Ilária Cristina Sgardioli

Atypical copy number abnormalities in 22q11.2 region: report of three cases.

The 22q11.2 Deletion Syndrome (22q11.2DS) is the most common microdeletion syndrome in humans, with a highly variable phenotype. This chromosomal region contains low copy repeat (LCR) sequences that mediate non-allelic homologous recombination which predispose to copy number abnormalities at this locus. This article describes three patients investigated for suspicion of 22q11.2DS presenting atypical copy number abnormalities overlapping or not with the common ∼3 Mb deletion. They were investigated by G-banding karyotype, Multiplex-ligation dependent probe amplification (MLPA) and array Genomic Hibridization (aGH). Clinical and molecular data were compared with literature, in order to contribute to genotype-phenotype correlation. Atypical chromosomal abnormalities were detected: 3.6 Mb deletion at 22q11.21-q11.23 between LCRs B-F in patient 1 and approximately 1.5 Mb deletion at 22q11.21-q11.22 between LCRs D-E in patients 2 and 3. The breakpoints detected in patient 1 have not been previously described. These findings exemplify the complexity and genetic heterogeneity observed in 22q11.2 region and corroborates the idea that genetic modifiers contribute to the phenotypic variability observed in proximal and distal 22q11.2 deletion syndromes.

Insertional Translocation of 15q25-q26 into 11p13 and Duplication at 8p23.1 Characterized by High Resolution Arrays in a Boy With Congenital Malformations and Aniridia

We report on a boy presenting submucous cleft palate, hydronephrosis, ventriculoseptal defect, aniridia, and developmental delay. Additional material on 11p13 was cytogenetically visible and array analyses identified a duplicated segment on 15q25‐26 chromosome region; further, array analyses revealed a small deletion (49 kb) at 11p13 region involving the ELP4 gene and a duplication at 8p23.1. Results were confirmed with both molecular and molecular cytogenetics techniques. Possibilities for etiological basis of clinical phenotype are discussed.

Improved Detection of Helicobacter pylori DNA in Formalin-fixed Paraffin-embedded (FFPE) Tissue of Patients with Hepatocellular Carcinoma Using Laser Capture Microdissection (LCM)

Dear Editor, Helicobacter DNA has been detected in hepatic tissues from patients with various hepatobiliary diseases, mainly cirrhosis and hepatocellular carcinoma (HCC). Although the role of Helicobacter spp. in pathogenesis of these diseases remains unclear, the available data suggest that Helicobacter infection may play a role in hepatic carcinogenesis [1]. Considering that HCC is one of the most common malignancies with more than 500.000 new tumors diagnosed annually [2], further studies related to H. pylori and development of HCC have fundamental importance on the understanding of its pathogenesis. Formalin-fixed paraffin-embedded (FFPE) tissue represents an extraordinary source for molecular studies as genomic DNA can be extracted from this sample. However, DNA extraction from FFPE tissues is challenging because nucleic acids are commonly fragmented and cross-linked with proteins. Furthermore, methods of DNA extraction from FFPE tissue are generally laborious and time-consuming. Laser capture microdissection (LCM) is a recently developed technique for isolation of pure populations of cells from tissue sections by microscopic visualization. Because of its high precision and accuracy, LCM has been employed in cancer-related studies. In this work, we used LCM technique to improve the detection of H. pylori in FFPE liver from patients with HCC. With this aim, six H. pylori-positive samples detected by polymerase chain reaction (PCR) with H. pylori-specific 16S rRNA primers were selected. The sequence of the sense primer (JW21) was 5′-GCGACCTGCTGGAACATTAC-3′(position 691-710) and the antisense primer (JW22) was 5′-CGTTAGCTCCATTACTGGAGA-3′ (position 829-809) [3]. Tissue samples were cut on 0.17 mm PEN membrane-covered slides (Carl Zeiss, MicroImaging GmbH, G€ ottingen, Germany) and then routine staining with carbol fuchsin was performed [4]. Thereafter, stained bacteria were microdissected using a PALM MicroBeam system (Carl Zeiss, MicroImaging GmbH, G€ ottingen, Germany) and then ejected into the Eppendorf tube cap by a single laser shot (Fig. 1C,D). After microdissection, a digestion buffer was added into Eppendorf for DNA extraction. The crude lysate was directly employed as template for PCR [4]. The samples were further amplified using H. pylori 16S rRNA primers as previously described [3], and amplicons were identified by sequence analysis. Microorganisms resembling H. pylori were observed in hepatic sinus from HCC samples (Fig. 1A,B). The number of cocci was greater than of bacilli as previously described [5]. PCR results showed that all six microdissected samples were positive for 16S rRNA gene and showed 98% similarity to 16S rRNA gene of H. pylori by sequence analysis (GeneBank accession number CP003419.1). Nevertheless, we cannot exclude the possibility of cross-reaction of these primers with other Helicobacter spp. These results demonstrated that LCM can be extensively applied for identification of H. pylori in FFPE liver tissue of HCC patients. Considering that bacteria were mainly found in peritumoral tissue, this technique was highly effective for obtaining a targeted bacterial population within a selected area in the HCC tissue. Beyond that, LCM simplified the H. pylori detection because extracted DNA was used directly as a template for PCR amplification. Further studies will be performed to isolate H. pylori from other tissues using LCM technique.

