Weimin Bi

Characterization of Potocki-Lupski Syndrome (dup(17)(p11.2p11.2)) and Delineation of a Dosage-Sensitive Critical Interval That Can Convey an Autism Phenotype

The duplication 17p11.2 syndrome, associated with dup(17)(p11.2p11.2), is a recently recognized syndrome of multiple congenital anomalies and mental retardation and is the first predicted reciprocal microduplication syndrome described--the homologous recombination reciprocal of the Smith-Magenis syndrome (SMS) microdeletion (del(17)(p11.2p11.2)). We previously described seven subjects with dup(17)(p11.2p11.2) and noted their relatively mild phenotype compared with that of individuals with SMS. Here, we molecularly analyzed 28 additional patients, using multiple independent assays, and also report the phenotypic characteristics obtained from extensive multidisciplinary clinical study of a subset of these patients. Whereas the majority of subjects (22 of 35) harbor the homologous recombination reciprocal product of the common SMS microdeletion (~3.7 Mb), 13 subjects (~37%) have nonrecurrent duplications ranging in size from 1.3 to 15.2 Mb. Molecular studies suggest potential mechanistic differences between nonrecurrent duplications and nonrecurrent genomic deletions. Clinical features observed in patients with the common dup(17)(p11.2p11.2) are distinct from those seen with SMS and include infantile hypotonia, failure to thrive, mental retardation, autistic features, sleep apnea, and structural cardiovascular anomalies. We narrow the critical region to a 1.3-Mb genomic interval that contains the dosage-sensitive RAI1 gene. Our results refine the critical region for Potocki-Lupski syndrome, provide information to assist in clinical diagnosis and management, and lend further support for the concept that genomic architecture incites genomic instability.

Chromosome Catastrophes Involve Replication Mechanisms Generating Complex Genomic Rearrangements

Complex genomic rearrangements (CGRs) consisting of two or more breakpoint junctions have been observed in genomic disorders. Recently, a chromosome catastrophe phenomenon termed chromothripsis, in which numerous genomic rearrangements are apparently acquired in one single catastrophic event, was described in multiple cancers. Here, we show that constitutionally acquired CGRs share similarities with cancer chromothripsis. In the 17 CGR cases investigated, we observed localization and multiple copy number changes including deletions, duplications, and/or triplications, as well as extensive translocations and inversions. Genomic rearrangements involved varied in size and complexities; in one case, array comparative genomic hybridization revealed 18 copy number changes. Breakpoint sequencing identified characteristic features, including small templated insertions at breakpoints and microhomology at breakpoint junctions, which have been attributed to replicative processes. The resemblance between CGR and chromothripsis suggests similar mechanistic underpinnings. Such chromosome catastrophic events appear to reflect basic DNA metabolism operative throughout an organisms life cycle.

Clinical use of array comparative genomic hybridization (aCGH) for prenatal diagnosis in 300 cases.

To evaluate the use of array comparative genomic hybridization (aCGH) for prenatal diagnosis, including assessment of variants of uncertain significance, and the ability to detect abnormalities not detected by karyotype, and vice versa.

Detection of Clinically Relevant Exonic Copy-Number Changes by Array CGH

Array comparative genomic hybridization (aCGH) is a powerful tool for the molecular elucidation and diagnosis of disorders resulting from genomic copy‐number variation (CNV). However, intragenic deletions or duplications—those including genomic intervals of a size smaller than a gene—have remained beyond the detection limit of most clinical aCGH analyses. Increasing array probe number improves genomic resolution, although higher cost may limit implementation, and enhanced detection of benign CNV can confound clinical interpretation. We designed an array with exonic coverage of selected disease and candidate genes and used it clinically to identify losses or gains throughout the genome involving at least one exon and as small as several hundred base pairs in size. In some patients, the detected copy‐number change occurs within a gene known to be causative of the observed clinical phenotype, demonstrating the ability of this array to detect clinically relevant CNVs with subkilobase resolution. In summary, we demonstrate the utility of a custom‐designed, exon‐targeted oligonucleotide array to detect intragenic copy‐number changes in patients with various clinical phenotypes. Hum Mutat 31:1–17, 2010.

