. Approximately 23% of patients with nested LCR22C-D deletions have CTDs, and inactivation of Crkl in mice causes CTDs, thus implicating this gene as a modifier. Rs178252 and rs6004160 are expression quantitative trait loci (eQTLs) of CRKL. Furthermore, set-based tests identified an enhancer that is predicted to target CRKL and is significantly associated with CTD risk (GH22J020946, sequence kernal association test (SKAT) p = 7.21 × 10-5) in the 22q11.2DS cohort. These findings suggest that variance in CTD penetrance in the 22q11.2DS population can be explained in part by variants affecting CRKL expression.

Common atrium/atrioventricular canal defect and postaxial polydactyly: A mild clinical subtype of Ellis-van Creveld syndrome caused by hypomorphic mutations in the EVC gene.

Clinical expression of Ellis-van Creveld syndrome (EvC) is variable and mild phenotypes have been described, including patients with mostly cardiac and limb involvement. Whether these cases are part of the EvC phenotypic spectrum or separate conditions is disputed. Herein, we describe a family with vertical transmission of atrioventricular canal defect (AVCD), common atrium, and postaxial polydactyly. Targeted sequencing of EVC, EVC2, WDR35, DYNC2LI1, and DYNC2H1 identified different compound heterozygosity in EVC genotypes in the two affected members, consisting of a nonsense (p.Arg622Ter) and a missense (p.Arg663Pro) variant in the father, and the same nonsense variant and a noncanonical splice-site in-frame change (c.1316-7A>G) in the daughter. Complementary DNA sequencing, immunoblot, and immunofluorescence experiments using patient-derived fibroblasts and Evc-/- mouse embryonic fibroblasts showed that p.Arg622Ter is a loss-of-function mutation, whereas p.Arg663Pro and the splice-site change c.1316-7A>G are hypomorphic variants resulting in proteins that retain, in part, the ability to complex with EVC2. Our molecular and functional data demonstrate that at least in some cases the condition characterized as "common atrium/AVCD with postaxial polydactyly" is a mild form of EvC due to hypomorphic EVC mutations, further supporting the occurrence of genotype-phenotype correlations in this syndrome.

Familial aggregation of "apple peel" intestinal atresia and cardiac left-sided obstructive lesions: A possible causal relationship with NOTCH1 gene mutations.

"Apple peel" intestinal atresia is a rare form of small bowel atresia, in which the duodenum or proximal jejunum ends in a blind pouch and the distal small bowel wraps around its vascular supply, in a spiral resembling an apple peel. The etiology of "apple peel" intestinal atresia is presently unknown, although a congenital or acquired intestinal vascular accident can have a role in the pathogenesis. We report a family in which the proband affected by "apple peel" intestinal atresia, had a sibling (an interrupted pregnancy), and a paternal cousin with cardiac left-sided obstructive lesions. Molecular testing for NOTCH1 gene was carried out in the proband, because pathogenic mutations in this gene have been associated with familial and sporadic cardiac left-sided obstructive lesions and vascular anomalies, both isolated or within the spectrum of the Adams-Oliver syndrome (AOS). The heterozygous c.2734C>T (p.Arg912Trp) NOTCH1 variant was found in the proband with "apple peel" intestinal atresia and in his father. This result argues for a possible causal relationship between NOTCH1 gene mutations and some forms of intestinal defects, through a vascular mechanism. The spectrum of NOTCH1-associated malformations is widened. Genetic counseling should take into account intrafamilial variable clinical expression and incomplete penetrance.

Hypoplastic left heart syndrome and 21q22.3 deletion.