A new case of partial 14q31.3-qter trisomy due to maternal pericentric inversion

Chromosome 14 is often involved in chromosome rearrangements, although pericentric inversions are rare. Here we report a mother carrying a pericentric inversion of chromosome 14, and her daughter with recombinant chromosome characterized by a partial distal 14q trisomy. Principal clinical findings of the child include facial anomalies, microcephaly, developmental delay, hypotonia and cardiac malformation. Her final karyotype was 46,XX,rec(14)dup(14q)inv(14)(p12q31)mat[20], arr 14q31.3qter(85,427,839-106,356,482)x3. This report brings new data about clinical features of partial 14q trisomy and molecular analysis enables the visualization of genes involved in the segment duplicated.

Genomic imbalances in syndromic congenital heart disease

OBJECTIVE To identify pathogenic genomic imbalances in patients presenting congenital heart disease (CHD) with extra cardiac anomalies and exclusion of 22q11.2 deletion syndrome (22q11.2 DS). METHODS 78 patients negative for the 22q11.2 deletion, previously screened by fluorescence in situ hybridization (FISH) and/or multiplex ligation probe amplification (MLPA) were tested by chromosomal microarray analysis (CMA). RESULTS Clinically significant copy number variations (CNVs ≥300kb) were identified in 10% (8/78) of cases. In addition, potentially relevant CNVs were detected in two cases (993kb duplication in 15q21.1 and 706kb duplication in 2p22.3). Genes inside the CNV regions found in this study, such as IRX4, BMPR1A, SORBS2, ID2, ROCK2, E2F6, GATA4, SOX7, SEMAD6D, FBN1, and LTPB1 are known to participate in cardiac development and could be candidate genes for CHD. CONCLUSION These data showed that patients presenting CHD with extra cardiac anomalies and exclusion of 22q11.2 DS should be investigated by CMA. The present study emphasizes the possible role of CNVs in CHD.

Distal 22q11.2 microduplication combined with typical 22q11.2 proximal deletion: a case report.

The 22q11 chromosomal region contains low copy repeats (LCRs) sequences that mediate non‐allelic homologous recombination, which predisposes to copy number variations (CNVs) at this locus. Hemizygous deletions of the proximal 22q11.2 region result in the 22q11.2 deletion syndrome (22q11.2 DS). In addition, 22q11.2 duplications involving the distal LCR22s have been reported. This article describes a patient presenting a 2.5‐Mb de novo deletion at proximal 22q11.21 region (between LCRs A‐D), combined with a 1.3‐Mb maternally inherited duplication at distal 22q11.23 region (between LCRs F‐H). The presence of concomitant chromosomal imbalances found in this patient has not been reported previously. Clinical and molecular data were compared with literature, in order to contribute to genotype‐phenotype correlation. These findings exemplify the complexity and genetic heterogeneity observed in 22q11.2 deletion syndrome and highlights the difficulty to make genetic counseling and predict phenotypic consequences in these situations.

