Erin K. Roney

Molecular mechanisms for subtelomeric rearrangements associated with the 9q34.3 microdeletion syndrome

We characterized at the molecular level the genomic rearrangements in 28 unrelated patients with 9q34.3 subtelomeric deletions. Four distinct categories were delineated: terminal deletions, interstitial deletions, derivative chromosomes and complex rearrangements; each results in haploinsufficiency of the EHMT1 gene and a characteristic phenotype. Interestingly, 25% of our patients had de novo interstitial deletions, 25% were found with derivative chromosomes and complex rearrangements and only 50% were bona fide terminal deletions. In contrast to genomic disorders that are often associated with recurrent rearrangements, breakpoints involving the 9q34.3 subtelomere region are highly variable. Molecular studies identified three regions of breakpoint grouping. Interspersed repetitive elements such as Alu, LINE, long-terminal repeats and simple tandem repeats are frequently observed at the breakpoints. Such repetitive elements may play an important role by providing substrates with a specific DNA secondary structure that stabilizes broken chromosomes or assist in either DNA double-strand break repair or repair of single double-strand DNA ends generated by collapsed forks. Sequence analyses of the breakpoint junctions suggest that subtelomeric deletions can be stabilized by both homologous and nonhomologous recombination mechanisms, through a telomere-capture event, by de novo telomere synthesis, or multistep breakage-fusion-bridge cycles.

Somatic mosaicism detected by exon-targeted, high-resolution aCGH in 10 362 consecutive cases

Somatic chromosomal mosaicism arising from post-zygotic errors is known to cause several well-defined genetic syndromes as well as contribute to phenotypic variation in diseases. However, somatic mosaicism is often under-diagnosed due to challenges in detection. We evaluated 10 362 patients with a custom-designed, exon-targeted whole-genome oligonucleotide array and detected somatic mosaicism in a total of 57 cases (0.55%). The mosaicism was characterized and confirmed by fluorescence in situ hybridization (FISH) and/or chromosome analysis. Different categories of abnormal cell lines were detected: (1) aneuploidy, including sex chromosome abnormalities and isochromosomes (22 cases), (2) ring or marker chromosomes (12 cases), (3) single deletion/duplication copy number variations (CNVs) (11 cases), (4) multiple deletion/duplication CNVs (5 cases), (5) exonic CNVs (4 cases), and (6) unbalanced translocations (3 cases). Levels of mosaicism calculated based on the array data were in good concordance with those observed by FISH (10–93%). Of the 14 cases evaluated concurrently by chromosome analysis, mosaicism was detected solely by the array in 4 cases (29%). In summary, our exon-targeted array further expands the diagnostic capability of high-resolution array comparative genomic hybridization in detecting mosaicism for cytogenetic abnormalities as well as small CNVs in disease-causing genes.

CHRNA7 triplication associated with cognitive impairment and neuropsychiatric phenotypes in a three-generation pedigree

Although deletions of CHRNA7 have been associated with intellectual disability (ID), seizures and neuropsychiatric phenotypes, the pathogenicity of CHRNA7 duplications has been uncertain. We present the first report of CHRNA7 triplication. Three generations of a family affected with various neuropsychiatric phenotypes, including anxiety, bipolar disorder, developmental delay and ID, were studied with array comparative genomic hybridization (aCGH). High-resolution aCGH revealed a 650-kb triplication at chromosome 15q13.3 encompassing the CHRNA7 gene, which encodes the alpha7 subunit of the neuronal nicotinic acetylcholine receptor. A small duplication precedes the triplication at the proximal breakpoint junction, and analysis of the breakpoint indicates that the triplicated segment is in an inverted orientation with respect to the duplication. CHRNA7 triplication appears to occur by a replication-based mechanism that produces inverted triplications embedded within duplications. Co-segregation of the CHRNA7 triplication with neuropsychiatric and cognitive phenotypes provides further evidence for dosage sensitivity of CHRNA7.

