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.

Genes and Pseudogenes: Complexity of the RCCX Locus and Disease

Copy Number Variations (CNVs) account for a large proportion of human genome and are a primary contributor to human phenotypic variation, in addition to being the molecular basis of a wide spectrum of disease. Multiallelic CNVs represent a considerable fraction of large CNVs and are strictly related to segmental duplications according to their prevalent duplicate alleles. RCCX CNV is a complex, multiallelic and tandem CNV located in the major histocompatibility complex (MHC) class III region. RCCX structure is typically defined by the copy number of a DNA segment containing a series of genes – the serine/threonine kinase 19 (STK19), the complement 4 (C4), the steroid 21-hydroxylase (CYP21), and the tenascin-X (TNX) – lie close to each other. In the Caucasian population, the most common RCCX haplotype (69%) consists of two segments containing the genes STK19-C4A-CYP21A1P-TNXA-STK19B-C4B-CYP21A2-TNXB, with a telomere-to-centromere orientation. Nonallelic homologous recombination (NAHR) plays a key role into the RCCX genetic diversity: unequal crossover facilitates large structural rearrangements and copy number changes, whereas gene conversion mediates relatively short sequence transfers. The results of these events increased the RCCX genetic diversity and are responsible of specific human diseases. This review provides an overview on RCCX complexity pointing out the molecular bases of Congenital Adrenal Hyperplasia (CAH) due to CYP21A2 deficiency, CAH-X Syndrome and disorders related to CNV of complement component C4.

Sequencing individual genomes with recurrent deletions reveals allelic architecture and disease loci for autosomal recessive traits

In medical genetics, discovery and characterization of new “disease genes” and alleles depend on patient ascertainment strategies to enrich previously uncharacterized alleles. Here, we present a novel strategy of new allele and gene discovery for recessive/biallelic disease traits. In this approach, patients with large recurrent genomic deletions mediated by nonallelic homologous recombination (NAHR) are sequenced, and new discoveries are revealed in the hemizygous chromosomal regions in trans to the large deletion, essentially enabling haploid genomic segment genetics. We demonstrate through computational analyses that a collection of 30 large recurrent genomic deletions scattered in the human genome contribute to more than 10% of individual disease load for 2.13% of all known “recessive disease genes”. We performed meta-analyses for all literature reported patients affected with the 13 genes whose carrier burden are predicted to be almost exclusively from large recurrent genomic deletions. The results suggest that current sequencing efforts for personal genomes with large recurrent deletions is under-appreciated. By retrospective analyses of previously undiagnostic exome sequencing (ES) data on 69 subjects harboring 26 types of recurrent deletions, probable diagnostic variants were uncovered in genes including COX10, ERCC6, PRRT2 and OTUD7A, demonstrating new disease allele/gene/mechanism characterization. Findings from this study support the contention that more whole genome sequencing (WGS) may further resolve molecular diagnoses and provide evidence for multi-locus pathogenic variation (MPV). Such analyses benefit all stakeholders in both research development and patient clinical care.

Screening of 22q11.2DS Using Multiplex Ligation-Dependent Probe Amplification as an Alternative Diagnostic Method

Background. The 22q11.2 deletion syndrome (22q11.2DS) is the most common form of deletion disorder in humans. Low copy repeats flanking the 22q11.2 region confers a substrate for nonallelic homologous recombination (NAHR) events leading to rearrangements which have been reported to be associated with highly variable and expansive phenotypes. The 22q11.2DS is reported as the most common genetic cause of congenital heart defects (CHDs). Methods. A total of 42 patients with congenital heart defects, as confirmed by echocardiography, were recruited. Genetic molecular analysis using a fluorescence in situ hybridization (FISH) technique was conducted as part of routine 22q11.2DS screening, followed by multiplex ligation-dependent probe amplification (MLPA), which serves as a confirmatory test. Results. Two of the 42 CHD cases (4.76%) indicated the presence of 22q11.2DS, and interestingly, both cases have conotruncal heart defects. In terms of concordance of techniques used, MLPA is superior since it can detect deletions within the 22q11.2 locus and outside of the typically deleted region (TDR) as well as duplications. Conclusion. The incidence of 22q11.2DS among patients with CHD in the east coast of Malaysia is 0.047. MLPA is a scalable and affordable alternative molecular diagnostic method in the screening of 22q11.2DS and can be routinely applied for the diagnosis of deletion syndromes.

An Adolescent with a Rare De Novo Distal Trisomy 6p and Distal Monosomy 6q Chromosomal Combination

We report on a 12-year-old female with both a partial duplication and deletion involving chromosome 6. The duplication involves 6p25.3p24.3 (7.585 Mb) while the deletion includes 6q27q27 (6.244 Mb). This chromosomal abnormality is also described as distal trisomy 6p and distal monosomy 6q. The patient has a Chiari II malformation, hydrocephalus, agenesis of the corpus callosum, microcephaly, bilateral renal duplicated collecting system, scoliosis, and myelomeningocele associated with a neurogenic bladder and bladder reflux. Additional features have included seizures, feeding dysfunction, failure to thrive, sleep apnea, global developmental delay, intellectual disability, and absent speech. To our knowledge, our report is just the sixth case in the literature with concomitant distal 6p duplication and distal 6q deletion. Although a majority of chromosomal duplication-deletion cases have resulted from a parental pericentric inversion, the parents of our case have normal chromosomes. This is the first reported de novo case of distal 6p duplication and distal 6q deletion. Alternate explanations for the origin of the patient’s chromosome abnormalities include parental gonadal mosaicism, nonallelic homologous recombination, or potentially intrachromosomal transposition of the telomeres of chromosome 6. Nonpaternity was considered but ruled out by whole exome sequencing analysis.

