Clinical Implications of Chromosome 16 Copy Number Variation

Chromosome 16 is one of the gene-rich chromosomes; however, approximately 10% of the chromosome 16 sequence is composed of segmental copies, which renders this chromosome instable and predisposes it to rearrangements via frequent nonallelic homologous recombination. Microarray technologies have enabled the analysis of copy number variations (CNV), which may be associated with the risk of developing complex diseases. Through comparative genomic hybridisation in 1,298 patients, we detected 18 cases with chromosome 16 CNV. We identified 2recurrent CNV regions, including 1 at 16p13.11 in 4 patients and another at 16p11.2 in 7 patients. We also detected atypical chromosome 16 rearrangements in 7 patients. Furthermore, we noted an increased frequency of co-occurring genomic changes, supporting the two-hit hypothesis to explain the phenotypic variability in the clinical presentation of CNV syndromes. Our findings can contribute to the creation of a chromosome 16 disease map based on regions that may be associated with disease development.

Chromosomes, Nuclear Genes and the Phylogenetic Placement Within the Reptilia of Sphenodon (Tuatara)

<p>Chromosomes were examined from five populations of Sphenodon (tuatara) using giemsa, Ag-NOR, C-, G- and RE- banding. There were no differences between species, populations or sexes, although one animal had a structural heteromorphism. Chromosome morphology homology to Testudines (turtles), Aves (birds) and to a lesser extent Crocodylia (crocodiles) allowed reconstruction ofa Reptilian proto-karyotype, dated to 300 million years ago. DNA sequence was isolated from the WT1, AMH, DMRT1, FoxG1 and 28S. No variation was present in Sphenodon 28S, FoxG1 or AMH sequence. 28S could be dated to a common ancestor with Testudines, similar to the archaic karyotype. FoxG1 and AMH reflect an Oligocene divergence, WT1 divides north-eastern North Island and Cook Strait, and can be dated to the Pleistocene or the Pliocene, and DMRT1appears a recent post- pliocene divergence. FISH localised DIG-labelled probes of AMH to chromosome 11 and WT1 to chromosome 13 or 14. Human telomeric probes localised to Sphenodon telomeric regions demonstrating the highly conserved nature of telomeric sequences. Comparative genomic hybridisation with chicken chromosomes did not produce any regions of homology, implying significant chromosomal and DNA changes since the Orders shared a common ancestor, although macrochromosome morphology has remained similar. Sphenodon chromosomal and nuclear DNA analyses demonstrate evolutionary decoupling, supporting recent mtDNA work.</p>

Chromosomes, Nuclear Genes and the Phylogenetic Placement Within the Reptilia of Sphenodon (Tuatara)

<p>Chromosomes were examined from five populations of Sphenodon (tuatara) using giemsa, Ag-NOR, C-, G- and RE- banding. There were no differences between species, populations or sexes, although one animal had a structural heteromorphism. Chromosome morphology homology to Testudines (turtles), Aves (birds) and to a lesser extent Crocodylia (crocodiles) allowed reconstruction ofa Reptilian proto-karyotype, dated to 300 million years ago. DNA sequence was isolated from the WT1, AMH, DMRT1, FoxG1 and 28S. No variation was present in Sphenodon 28S, FoxG1 or AMH sequence. 28S could be dated to a common ancestor with Testudines, similar to the archaic karyotype. FoxG1 and AMH reflect an Oligocene divergence, WT1 divides north-eastern North Island and Cook Strait, and can be dated to the Pleistocene or the Pliocene, and DMRT1appears a recent post- pliocene divergence. FISH localised DIG-labelled probes of AMH to chromosome 11 and WT1 to chromosome 13 or 14. Human telomeric probes localised to Sphenodon telomeric regions demonstrating the highly conserved nature of telomeric sequences. Comparative genomic hybridisation with chicken chromosomes did not produce any regions of homology, implying significant chromosomal and DNA changes since the Orders shared a common ancestor, although macrochromosome morphology has remained similar. Sphenodon chromosomal and nuclear DNA analyses demonstrate evolutionary decoupling, supporting recent mtDNA work.</p>

Xq21.1q21.31 Duplication in Two Male Siblings

Despite the increased use of array comparative genomic hybridisation, duplications of Xq remain rarely reported in the literature. Xq21.1q21.31 duplication has previously been reported only once in a boy with features of Prader Willi syndrome (PWS). We report 2 malesiblings with maternally inherited duplication of Xq21.1q21.31 who demonstrate a variable phenotype. The proband has Prader Willi-like features such as global developmental delay, autism, obesity, short hands, and small genitalia with a history of food seeking behaviour, while his younger brother has isolated speech delay with some autistic features under evaluation. Both siblings have features such as bitemporal narrowing and small hands. It is therefore likely that the phenotype of duplications in this region is broader than PWS phenocopy, and further cases would be required to elucidate this.