Mechanism, prevalence, and more severe neuropathy phenotype of the Charcot-Marie-Tooth type 1A triplication.

Copy-number variations cause genomic disorders. Triplications, unlike deletions and duplications, are poorly understood because of challenges in molecular identification, the choice of a proper model system for study, and awareness of their phenotypic consequences. We investigated the genomic disorder Charcot-Marie-Tooth disease type 1A (CMT1A), a dominant peripheral neuropathy caused by a 1.4 Mb recurrent duplication occurring by nonallelic homologous recombination. We identified CMT1A triplications in families in which the duplication segregates. The triplications arose de novo from maternally transmitted duplications and caused a more severe distal symmetric polyneuropathy phenotype. The recombination that generated the triplication occurred between sister chromatids on the duplication-bearing chromosome and could accompany gene conversions with the homologous chromosome. Diagnostic testing for CMT1A (n = 20,661 individuals) identified 13% (n = 2,752 individuals) with duplication and 0.024% (n = 5 individuals) with segmental tetrasomy, suggesting that triplications emerge from duplications at a rate as high as ~1:550, which is more frequent than the rate of de novo duplication. We propose that individuals with duplications are predisposed to acquiring triplications and that the population prevalence of triplication is underascertained.

Human subtelomeric copy number gains suggest a DNA replication mechanism for formation: beyond breakage – fusion - bridge for telomere stabilization

Constitutional deletions of distal 9q34 encompassing the EHMT1 (euchromatic histone methyltransferase 1) gene, or loss-of-function point mutations in EHMT1, are associated with the 9q34.3 microdeletion syndrome, also known as Kleefstra syndrome [MIM#610253]. We now report further evidence for genomic instability of the subtelomeric 9q34.3 region as evidenced by copy number gains of this genomic interval that include duplications, triplications, derivative chromosomes and complex rearrangements. Comparisons between the observed shared clinical features and molecular analyses in 20 subjects suggest that increased dosage of EHMT1 may be responsible for the neurodevelopmental impairment, speech delay, and autism spectrum disorders revealing the dosage sensitivity of yet another chromatin remodeling protein in human disease. Five patients had 9q34 genomic abnormalities resulting in complex deletion–duplication or duplication–triplication rearrangements; such complex triplications were also observed in six other subtelomeric intervals. Based on the specific structure of these complex genomic rearrangements (CGR) a DNA replication mechanism is proposed confirming recent findings in Caenorhabditis elegans telomere healing. The end-replication challenges of subtelomeric genomic intervals may make them particularly prone to rearrangements generated by errors in DNA replication.

NUDT21-spanning CNVs lead to neuropsychiatric disease and altered MeCP2 abundance via alternative polyadenylation

The brain is sensitive to the dose of MeCP2 such that small fluctuations in protein quantity lead to neuropsychiatric disease. Despite the importance of MeCP2 levels to brain function, little is known about its regulation. In this study, we report eleven individuals with neuropsychiatric disease and copy-number variations spanning NUDT21, which encodes a subunit of pre-mRNA cleavage factor Im. Investigations of MECP2 mRNA and protein abundance in patient-derived lymphoblastoid cells from one NUDT21 deletion and three duplication cases show that NUDT21 regulates MeCP2 protein quantity. Elevated NUDT21 increases usage of the distal polyadenylation site in the MECP2 3′ UTR, resulting in an enrichment of inefficiently translated long mRNA isoforms. Furthermore, normalization of NUDT21 via siRNA-mediated knockdown in duplication patient lymphoblasts restores MeCP2 to normal levels. Ultimately, we identify NUDT21 as a novel candidate for intellectual disability and neuropsychiatric disease, and elucidate a mechanism of pathogenesis by MeCP2 dysregulation via altered alternative polyadenylation. DOI: http://dx.doi.org/10.7554/eLife.10782.001

A Mosaic 2q24.2 Deletion Narrows the Critical Region to a 0.4 Mb Interval That Includes TBR1, TANK, and PSMD14