Hypoplastic left heart syndrome (HLHS) is a rare congenital heart defect (CHD), associated with extracardiac anomalies in the 15-28% of cases, in the setting of chromosomal anomalies, mendelian disorders, and organ defects. We report on a syndromic female newborn with HLHS and terminal 21q22.3 deletion (del 21q22.3), investigated by Fluorescence In Situ Hybridization (FISH) using a panel of 26 contiguous BAC probes. Although rare, del 21q22.3 has been described in two additional patients with HLHS. In order to investigate the frequency and role of this chromosomal imbalance in the pathogenesis of left-sided obstructive heart defects, we screened for del 21q22.3 a series of syndromic and non-syndromic children with HLHS, aortic coarctation and valvular aortic stenosis, consecutively admitted to our hospital in a three-year period. Although none of the 56 analyzed patients were hemizygous for this region, the present case report and published patients argue that del 21q22 should be added to the list of chromosomal imbalances associated with HLHS. Accordingly, the presence of a cardiac locus mapping in the critical region cannot be excluded.

Chromatin regulators in the TBX1 network confer risk for conotruncal heart defects in 22q11.2DS.

Congenital heart disease (CHD) affecting the conotruncal region of the heart, occurs in 40-50% of patients with 22q11.2 deletion syndrome (22q11.2DS). This syndrome is a rare disorder with relative genetic homogeneity that can facilitate identification of genetic modifiers. Haploinsufficiency of TBX1, encoding a T-box transcription factor, is one of the main genes responsible for the etiology of the syndrome. We suggest that genetic modifiers of conotruncal defects in patients with 22q11.2DS may be in the TBX1 gene network. To identify genetic modifiers, we analyzed rare, predicted damaging variants in whole genome sequence of 456 cases with conotruncal defects and 537 controls, with 22q11.2DS. We then performed gene set approaches and identified chromatin regulatory genes as modifiers. Chromatin genes with recurrent damaging variants include EP400, KAT6A, KMT2C, KMT2D, NSD1, CHD7 and PHF21A. In total, we identified 37 chromatin regulatory genes, that may increase risk for conotruncal heart defects in 8.5% of 22q11.2DS cases. Many of these genes were identified as risk factors for sporadic CHD in the general population. These genes are co-expressed in cardiac progenitor cells with TBX1, suggesting that they may be in the same genetic network. The genes KAT6A, KMT2C, CHD7 and EZH2, have been previously shown to genetically interact with TBX1 in mouse models. Our findings indicate that disturbance of chromatin regulatory genes impact the TBX1 gene network serving as genetic modifiers of 22q11.2DS and sporadic CHD, suggesting that there are some shared mechanisms involving the TBX1 gene network in the etiology of CHD.

Overt cleft palate phenotype and TBX1 genotype correlations in velo-cardio-facial/DiGeorge/22q11.2 deletion syndrome patients.

Velo-cardio-facial syndrome/DiGeorge syndrome, also known as 22q11.2 deletion syndrome (22q11DS) is the most common microdeletion syndrome, with an estimated incidence of 1/2,000-1/4,000 live births. Approximately 9-11% of patients with this disorder have an overt cleft palate (CP), but the genetic factors responsible for CP in the 22q11DS subset are unknown. The TBX1 gene, a member of the T-box transcription factor gene family, lies within the 22q11.2 region that is hemizygous in patients with 22q11DS. Inactivation of one allele of Tbx1 in the mouse does not result in CP, but inactivation of both alleles does. Based on these data, we hypothesized that DNA variants in the remaining allele of TBX1 may confer risk to CP in patients with 22q11DS. To test the hypothesis, we evaluated TBX1 exon sequencing (n = 360) and genotyping data (n = 737) with respect to presence (n = 54) or absence (n = 683) of CP in patients with 22q11DS. Two upstream SNPs (rs4819835 and rs5748410) showed individual evidence for association but they were not significant after correction for multiple testing. Associations were not identified between DNA variants and haplotypes in 22q11DS patients with CP. Overall, this study indicates that common DNA variants in TBX1 may be nominally causative for CP in patients with 22q11DS. This raises the possibility that genes elsewhere on the remaining allele of 22q11.2 or in the genome could be relevant.

Genetic dosage compensation in a family with velo-cardio-facial/DiGeorge/22q11.2 deletion syndrome.

