Renius Owen

Frequency and Complexity of De Novo Structural Mutation in Autism

Genetic studies of autism spectrum disorder (ASD) have established that de novo duplications and deletions contribute to risk. However, ascertainment of structural variants (SVs) has been restricted by the coarse resolution of current approaches. By applying a custom pipeline for SV discovery, genotyping, and de novo assembly to genome sequencing of 235 subjects (71 affected individuals, 26 healthy siblings, and their parents), we compiled an atlas of 29,719 SV loci (5,213/genome), comprising 11 different classes. We found a high diversity of de novo mutations, the majority of which were undetectable by previous methods. In addition, we observed complex mutation clusters where combinations of de novo SVs, nucleotide substitutions, and indels occurred as a single event. We estimate a high rate of structural mutation in humans (20%) and propose that genetic risk for ASD is attributable to an elevated frequency of gene-disrupting de novo SVs, but not an elevated rate of genome rearrangement.

Regions of homozygosity identified by oligonucleotide SNP arrays: evaluating the incidence and clinical utility

Copy neutral segments with allelic homozygosity, also known as regions of homozygosity (ROHs), are frequently identified in cases interrogated by oligonucleotide single-nucleotide polymorphism (oligo-SNP) microarrays. Presence of ROHs may be because of parental relatedness, chromosomal recombination or rearrangements and provides important clues regarding ancestral homozygosity, consanguinity or uniparental disomy. In this study of 14 574 consecutive cases, 832 (6%) were found to harbor one or more ROHs over 10 Mb, of which 651 cases (78%) had multiple ROHs, likely because of identity by descent (IBD), and 181 cases (22%) with ROHs involving a single chromosome. Parental relatedness was predicted to be first degree or closer in 5%, second in 9% and third in 19%. Of the 181 cases, 19 had ROHs for a whole chromosome revealing uniparental isodisomy (isoUPD). In all, 25 cases had significant ROHs involving a single chromosome; 5 cases were molecularly confirmed to have a mixed iso- and heteroUPD15 and 1 case each with segmental UPD9pat and segmental UPD22mat; 17 cases were suspected to have a mixed iso- and heteroUPD including 2 cases with small supernumerary marker and 2 cases with mosaic trisomy. For chromosome 15, 12 (92%) of 13 molecularly studied cases had either Prader–Willi or Angelman syndrome. Autosomal recessive disorders were confirmed in seven of nine cases from eight families because of the finding of suspected gene within a ROH. This study demonstrates that ROHs are much more frequent than previously recognized and often reflect parental relatedness, ascertain autosomal recessive diseases or unravel UPD in many cases.

Abnormalities in spontaneous abortions detected by G-banding and chromosomal microarray analysis (CMA) at a national reference laboratory

BackgroundCytogenetic evaluation of products of conception (POC) for chromosomal abnormalities is central to determining the cause of pregnancy loss. We compared the test success rates in various specimen types and the frequencies of chromosomal abnormalities detected by G-banding analysis with those found by Oligo-SNP chromosomal microarray analysis (CMA). We evaluated the benefit of CMA testing in cases of failed culture growth.MethodsConventional cytogenetic results of 5457 consecutive POC specimens were reviewed and categorized as placental villi, fetal parts, and unspecified POC tissue. The CMA was performed on 268 cases. Of those, 32 cases had concurrent G-banding results. The remaining 236 cases included 107 cases with culture failure and 129 cases evaluated by CMA alone.ResultsThe overall POC culture success rate was 75%, with the lowest for fetal parts (37.4%) and the highest for placental villi (81%). The abnormality rate was 58% for placental villi, but only 25% for fetal parts. Of the abnormalities detected, the most common were aneuploidies, including trisomy 16, triploidy, monosomy X, trisomy 22, trisomy 21 and trisomy 15, while the least encountered aneuploidies were trisomy 1, trisomy 19 and monosomies (except monosomy 21). Overall, POC specimens studied by CMA were successful in 89.6% of cases and yielded a 44.6% abnormality rate.ConclusionsPlacental villi yielded higher rates of culture success and a higher percentage of abnormal karyotypes than did other specimen types. The Oligo-SNP CMA method has demonstrated a viable alternative to the G-banding method in view of its advantages in detection of submicroscopic genomic aberrations, shorter turnaround time due to elimination of time required for culture and a higher test success rate.