Interstitial deletions involving 2q24 have been associated with a wide range of phenotypes including intellectual disability and short stature. To date, the smallest common region among reported cases of deletions in this region is approximately 2.65 Mb and contains 15 genes. In the present case report, we describe an 18‐year‐old male with mild intellectual disability, short stature, and mosaicism for a 0.422 Mb deletion on 2q24.2 that was diagnosed by comparative genomic hybridization and confirmed with fluorescent in situ hybridization (FISH). This deletion, which is present in approximately 61% of cells, includes three genes: TBR1, TANK, and PSMD14. The findings suggest that the critical region for intellectual disability and short stature in 2q24.2 can be narrowed to a 0.422 Mb segment. TBR1, a transcription factor involved in early cortical development, is a strong candidate for the intellectual disability phenotype seen in our patient and in patients with larger deletions in this region of the genome.

Co-occurrence of recurrent duplications of the DiGeorge syndrome region on both chromosome 22 homologues due to inherited and de novo events

Background Genomic rearrangements usually involve one of the two chromosome homologues. Homozygous microdeletion/duplication is very rare. The chromosome 22q11.2 region is prone to recurrent rearrangements due to the presence of low-copy repeats. A common 3 Mb microdeletion causes the well-characterised DiGeorge syndrome (DGS). The reciprocal duplication is associated with an extremely variable phenotype, ranging from apparently normal to learning disabilities and multiple congenital anomalies. Methods and results We describe duplications of the DGS region on both homologues in five patients from three families, detected by array CGH and confirmed by both fluorescence in situ hybridisation and single nucleotide polymorphism arrays. The proband in the first family is homozygous for the common duplication; one maternally inherited and the other a de novo duplication that was generated by nonallelic homologous recombination during spermatogenesis. The 22q11.2 duplications in the four individuals from the other two families are recurrent duplications on both homologues, one inherited from the mother and the other from the father. The phenotype in the patients with a 22q11.2 tetrasomy is similar to the features seen in duplication patients, including cognitive deficits and variable congenital defects. Conclusions Our studies that reveal phenotypic variability in patients with four copies of the 22q11.2 genomic segment, demonstrate that both inherited and de novo events can result in the generation of homozygous duplications, and further document how multiple seemingly rare events can occur in a single individual.

Delineation of a 1.65 Mb Critical Region for Hemihyperplasia and Digital Anomalies on Xq25

Duplications involving portions of the long arm of the X‐chromosome can be associated with mental retardation, short stature, microcephaly, panhypopituitarism, and a wide range of physical findings. Less common are duplications in distal Xq associated with hemihyperplasia and digital anomalies. We report on a 4‐year‐old female with hemihyperplasia, syndactyly of fingers and toes, bilateral 5th finger clinodactyly, short stature, developmental delay, and microcephaly associated with an 11.2 Mb duplication of Xq25–Xq27.1. The boundaries of this duplication were mapped using high resolution array comparative genome hybridization and follow‐up studies revealed that the same duplication was carried by the patients mother who has short stature and cognitive disabilities. Using the duplication boundaries from this case, and data from previously published reports, we have delineated a 1.65 Mb critical region for hemihyperplasia and digital anomalies on chromosome Xq25. Based on these findings physicians should consider obtaining array comparative genome hybridization studies on individuals with hemihyperplasia especially when accompanied by digital findings since identification of an Xq25 duplication can dramatically change recurrence risk estimations and may also provide insight into the possible comorbidities.