Cytogenetic studies of a male child carrying the 22q11.2 deletion common in patients with velo-cardio-facial/DiGeorge syndrome showed an unexpected rearrangement of the 22q11.2 region in his normal appearing mother. The mother carried a 3 Mb deletion on one copy and a reciprocal, similar sized duplication on the other copy of chromosome 22q11.2 as shown by fluorescence in situ hybridization and array comparative genome hybridization analyses. The most parsimonious mechanism for the rearrangement is a mitotic non-allelic homologous recombination event in a cell in the early embryo soon after fertilization. The normal phenotype of the mother can be explained by the theory of genetic dosage compensation. This is the second documented case of such an event for this or any genomic disorder. This finding helps to reinforce this phenomenon in a human model, and has significant implications for recurrence risks for the dose-compensated mother.

Three patients with oculo-auriculo-vertebral spectrum and microdeletion 22q11.2.

We report on three unrelated patients with the 22q11.2 microdeletion syndrome (del22q11) who have phenotypic anomalies compatible with oculo-auriculo-vertebral spectrum (OAVS). Hemifacial microsomia, unilateral microtia, hearing loss, congenital heart/aortic arch arteries defects, and feeding difficulties were present in all three patients. Additional anomalies occasionally diagnosed included coloboma of the upper eyelid, microphthalmia, cerebral malformation, palatal anomalies, neonatal hypocalcemia, developmental delay, and laryngomalacia. Several clinical features characteristic of OAVS have been described in patients with del22q11 from the literature, including ear anomalies, hearing loss, cervical vertebral malformations, conotruncal cardiac defects, renal malformations, feeding and respiratory difficulties. Atretic ear with facial asymmetry has been previously described in one patient. Thus, clinical expression of hemifacial microsomia and microtia resembling OAVS should now be included within the wide phenotypic expression of del22q11. The occurrence of this manifestation in del22q11 is currently low. Nevertheless, patients with hemifacial microsomia and microtia associated with clinical features typically associated with del22q11 should now have for specific cytogenetic testing.

Congenital heart defects in patients with oculo-auriculo-vertebral spectrum (Goldenhar syndrome).

The oculo-auriculo-vertebral spectrum (OAVS) is a non-random association of microtia, hemifacial microsomia with mandibular hypoplasia, ocular epibulbar dermoid, and cervical vertebral malformations. Congenital heart defects (CHDs) have been reported in 5-58% of the patients. We analyze the frequency and anatomic features of CHD in a series of 87 patients with OAVS examined between January 1990 and February 2007 with normal chromosomes, ranging in age between 0.1 and 16.8 years. A twin pregnancy occurred in eight cases (dizygotic in six cases and monozygotic in two). CHDs were diagnosed in 28/87 (32%) patients, and classified into categories of postulated developmental mechanisms including 9 (32%) atrial and ventricular septal defects, 11 (39%) conotruncal defects, 4 (14%) targeted growth defects, two (7%) with situs and looping defects, one (4%) with a left-sided obstructive lesion and one (4%) with patent ductus arteriosus. As noted in other series, the most common individual CHDs were ventricular septal defect (six patients) and tetralogy of Fallot (TOF) (classic or with pulmonary atresia) (six patients). Comparing the frequencies of CHDs groups observed in the OAVS patients with the findings of the Emilia-Romagna Registry which ascertained CHDs prevalence in the general population, conotruncal defects, targeted growth defects, and heterotaxia were significantly associated with OAVS.

Cardiac defects, morbidity and mortality in patients affected by RASopathies. CARNET study results.