Disruption of ROBO2 is associated with urinary tract anomalies and confers risk of vesicoureteral reflux

Congenital anomalies of the kidney and urinary tract (CAKUT) include vesicoureteral reflux (VUR). VUR is a complex, genetically heterogeneous developmental disorder characterized by the retrograde flow of urine from the bladder into the ureter and is associated with reflux nephropathy, the cause of 15% of end-stage renal disease in children and young adults. We investigated a man with a de novo translocation, 46,X,t(Y;3)(p11;p12)dn, who exhibits multiple congenital abnormalities, including severe bilateral VUR with ureterovesical junction defects. This translocation disrupts ROBO2, which encodes a transmembrane receptor for SLIT ligand, and produces dominant-negative ROBO2 proteins that abrogate SLIT-ROBO signaling in vitro. In addition, we identified two novel ROBO2 intracellular missense variants that segregate with CAKUT and VUR in two unrelated families. Adult heterozygous and mosaic mutant mice with reduced Robo2 gene dosage also exhibit striking CAKUT-VUR phenotypes. Collectively, these results implicate the SLIT-ROBO signaling pathway in the pathogenesis of a subset of human VUR.

Resolution of Disease Phenotypes Resulting from Multilocus Genomic Variation

BACKGROUND Whole‐exome sequencing can provide insight into the relationship between observed clinical phenotypes and underlying genotypes. METHODS We conducted a retrospective analysis of data from a series of 7374 consecutive unrelated patients who had been referred to a clinical diagnostic laboratory for whole‐exome sequencing; our goal was to determine the frequency and clinical characteristics of patients for whom more than one molecular diagnosis was reported. The phenotypic similarity between molecularly diagnosed pairs of diseases was calculated with the use of terms from the Human Phenotype Ontology. RESULTS A molecular diagnosis was rendered for 2076 of 7374 patients (28.2%); among these patients, 101 (4.9%) had diagnoses that involved two or more disease loci. We also analyzed parental samples, when available, and found that de novo variants accounted for 67.8% (61 of 90) of pathogenic variants in autosomal dominant disease genes and 51.7% (15 of 29) of pathogenic variants in X‐linked disease genes; both variants were de novo in 44.7% (17 of 38) of patients with two monoallelic variants. Causal copy‐number variants were found in 12 patients (11.9%) with multiple diagnoses. Phenotypic similarity scores were significantly lower among patients in whom the phenotype resulted from two distinct mendelian disorders that affected different organ systems (50 patients) than among patients with disorders that had overlapping phenotypic features (30 patients) (median score, 0.21 vs. 0.36; P=1.77×10‐7). CONCLUSIONS In our study, we found multiple molecular diagnoses in 4.9% of cases in which whole‐exome sequencing was informative. Our results show that structured clinical ontologies can be used to determine the degree of overlap between two mendelian diseases in the same patient; the diseases can be distinct or overlapping. Distinct disease phenotypes affect different organ systems, whereas overlapping disease phenotypes are more likely to be caused by two genes encoding proteins that interact within the same pathway. (Funded by the National Institutes of Health and the Ting Tsung and Wei Fong Chao Foundation.)

DNA sequence of human chromosome 17 and analysis of rearrangement in the human lineage

Chromosome 17 is unusual among the human chromosomes in many respects. It is the largest human autosome with orthology to only a single mouse chromosome, mapping entirely to the distal half of mouse chromosome 11. Chromosome 17 is rich in protein-coding genes, having the second highest gene density in the genome. It is also enriched in segmental duplications, ranking third in density among the autosomes. Here we report a finished sequence for human chromosome 17, as well as a structural comparison with the finished sequence for mouse chromosome 11, the first finished mouse chromosome. Comparison of the orthologous regions reveals striking differences. In contrast to the typical pattern seen in mammalian evolution, the human sequence has undergone extensive intrachromosomal rearrangement, whereas the mouse sequence has been remarkably stable. Moreover, although the human sequence has a high density of segmental duplication, the mouse sequence has a very low density. Notably, these segmental duplications correspond closely to the sites of structural rearrangement, demonstrating a link between duplication and rearrangement. Examination of the main classes of duplicated segments provides insight into the dynamics underlying expansion of chromosome-specific, low-copy repeats in the human genome.