Comprehensive Genetic Evaluation of Children With Syndromic Craniosynostosis by a Combination of Cytogenetics, Multiplex Ligation-dependent Probe Amplification and Array-based Comparative Genomic Hybridisation

Syndromic craniosynostosis (SC) is a genetically determined premature closure of one or more of the cranial sutures, which may result in severe dysmorphism, increased intracranial pressure along with many other clinical manifestations. The considerable risk of complications along with significant incidence makes these cranial deformations an important medical problem. Despite the efforts to clarify the pathogenesis of SC in recent years, its genetic aspects remain largely unknown.Aiming to elucidate the complex genetic etiology of syndromic craniosynostosis, we conducted an investigation of 39 children, screened systematically with a combination of conventional cytogenetic analysis, multiplex ligation-dependent probe amplification (MLPA) and array-based comparative genomic hybridisation (aCGH).Pathological findings were established in 15.3% (6/39) of the cases using aCGH, in 7.7% (3/39) using MLPA and 2.5% (1/39) using conventional karyotyping. About 12.8% (5/39) of the patients with normal karyotype carried submicroscopic chromosomal rearrangements. Duplications were found to be more common than deletions.Conclusion: The systematic genetic evaluation of children with SC revealed a high prevalence of submicrosopic chromosomal rearrangements (most commonly duplications and gain-of-function variations). This suggests the leading role of those defects in the pathogenesis of syndromic craniosynostosis. The genetic complexity of SC was reaffirmed by the discovery of pathological findings in various chromosomal regions. Certain genes were discussed in conjunction with craniosynostosis.

Novel clinical presentation of a CRX rod-cone dystrophy

We describe a novel clinical presentation of a CRX rod-cone dystrophy in a single family. Two boys ages 6 and 12 years presented with clinical and optical coherence tomography features suggestive of X-linked retinoschisis, but with optic nerve swelling without increased intracranial pressure. One patient had an electronegative electroretinogram (ERG) and the other had rod-cone dysfunction. Neither had retinoschisin (RS1) gene mutations. Biological mother and sister presented with retinal pigment epithelium (RPE) changes and abnormal cone-rod ERG responses. On further testing, next generation sequencing with array comparative genomic hybridisation showed a deletion in exon 4 of the CRX gene. Cystoid maculopathy in young male children can be difficult to distinguish from RS1-associated schisis. Phenotypic variants within a family must prompt a thorough retinal dystrophy evaluation even with electronegative ERG in the presenting child. This novel phenotype for CRX presents with optic nerve swelling and cystoid maculopathy in men, and RPE changes in women.

Genomic characterisation of multiple myeloma: study of a Portuguese cohort

Multiple myeloma (MM) genomic complexity reflects in the variable patients’ clinical presentation. Genome-wide studies seem to be a reasonable alternative to identify critical genomic lesions. In the current study, we have performed the genomic characterisation of a Portuguese cohort of patients with MM by array comparative genomic hybridisation. Overall, the most frequently detected alterations were 13q deletions, gains of 1q, 19p, 15q, 5p and 7p and trisomy 9. Even though some identified genomic alterations were previously associated with a prognostic value, other abnormalities remain with unknown, but putative significance for patients’ clinical practice. These genomic alterations should be further assessed as possible biomarkers.

Development of a genomic predictive model for cholangiocarcinoma using copy number alteration data