BACKGROUND: RASopathies are developmental disease caused by mutations in genes encoding for signal transducers of the RAS-MAPK cascade. The aim of the present study was to provide a comprehensive description of morbidity and mortality in patients with molecularly confirmed RASopathy. METHODS: A multicentric, observational, retrospective study was conducted in seven European cardiac centres participating to the CArdiac Rasopathy NETwork (CARNET). Clinical records of 371 patients with confirmed molecular diagnosis of RASopathy were reviewed. Mortality was described as crude mortality, cumulative survival and restricted estimated mean survival. Multivariable regression analysis was used to assess the impact of mutated genes on number of interventions and overall prognosis. RESULTS: Cardiac defects occurred in 80.3% of cases, almost half of them underwent at least one intervention. Overall, crude mortality was 0.29/100 patients-year. Cumulative survival was 98.8%, 98.2%, 97.7%, 94.3%, at 1, 5, 10, and 20years, respectively. Restricted estimated mean survival at 20years follow-up was 19.6years. Ten patients died (2.7% of the entire cohort; 3.4% of patients with cardiac defect). Patients with hypertrophic cardiomyopathy (HCM) and age <2years or young adults, as well as subjects with biventricular obstruction and PTPN11 mutations had a higher risk of cardiac death. CONCLUSIONS: The risk of intervention was higher in individuals with Noonan syndrome and pulmonary stenosis carrying PTPN11 mutations. Overall, mortality was relatively low, even though the specific association between HCM, biventricular outflow tract obstructions and PTPN11 mutations appeared to be associated with early mortality, including immediate post-operative events and sudden death.

Genome-Wide Association Study to Find Modifiers for Tetralogy of Fallot in the 22q11.2 Deletion Syndrome Identifies Variants in the GPR98 Locus on 5q14.3.

BACKGROUND: The 22q11.2 deletion syndrome (22q11.2DS; DiGeorge syndrome/velocardiofacial syndrome) occurs in 1 of 4000 live births, and 60% to 70% of affected individuals have congenital heart disease, ranging from mild to severe. In our cohort of 1472 subjects with 22q11.2DS, a total of 62% (n=906) have congenital heart disease and 36% (n=326) of these have tetralogy of Fallot (TOF), comprising the largest subset of severe congenital heart disease in the cohort. METHODS AND RESULTS: To identify common genetic variants associated with TOF in individuals with 22q11.2DS, we performed a genome-wide association study using Affymetrix 6.0 array and imputed genotype data. In our cohort, TOF was significantly associated with a genotyped single-nucleotide polymorphism (rs12519770, P=2.98×10-8) in an intron of the adhesion GPR98 (G-protein-coupled receptor V1) gene on chromosome 5q14.3. There was also suggestive evidence of association between TOF and several additional single-nucleotide polymorphisms in this region. Some genome-wide significant loci in introns or noncoding regions could affect regulation of genes nearby or at a distance. On the basis of this possibility, we examined existing Hi-C chromatin conformation data to identify genes that might be under shared transcriptional regulation within the region on 5q14.3. There are 6 genes in a topologically associated domain of chromatin with GPR98, including MEF2C (Myocyte-specific enhancer factor 2C). MEF2C is the only gene that is known to affect heart development in mammals and might be of interest with respect to 22q11.2DS. CONCLUSIONS: In conclusion, common variants may contribute to TOF in 22q11.2DS and may function in cardiac outflow tract development.

Narrowing the critical region for overgrowth within 13q14.2-q14.3 microdeletions.

Large chromosomal deletions from 13q13.3 to 13q21.3 have previously been associated with overgrowth. We present two patients with deletions at 13q14.2q14.3 who have macrocephaly, tall stature relative to their parents, cardiac phenotypes, and intellectual disability. This report narrows the critical region for tall stature, macrocephaly, and possibly cardiac disease.

Haploinsufficiency of the NOTCH1 Receptor as a Cause of Adams-Oliver Syndrome With Variable Cardiac Anomalies.