22q11.2 Deletion Syndrome: Laboratory Diagnosis and TBX1 and FGF8 Mutation Screening.

Velocardiofacial syndrome is one of the recognized forms of chromosome 22q11.2 deletion syndrome (22q11.2 DS) and has an incidence of 1 of 4,000 to 1 of 6,000 births. Nevertheless, the 22q11 deletion is not found in several patients with a 22q11.2 DS phenotype. In this situation, other chromosomal aberrations and/or mutations in the T-box 1 transcription factor C (TBX1) gene have been detected in some patients. A similar phenotype to that of the 22q11.2 DS has been reported in animal models with mutations in fibroblast growth factor 8 (Fgf8) gene. To date, FGF8 mutations have not been investigated in humans. We tested a strategy to perform laboratory testing to reduce costs in the investigation of patients presenting with the 22q11.2 DS phenotype. A total of 109 individuals with clinical suspicion were investigated using GTG-banding karyotype, fluorescence in situ hybridization, and/or multiplex ligation-dependent probe amplification. A conclusive diagnosis was achieved in 33 of 109 (30.2%) cases. In addition, mutations in the coding regions of TBX1 and FGF8 genes were investigated in selected cases where 22q11.2 deletion had been excluded, and no pathogenic mutations were detected in both genes. This study presents a strategy for molecular genetic characterization of patients presenting with the 22q11.2 DS using different laboratory techniques. This strategy could be useful in different countries, according to local resources. Also, to our knowledge, this is the first investigation of FGF8 gene in humans with this clinical suspicion.

A Rare Case of Concomitant Deletions in 15q11.2 and 19p13.3

A female individual with concomitant deletions in 15q11.2 and 19p13.3 is reported. She presents facial dysmorphisms, motor delay, learning difficulties, and mild behavioral impairment. After chromosomal microarray analysis, the final karyotype was established as 46,XX.arr[GRCh37] 15q11.2 (22770421_23282798)×1,19p13.3(3793904_4816330)×1. The deletion in 15q11.2 is 507 kb in size involving 7 non-imprinted genes, 4 of which are registered in the OMIM database and are implicated in neuropsychiatric or neurodevelopmental disorders. The deletion in 19p13.3 is 1,022 kb in size and encompasses 47 genes, most of which do not have a well-known function. The genotype-phenotype correlation is discussed, and most of the features could be related to the 19p13.3 deletion, except for velopharyngeal insufficiency. Other genes encompassed in the deleted region, as well as unrecognized epistatic factors could also be involved. Nevertheless, the two-hit model related to the 15q11.2 deletion would be an important hypothesis to be considered.

Pure 21q22.3 deletion identified in a patient with mild phenotypic features: A pure 21q22.3 deletion