AimsCholangiocarcinoma (CC) is a rare tumour arising from the biliary tract epithelium. The aim of this study was to perform a genomic characterisation of CC tumours and to implement a model to differentiate extrahepatic (ECC) and intrahepatic (ICC) cholangiocarcinoma.MethodsDNA extracted from tumour samples of 23 patients with CC, namely 10 patients with ECC and 13 patients with ICC, was analysed by array comparative genomic hybridisation. A support vector machine algorithm for classification was applied to the genomic data to distinguish between ICC and ECC. A survival analysis comparing both groups of patients was also performed.ResultsWith these whole genome results, we observed several common alterations between tumour samples of the same CC anatomical type, namely gain of Xp and loss of 3p, 11q11, 14q, 16q, Yp and Yq in ICC tumours, and gain of 16p25.3 and loss of 3q26.1, 6p25.3–22.3, 12p13.31, 17p, 18q and Yp in ECC tumours. Gain of 2q37.3 was observed in the samples of both tumour subtypes, ICC and ECC. The developed genomic model comprised four chromosomal regions that seem to enable the distinction between ICC and ECC, with an accuracy of 71.43% (95% CI 43% to 100%). Survival analysis revealed that in our cohort, patients with ECC survived on average 8 months less than patients with ICC.ConclusionsThis genomic characterisation and the introduction of genomic models to clinical practice could be important for patient management and for the development of targeted therapies. The power of this genomic model should be evaluated in other CC populations.

Oncogenetic landscape of lymphomagenesis in coeliac disease

ObjectiveEnteropathy-associated T-cell lymphoma (EATL) is a rare but severe complication of coeliac disease (CeD), often preceded by low-grade clonal intraepithelial lymphoproliferation, referred to as type II refractory CeD (RCDII). Knowledge on underlying oncogenic mechanisms remains scarce. Here, we analysed and compared the mutational landscape of RCDII and EATL in order to identify genetic drivers of CeD-associated lymphomagenesis.DesignPure populations of RCDII-cells derived from intestinal biopsies (n=9) or sorted from blood (n=2) were analysed by whole exome sequencing, comparative genomic hybridisation and RNA sequencing. Biopsies from RCDII (n=50), EATL (n=19), type I refractory CeD (n=7) and uncomplicated CeD (n=18) were analysed by targeted next-generation sequencing. Moreover, functional in vitro studies and drug testing were performed in RCDII-derived cell lines.Results80% of RCDII and 90% of EATL displayed somatic gain-of-functions mutations in the JAK1-STAT3 pathway, including a remarkable p.G1097 hotspot mutation in the JAK1 kinase domain in approximately 50% of cases. Other recurrent somatic events were deleterious mutations in nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB) regulators TNFAIP3 and TNIP3 and potentially oncogenic mutations in TET2, KMT2D and DDX3X. JAK1 inhibitors, and the proteasome inhibitor bortezomib could block survival and proliferation of malignant RCDII-cell lines.ConclusionMutations activating the JAK1-STAT3 pathway appear to be the main drivers of CeD-associated lymphomagenesis. In concert with mutations in negative regulators of NF-κB, they may favour the clonal emergence of malignant lymphocytes in the cytokine-rich coeliac intestine. The identified mutations are attractive therapeutic targets to treat RCDII and block progression towards EATL.

Rare CNVs provide novel insights into the molecular basis of GH and IGF-1 insensitivity

Objective Copy number variation (CNV) has been associated with idiopathic short stature, small for gestational age and Silver-Russell syndrome (SRS). It has not been extensively investigated in growth hormone insensitivity (GHI; short stature, IGF-1 deficiency and normal/high GH) or previously in IGF-1 insensitivity (short stature, high/normal GH and IGF-1). Design and methods Array comparative genomic hybridisation was performed with ~60 000 probe oligonucleotide array in GHI (n = 53) and IGF-1 insensitivity (n = 10) subjects. Published literature, mouse models, DECIPHER CNV tracks, growth associated GWAS loci and pathway enrichment analyses were used to identify key biological pathways/novel candidate growth genes within the CNV regions. Results Both cohorts were enriched for class 3–5 CNVs (7/53 (13%) GHI and 3/10 (30%) IGF-1 insensitivity patients). Interestingly, 6/10 (60%) CNV subjects had diagnostic/associated clinical features of SRS. 5/10 subjects (50%) had CNVs previously reported in suspected SRS: 1q21 (n = 2), 12q14 (n = 1) deletions and Xp22 (n = 1), Xq26 (n = 1) duplications. A novel 15q11 deletion, previously associated with growth failure but not SRS/GHI was identified. Bioinformatic analysis identified 45 novel candidate growth genes, 15 being associated with growth in GWAS. The WNT canonical pathway was enriched in the GHI cohort and CLOCK was identified as an upstream regulator in the IGF-1 insensitivity cohorts. Conclusions Our cohort was enriched for low frequency CNVs. Our study emphasises the importance of CNV testing in GHI and IGF-1 insensitivity patients, particularly GHI subjects with SRS features. Functional experimental evidence is now required to validate the novel candidate growth genes, interactions and biological pathways identified.