Identification of complex chromosome 18 rearrangements by FISH and array CGH in two patients with apparent isochromosome 18q

TO THE EDITOR: Most cases of Edwards syndrome result from an extra copy of chromosome 18 (Trisomy 18) [Edwards et al., 1960], however, a small fraction of individuals have unbalanced translocations or isochromosome 18q (i(18q)) [Bass et al., 1979; Froster-Iskenius et al., 1984], reviewed in Bugge et al. [2004]. The observation of i(18q) generally indicates a deletion of the entire short arm of chromosome 18 and a gain of the entire long arm; however, we have identified two unique cases in which cytogenetically apparent isochromosomes 18q were in fact complex rearrangements involving portions of both 18p and 18q. In one case, chromosome studies were initially performed in the newborn period, and low resolution banding failed to identify the complex nature of the rearrangement. In the second case, array CGH initially identified the complex rearrangement, however, follow up chromosome studies revealed an isochromosome-like appearance of the derivative 18. Thus, the results presented here demonstrate that patients with isochromosomes identified by conventional chromosome analysis could benefit from more detailed molecular cytogenetic studies such as FISH and array CGH. The first patient was delivered at 33.5 weeks gestation by cesarean section for abnormalities in fetal heart monitoring. A routine fetal ultrasound revealed an enlarged right atrium, and a fetal echocardiogram revealed a mobile mass in the left ventricular outlet. Birth weight was 1,353 g and head circumference was 23 cm, both well below the 5th percentile for gestational age. Features included prominent occiput with a large posterior fontanelle, posteriorly rotated ears, clenched hands, hypoplasia of the nails, a short sternum, and mild rocker-bottom feet. An echocardiogram revealed multiple abnormalities, including a large patent ductus arteriosus, a large muscular ventricular septal defect, and aneurismal redundant tricuspid valve tissue. G-tube/Nissen fundoplication, bilateral inguinal hernia repair, and tracheostomy placement were performed at 4½ months of age. At 4 years of age, the child was nonverbal and could sit but not crawl or walk. He weighed 13.1 kg and received a mixture of G-tube feeds and oral feeds. He no longer required mechanical ventilation; however, he had a tracheostomy in place and required oxygen at night. The initial low-resolution GTG-banded chromosome analysis performed in the newborn period identified an abnormal male karyotype with one normal chromosome 18 and one apparent isochromosome 18q, consistent with the clinical diagnosis of Edwards syndrome (Fig. 1a). Follow-up high-resolution studies of the maternal chromosomes were performed which identified a pericentric inversion of one chromosome 18 between bands 18p11.31 and 18q12.2 (Fig. 1b). Given this result, a repeat high-resolution chromosome analysis combined with FISH studies was performed on Patient 1, which revealed that the child had in fact one normal chromosome 18 and one complex chromosome 18 made up of one q arm and one arm consisting of the proximal portion of 18p from 18p11.31 to the centromere and the distal portion of 18q from 18q12.2 to qter (Fig. 1c,e). Based on this data, the karyotype was redefined as 46,XY,rec(18)dup(18q)inv(18)(p11.31q12.2). Following a genetics consultation, it was noted that the mother had 5 previous pregnancies, 4 of which resulted in spontaneous abortions and 1 that produced a child with dextrocardia and congenital diaphragmatic hernia who died in the first 24 hr of life. Karyotype analysis on that child revealed an abnormal chromosome 18, reported as 46,XY,i(18)(q10) by an outside lab. A third child (sibling 2) has since been born following an abnormal prenatal chromosome analysis which revealed the same recombinant chromosome 18 as the proband (Fig. 1d). FIG. 1 Partial karyotypes and ideograms of the three family members included in Case 1. Red and green highlighted areas on the ideograms correspond to FISH probes in (e). a: Initial low-resolution chromosomes 18 from Patient 1. b: Maternal chromosomes 18 showing ... The second patient was born prematurely at 34 weeks gestation to a G1P0→1 mother via cesarean. Birth weight was 1,490 g (10th centile) and head circumference was 29.5 cm (10th–25th centile). Clinical