Partial deletions at chromosome 21 have been rarely reported and in the majority of descriptions are associated with ring chromosome 21, resulting in terminal deletions of 21q22.3 and a large phenotypic variability (Roberson et al. 2011). This article reports a female patient with a de novo 21q22.3 deletion that was referred at 11 years because of speech delay and learning disability. She also presented a history of tonic–clonic seizures during her childhood and was already controlled by medications. She had a long and triangular face, low-set ears, ocular hypertelorism, upslanting palpebral fissures, epicanthal folds, malar hypoplasia, nose with broad bridge, bulbous and mild grooved tip and alar hypoplasia, long philtrum, furrowed tongue, high palate, cleft uvula, and microrretrognathia (Fig. 1A). She also exhibited joint hyperextensibility, long hands and fingers with mild cutaneous syndactyly, and clinodactyly of fifth and proximal implantation of thumbs. Dysphagia and hypernasal tone in the voice were also detected. Echocardiogram showed mitral valve prolapse with discrete valve regurgitation and laryngoscopy revealed nasal septum deviation and velopharyngeal insufficiency. At 29 years, she had mild mental retardation and physical examination was essentially the same. GTG-banded karyotype was normal and chromosomal microarray analysis (CMA) with CytoScan HD Array (Affymetrix, Santa Clara, CA, USA) revealed a terminal deletion of 4787 kb at 21q22.3 region (43,310,796–48,097,372 – GRCh37/hg19; Fig. 1B) and an interstitial duplication of 1068 kb at 20q12-q13.12 region (41,500,568–42,568,152 – GRCh37/hg19). CMA with the 8x60K array Agilent (Agilent Technologies, Santa Clara, CA, USA) confirmed the findings of the patient and revealed the same duplication at 20q12-q13.12 region in her mother. However, cases with the same duplication were not found in databases. As her mother is phenotypically normal, this alteration was considered a rare variant of unknown significance, probably benign. Fluorescence in situ hybridization (FISH) confirmed the deletion at 21q22.3 region in the patient and revealed normal results for both parents, confirming a de novo alteration. The patient herein reported has a rare pure monosomy 21q22.3qter with mild phenotype. Table S1 (Supporting information) compares the reported patient with similar cases from the literature and the graphic design (Fig. 1C) allows the comparison of the deletion sizes and the involved Online Mendelian Inheritance in Man (OMIM) genes. The reported case presents a 4.7-Mb terminal deletion which involves 94 genes, many of them do not have a known function and 53 are registered in the OMIM. The dosage sensitivity of most of these genes are not well established according to ClinGen Dosage Sensitivity Map. However, the haploinsufficiency of some genes in the 21q22.3-qter region could be responsible for the phenotype presentation, as well as unrecognized epistatic factors should be considered. Among the genes inside the deleted region, the PRMT2 gene has been rated as an important factor that acts as a negative regulator of the nuclear factor-kB (NF-kB) pathway and could participate in the regulation of chromatin (Besson et al. 2007). Another study suggests that arginine methyltransferases, as PRMT2, when abnormally regulated could lead transcripts for chromatinremodeling enzymes associated with reproductive system diseases and cancer (Baumann et al. 2015). Also, previous reports showed that individuals with 21q deletion can be infertile, presenting azoospermia or irregular menstrual cycles (Marquet et al. 2015). The present patient has no history of other reproductive disorders or neoplasias until now, but she developed a severe polymenorrhea, which has lead to hysterectomy. Heterozygous mutations in SIK1 gene were associated to epileptic encephalopathy probably caused by variant and truncated SIK1 proteins (Hansen et al. 2015). Also, sequence variations in the SIK1 gene resulted in significant abnormalities of synapse regulation and neuronal morphology in in vitro human neuronal model, causing epilepsy in some cases (Proschel et al. 2017), a feature described in the present case. Despite of many genes mapped in the 21q22.3 region do not have a well-known function and epistatic factors could not be excluded, PRMT2 and SIK1 genes are possibly related to this mild phenotype. Also, the present case contributes to a better clinical recognition of this rare chromosomal abnormality.

A recognizable phenotype related to 19p13.12 microdeletion

Submicroscopic deletions in chromosome 19 have been rarely reported. We reported a male patient presenting with neurodevelopmental delay and facial dysmorphisms with a de novo 19p13.11p13.12 deletion of approximately 1.4 Mb. To date, there are seven cases with deletions overlapping the 19p13.11–p13.12 region described in the literature. A region of 800 kb for branchial arch defects in the proximal region of 19p13.12, and another minimal critical region of 305 kb for hypertrichosis, synophrys, and protruding front teeth have been proposed previously. We suggest that the shortest region of overlap could be refined to an approximately 53 kb region shared within all patients, encompassing part of BRD4 and AKAP8L genes and the AKAP8 gene. Based on the genotype‐phenotype correlation of the present case and cases with overlapping deletions described in the literature, it was possible to recognize a consistent phenotype characterized by microcephaly, ear abnormalities, rounded face, synophrys, arched or upwardly angulated eyebrows, short nose, anteverted nares, prominent cheeks, teeth abnormalities, and developmental delay.