Parental Somatic Mosaicism Is Underrecognized and Influences Recurrence Risk of Genomic Disorders

New human mutations are thought to originate in germ cells, thus making a recurrence of the same mutation in a sibling exceedingly rare. However, increasing sensitivity of genomic technologies has anecdotally revealed mosaicism for mutations in somatic tissues of apparently healthy parents. Such somatically mosaic parents might also have germline mosaicism that can potentially cause unexpected intergenerational recurrences. Here, we show that somatic mosaicism for transmitted mutations among parents of children with simplex genetic disease is more common than currently appreciated. Using the sensitivity of individual-specific breakpoint PCR, we prospectively screened 100 families with children affected by genomic disorders due to rare deletion copy-number variants (CNVs) determined to be de novo by clinical analysis of parental DNA. Surprisingly, we identified four cases of low-level somatic mosaicism for the transmitted CNV in DNA isolated from parental blood. Integrated probabilistic modeling of gametogenesis developed in response to our observations predicts that mutations in parental blood increase recurrence risk substantially more than parental mutations confined to the germline. Moreover, despite the fact that maternally transmitted mutations are the minority of alleles, our model suggests that sexual dimorphisms in gametogenesis result in a greater proportion of somatically mosaic transmitting mothers who are thus at increased risk of recurrence. Therefore, somatic mosaicism together with sexual differences in gametogenesis might explain a considerable fraction of unexpected recurrences of X-linked recessive disease. Overall, our results underscore an important role for somatic mosaicism and mitotic replicative mutational mechanisms in transmission genetics.

Aneuploidy as a mechanism for stress-induced liver adaptation

Over half of the mature hepatocytes in mice and humans are aneuploid and yet retain full ability to undergo mitosis. This observation has raised the question of whether this unusual somatic genetic variation evolved as an adaptive mechanism in response to hepatic injury. According to this model, hepatotoxic insults select for hepatocytes with specific numerical chromosome abnormalities, rendering them differentially resistant to injury. To test this hypothesis, we utilized a strain of mice heterozygous for a mutation in the homogentisic acid dioxygenase (Hgd) gene located on chromosome 16. Loss of the remaining Hgd allele protects from fumarylacetoacetate hydrolase (Fah) deficiency, a genetic liver disease model. When adult mice heterozygous for Hgd and lacking Fah were exposed to chronic liver damage, injury-resistant nodules consisting of Hgd-null hepatocytes rapidly emerged. To determine whether aneuploidy played a role in this phenomenon, array comparative genomic hybridization (aCGH) and metaphase karyotyping were performed. Strikingly, loss of chromosome 16 was dramatically enriched in all mice that became completely resistant to tyrosinemia-induced hepatic injury. The frequency of chromosome 16-specific aneuploidy was approximately 50%. This result indicates that selection of a specific aneuploid karyotype can result in the adaptation of hepatocytes to chronic liver injury. The extent to which aneuploidy promotes hepatic adaptation in humans remains under investigation.

Mutations of RAI1, a PHD-containing protein, in nondeletion patients with Smith-Magenis syndrome

Smith-Magenis syndrome (SMS) is a mental retardation/multiple congenital anomalies disorder associated with a heterozygous ~4-Mb deletion in 17p11.2. Patients with SMS show variability in clinical phenotype despite a common deletion found in >75–80% of patients. Recently, point mutations in the retinoic acid induced 1 (RAI1) gene, which lies within the SMS critical interval, were identified in three patients with many SMS features in whom no deletion was detected. It is not clear if the entire SMS phenotype can be accounted for by RAI1 haploinsufficiency, nor has the precise function of RAI1 been delineated. We report two novel RAI1 mutations, one frameshift and one nonsense allele, in nondeletion SMS patients. Comparisons of the clinical features in these two patients, three of the previously reported RAI1 point mutation cases, and the patients with a common deletion suggest that the majority of the clinical features in SMS result from RAI1 mutation, although phenotypic variability exists even among the individuals with RAI1 point mutations. Bioinformatics analyses of RAI1 and comparative genomics between human and mouse orthologues revealed a zinc finger-like plant homeo domain (PHD) at the carboxyl terminus that is conserved in the trithorax group of chromatin-based transcription regulators. These findings suggest RAI1 is involved in transcriptional control through a multi-protein complex whose function may be altered in individuals with SMS.