Array-CGH analysis in patients with intellectual disability and/or congenital malformations in Brazil.

In several patients, intellectual disability and/or congenital malformation may be attributed to chromosomal changes. In this study, we conducted an array-CGH test of 200 patients from the Northeast of Brazil with intellectual disability and/or congenital malformation. Blood samples were collected from the proband and from their parents when possible. DNA was extracted and investigated using the array-CGH test. Findings were evaluated for the pathogenicity in databases of benign and pathogenic changes (ISCA, UCSC, DGV, and DECIPHER). Forty-seven copy number variations (CNVs) were identified in 43/200 (21.5%) patients, including 25/98 (25.5%) in males and 22/102 (21.57%) in females. We considered 33 of these to be clinically significant, reaching a diagnosis rate of 16.5%. The sizes of the CNVs varied from 102 kb to 24 Mb in deletions and from 115 kb to 140 Mb in duplications. In 10/47 (21.3%) patients, the rearrangement involved a sex chromosome. Thirty-nine patients had one chromosomal aberration, while 2 concomitant abnormalities were detected in 4 patients. Ten of 47 CNVs (21.3%) were > 5Mb in size. Fifteen patients had CNVs related to known syndromes. This research highlights the contribution of submicroscopic chromosomal changes to the etiology of intellectual disability and/or congenital malformation, particularly the implication of chromosomal abnormalities detected using an array-CGH test, with a high rate of 16.5%. Thus, our results support the use of array-CGH replacing standard karyotype as the first-tier cytogenetic diagnostic test for patients with multiple congenital anomalies and/or intellectual disability.

Methylation profile of SNRPN gene and its correlation with weight and chronological age.

Genomic imprinting is an important epigenetic phenomenon, wherein genes or gene clusters are marked by DNA methylation during gametogenesis. This plays a major role in several functions of normal cells, including cell differentiation, X chromosome inactivation, and the maintenance of chromatin structure, in mammalian development. The aim of this study was to investigate the possible differences in SNRPN gene methylation profiles in non-obese and obese individuals, and in children and adults. Our results did not reveal any statistical correlations between the DNA methylation profiles of the SNRPN gene in children or adult obese and non-obese groups. However, a comparison of the methylation levels with the chronological age revealed statistically significant differences between the means of methylation in adults and children (46.20 ± 5.88 and 39.40 ± 2.87, respectively; P < 0.001). Pearson's correlation analysis indicated a positive association between the level of DNA methylation and the chronological age (R2 = 0.326; P < 0.001). Therefore, we concluded that the methylation profile of the SNRPN promoter (in blood) is not a useful biomarker for determining the predisposition of an individual to obesity. Additionally, we have confirmed that SNRPN methylation increases with age, which raises further questions regarding the role of SNRPN expression during the aging process.

Manipulation of primer affinity improves high-resolution melting accuracy for imprinted genes.

High-resolution melting (HRM) is considered an inexpensive, rapid, and attractive methodology for methylation analysis. In the application of the polymerase chain reaction (PCR) to methylation analysis, amplification efficiencies are biased towards unmethylated, rather than methylated, templates: a phenomenon known as PCR bias. To overcome PCR bias, primers that include CpG site(s) and are fully complementary to the methylated sequence have been proposed. However, genes mapped within imprinted regions usually present higher methylation levels, and an unusual PCR bias towards the methylated template can therefore arise. The manipulation of primer affinity attempts to overcome this problem. We attempted to show that mismatches at the primer's methylated binding sites increase the area between the 50 and 100% methylation plots on the melting curves, and may increase HRM accuracy for samples that have high methylation levels. Sets of primers for imprinted genes that included CpG sites at their binding sequences were designed, and were complementary to methylated or unmethylated templates. Primers fully complementary to methylated templates produced a very small area between the 50 and 100% methylation plots. When using primers that were fully complementary to the unmethylated sequence, we were able to increase the area between the 50 and 100% methylation plots. Therefore, when samples are highly methylated, such as targets in genes mapped in imprinted regions, we propose that primers should favor amplification of the rarest, unmethylated sequence. Primers may be designed to include one CpG at its binding site and be fully complementary to the unmethylated template.