Established and Novel Mechanisms Leading to de novo Genomic Rearrangements in the Human Germline

During gametogenesis, the human genome can acquire various de novo rearrangements. Most constitutional genomic rearrangements are created through 1 of the 4 well-known mechanisms, i.e., nonallelic homologous recombination, erroneous repair after double-strand DNA breaks, replication errors, and retrotransposition. However, recent studies have identified 2 types of extremely complex rearrangements that cannot be simply explained by these mechanisms. The first type consists of chaotic structural changes in 1 or a few chromosomes that result from “chromoanagenesis (an umbrella term that covers chromothripsis, chromoanasynthesis, and chromoplexy).” The other type is large independent rearrangements in multiple chromosomes indicative of “transient multifocal genomic crisis.” Germline chromoanagenesis (chromothripsis) likely occurs predominantly during spermatogenesis or postzygotic embryogenesis, while multifocal genomic crisis appears to be limited to a specific time window during oogenesis and early embryogenesis or during spermatogenesis. This review article introduces the current understanding of the molecular basis of de novo rearrangements in the germline.

Historical recombination variability contributes to deciphering the genetic basis of phenotypic traits

Recombination is a main source of genetic variability. However, the potential role of the variation generated by recombination in phenotypic traits, including diseases, remains unexplored as there is currently no method to infer chromosomal subpopulations based on recombination patterns differences. We developed recombClust, a method that uses SNP-phased data to detect differences in historic recombination in a chromosome population. We validated our method by performing simulations and by using real data to accurately predict the alleles of well known recombination modifiers, including common inversions in Drosophila melanogaster and human, and the chromosomes under selective pressure at the lactase locus in humans. We then applied recombClust to the complex human 1q21.1 region, where nonallelic homologous recombination produces deleterious phenotypes. We discovered and validated the presence of two different recombination histories in these regions that significantly associated with the differential expression of ANKRD35 in whole blood and that were in high linkage with variants previously associated with hypertension. By detecting differences in historic recombination, our method opens a way to assess the influence of recombination variation in phenotypic traits.

Fine-scale characterization of genomic structural variation in the human genome reveals adaptive and biomedically relevant hotspots

Genomic structural variants (SVs) are distributed nonrandomly across the human genome. These “hotspots” have been implicated in critical evolutionary innovations, as well as serious medical conditions. However, the evolutionary and biomedical features of these hotspots remain incompletely understood. In this study, we analyzed data from 2,504 genomes from the 1000 Genomes Project Consortium and constructed a refined map of 1,148 SV hotspots in human genomes. By studying the genomic architecture of these hotspots, we found that both nonallelic homologous recombination and non-homologous mechanisms act as mechanistic drivers of SV formation. We found that the majority of SV hotspots are within gene-poor regions and evolve under relaxed negative selection or neutrality. However, we found that a small subset of SV hotspots harbor genes that are enriched for anthropologically crucial functions, including blood oxygen transport, olfaction, synapse assembly, and antigen binding. We provide evidence that balancing selection may have maintained these SV hotspots, which include two independent hotspots on different chromosomes affecting alpha and beta hemoglobin gene clusters. Biomedically, we found that the SV hotspots coincide with breakpoints of clinically relevant, large de novo SVs, significantly more often than genome-wide expectations. As an example, we showed that the breakpoints of multiple large de novo SVs, which lead to idiopathic short stature, coincide with SV hotspots. As such, the mutational instability in SV hotpots likely enables chromosomal breaks that lead to pathogenic structural variation formations. Our study contributes to a better understanding of the mutational landscape of the genome and implicates both mechanistic and adaptive forces in the formation and maintenance of SV hotspots.

Partial 5p Deletion and Partial 5q Duplication in a Patient with Multiple Congenital Anomalies: A Two-Step Mechanism in Chromosomal Rearrangement Mediated by Non-Allelic Homologous Recombination

We describe a 5-month-old female who presented with clinical features of 5p deletion syndrome, including high-pitched cry, microcephaly, micrognathia, bilateral preauricular tags, bifid uvula, abnormal palmar creases, bilateral hypoplastic nipples, feeding difficulties, and developmental delay. In addition, the patient also had a cardiac defect, proximal esophageal atresia, and distal tracheoesophageal fistula. aCGH of the patient revealed a 22.9-Mb deletion of chromosome 5p15.33p14.3 and an 8.28-Mb duplication of chromosome 5q12.1q13.2. Parental chromosome analysis indicated that these alterations are de novo. Chromosome and FISH analysis demonstrated that the 5q12.1q13.2 duplicated segment was attached to the 5p14.3 region with the band 5q12.1 more distal to the centromere than the band 5q13.2. Based on the bioinformatic analysis, we postulate a mechanism for the formation of this complex rearrangement of chromosome 5 by 2-step-wise events mediate by nonallelic homologous recombination between low copy repeats. To the best of our knowledge this rearrangement found in our patient has not been reported in the literature. This report demonstrates the value of chromosome analysis in conjunction with FISH and aCGH for identification of complex rearrangements which cannot be revealed by array analysis alone.