features included a triangular facies, prominence of the forehead, intact high-arched palate, malformed ears with incomplete folding of bilateral helices, abnormal tragus and a cup-shaped right pinna. She had a III/VI harsh holosystolic cardiac murmur, patent foramen ovale, atrial septal defect and a large subaortic ventriculoseptal defect. The digits were without the typical overlapping seen in complete Trisomy 18 patients. A weak suck was noted, and swallow studies demonstrated moderate-to-severe dysphagia with nasopharyngeal aspiration. The perinatal course was complicated by prematurity, intrauterine growth retardation, respiratory distress, periodic breathing, hyperbilirubinemia, metabolic acidosis, hyponatremia, hypokalemia, and feeding intolerance. At 3 months of age, the child passed away due to complications related to congestive heart failure. Array-based comparative genomic hybridization (array CGH) analysis was performed at 40 days of age and revealed a gain in copy number in the subtelomeric region of the short arm of chromosome 18 at band 18p11.32 to18p11.31 (Fig. 2a,b). The duplication spanned a minimum of 4.7 Mb and a maximum of 4.8 Mb and was confirmed by FISH analysis. Additionally, there was a gain in copy number in the long arm of chromosome 18 at band 18q12.3 to 18q23, encompassing a segment of 38.7 Mb in size. GTG-banded chromosome analysis was subsequently performed to further characterize the chromosomal rearrangements. This analysis revealed the presence of one normal chromosome 18 and one apparent isochromosome 18q which, based on array CGH data, was in fact a derivative chromosome 18 consists of a duplication of band 18p11.32 to 18p11.31 and a duplication of band 18q12.3 to 18q23. FISH studies using probes specific to the regions 18p11.31, 18q12.3, 18q23, and 18cen confirmed the inverted nature of the dup (18p) and the dup (18q) (Fig. 3b–d). Based on this data, the infant’s karyotype was designated according to ISCN 2009 and the hg18 build of the human genome as 46,XX,der-(18)dup(18)(p11.32p11.31)dup(18)(q12.3q23).nuc ish 18p11. 31(RP11-838N2×3). arr 18p11.32p11.31(121700–4832613)×3, 18q12.3q23(37401561–76103255)×3. Paternal partial chromosome analysis showed no evidence of a rearrangement involving chromosome 18; however, the maternal sample was not available for analysis. FIG. 2 Array CGH analysis of Patient 2. a: Whole genome plot (left) depicting gain in copy number at 18p11.32-p11.31 and 18q12.3-q23. The enlarged segment of both 18p11.32-p11.31 (~5 Mb) and 18q12.3-q23 (38.7 Mb) is shown on the right. Each point represents ... FIG. 3 Cytogenetic and FISH analyses of Patient 2. a: Partial karyotype showing the normal chromosome 18 and the derivative 18 (arrows). b: Interphase FISH using probes RP11-183C12 (18p11.31, green, 4.4–4.6 Mb) and RP11-838N2 (18p11.31, red, 3.3–3.5 ... In summary, the cases presented here demonstrate the extent to which molecular studies can further elucidate chromosomal abnormalities that are detected by karyotype. In the first case, molecular studies allowed for the identification of a recombinant chromosome 18 found in both the proband (Patient 1) and his sibling, resulting from a maternal inversion (Fig. 1). This is consistent with previously reported chromosome 18 rearrangements in individuals born to normal parents, one of whom carries a pericentric inversion of chromosome 18 [Vianna-Morgante et al., 1976]. In the second case (Patient 2), array CGH identified multiple duplications of chromosome 18 material, and FISH studies revealed the nature of the complex rearrangement (Figs. 2 and ​and3).3). It is important to note that, cytogenetically, both patients had an apparent i(18q). Low-resolution GTG-banded chromosome analysis, which is common for newborn blood specimens or prenatal specimens, could easily have failed to detect these complex rearrangements. Given their cryptic nature, it is easy to predict that similar rearrangements may be more common in ‘‘apparent’’ isochromosomes than previously appreciated. For this reason, cases of i(18q) and other isochromosomes should be interpreted with caution, especially in the case of prenatal chromosome analysis when band resolution is low. Detailed molecular studies such as array CGH and FISH analysis may provide valuable information about the composition of an apparent isochromosome and should be used whenever possible.