High frequency of submicroscopic chromosomal imbalances in patients with syndromic craniosynostosis detected by a combined approach of microsatellite segregation analysis, multiplex ligation-dependent probe amplification and array-based comparative genome hybridisation.

We present the first comprehensive study, to our knowledge, on genomic chromosomal analysis in syndromic craniosynostosis. In total, 45 patients with craniosynostotic disorders were screened with a variety of methods including conventional karyotype, microsatellite segregation analysis, subtelomeric multiplex ligation-dependent probe amplification) and whole-genome array-based comparative genome hybridisation. Causative abnormalities were present in 42.2% (19/45) of the samples, and 27.8% (10/36) of the patients with normal conventional karyotype carried submicroscopic imbalances. Our results include a wide variety of imbalances and point to novel chromosomal regions associated with craniosynostosis. The high incidence of pure duplications or trisomies suggests that these are important mechanisms in craniosynostosis, particularly in cases involving the metopic suture.

Whole-genome array-CGH screening in undiagnosed syndromic patients: old syndromes revisited and new alterations.

We report array-CGH screening of 95 syndromic patients with normal G-banded karyotypes and at least one of the following features: mental retardation, heart defects, deafness, obesity, craniofacial dysmorphisms or urogenital tract malformations. Chromosome imbalances not previously detected in normal controls were found in 30 patients (31%) and at least 16 of them (17%) seem to be causally related to the abnormal phenotypes. Eight of the causative imbalances had not been described previously and pointed to new chromosome regions and candidate genes for specific phenotypes, including a connective tissue disease locus on 2p16.3, another for obesity on 7q22.1-->q22.3, and a candidate gene for the 3q29 deletion syndrome manifestations. The other causative alterations had already been associated with well-defined phenotypes including Sotos syndrome, and the 1p36 and 22q11.21 microdeletion syndromes. However, the clinical features of these latter patients were either not typical or specific enough to allow diagnosis before detection of chromosome imbalances. For instance, three patients with overlapping deletions in 22q11.21 were ascertained through entirely different clinical features, i.e., heart defect, utero-vaginal aplasia, and mental retardation associated with psychotic disease. Our results demonstrate that ascertainment through whole-genome screening of syndromic patients by array-CGH leads not only to the description of new syndromes, but also to the recognition of a broader spectrum of features for already described syndromes. Furthermore, on the technical side, we have significantly reduced the amount of reagents used and costs involved in the array-CGH protocol, without evident reduction in efficiency, bringing the method more within reach of centers with limited budgets.

Molecular screening for microdeletions at 9p22-p24 and 11q23-q24 in a large cohort of patients with trigonocephaly.

Trigonocephaly is a rare form of craniosynostosis characterized by the premature closure of the metopic suture. To contribute to a better understanding of the genetic basis of metopic synostosis and in an attempt to restrict the candidate regions related to metopic suture fusion, we studied 76 unrelated patients with syndromic and non-syndromic trigonocephaly. We found a larger proportion of syndromic cases in our population and the ratio of affected male to female was 1.8 : 1 and 5 : 1 in the non-syndromic and syndromic groups, respectively. A microdeletion screening at 9p22-p24 and 11q23-q24 was carried out for all patients and deletions in seven of them were detected, corresponding to 19.4% of all syndromic cases. Deletions were not found in non-syndromic patients. We suggest that a molecular screening for microdeletions at 9p22-p24 and 11q23-q24 should be offered to all syndromic cases with an apparently normal karyotype because it can potentially elucidate the cause of trigonocephaly in this subset of patients. We also suggest that genes on the X-chromosome play a major role in syndromic trigonocephaly.