BACKGROUND: Adams-Oliver syndrome (AOS) is a rare disorder characterized by congenital limb defects and scalp cutis aplasia. In a proportion of cases, notable cardiac involvement is also apparent. Despite recent advances in the understanding of the genetic basis of AOS, for the majority of affected subjects, the underlying molecular defect remains unresolved. This study aimed to identify novel genetic determinants of AOS. METHODS AND RESULTS: Whole-exome sequencing was performed for 12 probands, each with a clinical diagnosis of AOS. Analyses led to the identification of novel heterozygous truncating NOTCH1 mutations (c.1649dupA and c.6049_6050delTC) in 2 kindreds in which AOS was segregating as an autosomal dominant trait. Screening a cohort of 52 unrelated AOS subjects, we detected 8 additional unique NOTCH1 mutations, including 3 de novo amino acid substitutions, all within the ligand-binding domain. Congenital heart anomalies were noted in 47% (8/17) of NOTCH1-positive probands and affected family members. In leukocyte-derived RNA from subjects harboring NOTCH1 extracellular domain mutations, we observed significant reduction of NOTCH1 expression, suggesting instability and degradation of mutant mRNA transcripts by the cellular machinery. Transient transfection of mutagenized NOTCH1 missense constructs also revealed significant reduction in gene expression. Mutant NOTCH1 expression was associated with downregulation of the Notch target genes HEY1 and HES1, indicating that NOTCH1-related AOS arises through dysregulation of the Notch signaling pathway. CONCLUSIONS: These findings highlight a key role for NOTCH1 across a range of developmental anomalies that include cardiac defects and implicate NOTCH1 haploinsufficiency as a likely molecular mechanism for this group of disorders.

Six novel mutations of the RUNX2 gene in Italian patients with cleidocranial dysplasia.

We report clinical and molecular findings in 14 patients with cleidocranial dysplasia (CCD), a well defined skeletal disorder with characteristic clinical findings and autosomal dominant inheritance. We identified ten heterozygous base changes in the RUNX2 gene, including six novel mutations [c.522insA, c.389G>A (W130X), c.662T>G (V221G), IVS2+T>A, c.1111_1129del19, and c.873_874delCA]. We did not establish a clear correlation between clinical features and genotype, the phenotypes of all patients analyzed falling within the range of variation described in CCD without an effect related to the length of the predicted protein. In two cases, however, a limb-girdle myopathy affecting the shoulder muscles was also identified. Our data add new variants to the repertoire of RUNX2 mutations in CCD.

[Transposition of great arteries. Understanding its pathogenesis]. / Trasposizione delle grandi arterie. Qualche spiraglio nell'inquadramento patogenetico.

Transposition of the great arteries (TGA) is a frequent and severe cardiac defect. In patients with this malformation, diagnostic and surgical results and the long-term prognosis significantly improved in the last years. From the embryological point of view there are two main theories: 1) the anomalous infundibular rotation, and 2) the anomaly of the aortico-pulmonary septum. Both of them still present important limits. Moreover, TGA is difficult to reproduce by animal experiments, but interesting data, using retinoid acid in pregnant rats, are nowadays available, as well as there are interesting data from the epidemiologic studies on human teratologic agents. TGA is rarely associated with genetic syndromes and with additional extracardiac anomalies. A few cases are in relation with DiGeorge syndrome with deletion of chromosome 22q11. On the contrary TGA is significantly prevalent, in association with other cardiac and extracardiac anomalies, in children with lateralization defects, heterotaxy and asplenia syndrome (right isomerism). However in patients with heterotaxy and polysplenia syndrome (left isomerism) TGA is significantly more rare. In mice with mutation of Smad2 and NODAL, two genes involved in the lateralization process, some cases of TGA, with or without right isomerism of the lungs, were reported. Moreover, in families with heterotaxy some cases with congenitally corrected TGA were reported and a new gene associated with heterotaxy, CRYPTIC, can present mutations in patients with "isolated" TGA. A recent study on familiar recurrence of TGA shows in the same family some cases of TGA and of corrected TGA so that a monogenic inheritance (autosomic dominant or recessive) with variable phenotypic expression can be suggested. The normal righthand spiralization of the heart is genetically determined in cases of situs solitus and d-loop of the ventricles. This pattern is not present in cases of TGA presenting a parallel position of the great arteries. On the basis of these observations and according to new epidemiologic and genetic data some cases of TGA should be classified in the group of the anomalies of lateralization and ventricular loop. The mystery is still present but perhaps some gleams of light are appearing.