Amelioration of the typical cognitive phenotype in a patient with the 5pter deletion associated with Cri-du-chat syndrome in addition to a partial duplication of CTNND2

Cri‐du‐chat is a rare congenital syndrome characterized by intellectual disability, severe speech/developmental delay, dysmorphic features, and additional syndromic findings. The etiology of this disorder is well known, and is attributed to a large deletion on chromosome 5 that typically ranges from band 5p15.2 to the short arm terminus. This region contains CTNND2, a gene encoding a neuronal‐specific protein, delta‐catenin, which plays a critical role in cellular motility and brain function. The exact involvement of CTNND2 in the cognitive functionality of individuals with Cri‐du‐chat has not been fully deciphered, but it is thought to be significant. This report describes an 8‐year‐old African–American female with a complex chromosome 5 abnormality and a relatively mild case of cri‐du‐chat syndrome. Because of the surprisingly mild cognitive phenotype, although a karyotype had confirmed the 5p deletion at birth, an oligo‐SNP microarray was obtained to further characterize her deletion. The array revealed a complex rearrangement, including a breakpoint in the middle of CTNND2, which resulted in a partial deletion and partial duplication of that gene. The array also verified the expected 5p terminal deletion. Although the patient has a significant deletion in CTNND2, half of the gene (including the promoter region) is not only preserved, but is duplicated. The patients milder cognitive and behavioral presentation, in conjunction with her atypical 5p alteration, provides additional evidence for the role of CTNND2 in the cognitive phenotype of individuals with Cri‐du‐chat.

Genotype-phenotype analysis of recombinant chromosome 4 syndrome: an array-CGH study and literature review

BackgroundRecombinant chromosome 4, a rare constitutional rearrangement arising from pericentric inversion, comprises a duplicated segment of 4p13~p15→4pter and a deleted segment of 4q35→4qter. To date, 10 cases of recombinant chromosome 4 have been reported.ResultWe describe the second case in which array-CGH was used to characterize recombinant chromosome 4 syndrome. The patient was a one-year old boy with consistent clinical features. Conventional cytogenetics and FISH documented a recombinant chromosome 4, derived from a paternal pericentric inversion, leading to partial trisomy 4p and partial monosomy of 4q. Array-CGH, performed to further characterize the rearranged chromosome 4 and delineate the breakpoints, documented a small (4.36 Mb) 4q35.1 terminal deletion and a large (23.81 Mb) 4p15.1 terminal duplication. Genotype-phenotype analysis of 10 previously reported cases and the present case indicated relatively consistent clinical features and breakpoints. This consistency was more evident in our case and another characterized by array-CGH, where both showed the common breakpoints of p15.1 and q35.1. A genotype-phenotype correlation study between rec(4), dup(4p), and del(4q) syndromes revealed that urogenital and cardiac defects are probably due to the deletion of 4q whereas the other clinical features are likely due to 4p duplication.ConclusionOur findings support that the clinical features of patients with rec(4) are relatively consistent and specific to the regions of duplication or deletion. Recombinant chromosome 4 syndrome thus appears to be a discrete entity that can be suspected on the basis of clinical features or specific deleted and duplicated chromosomal regions.

Characterization of a complex rearrangement involving duplication and deletion of 9p in an infant with craniofacial dysmorphism and cardiac anomalies

Partial duplication and partial deletion of the short arm of chromosome 9 have each been reported in the literature as clinically recognizable syndromes. We present clinical, cytogenetic, and molecular findings on a five-week-old female infant with concomitant duplication and terminal deletion of the short arm of chromosome 9. To our knowledge ten such cases have previously been reported. Conventional cytogenetic analysis identified additional material on chromosome 9 at band p23. FISH analysis aided in determining the additional material consisted of an inverted duplication with a terminal deletion of the short arm. Microarray analysis confirmed this interpretation and further characterized the abnormality as a duplication of about 32.7 Mb, from 9p23 to 9p11.2, and a terminal deletion of about 11.5 Mb, from 9p24.3 to 9p23. The infant displayed characteristic features of Duplication 9p Syndrome (hypotonia, bulbous nose, single transverse palmar crease, cranial anomalies), as well as features associated with Deletion 9p Syndrome (flat nasal bridge, long philtrum, cardiac anomalies) despite the deletion being distal to the reported critical region for this syndrome. This case suggests that there are genes or regulatory elements that lie outside of the reported critical region responsible for certain phenotypic features associated with Deletion 9p Syndrome. It also underscores the importance of utilizing array technology to precisely define abnormalities involving the short arm of 9p in order to further refine genotype/phenotype associations and to identify additional cases of duplication/deletion.

Molecular combing compared to Southern blot for measuring D4Z4 contractions in FSHD

We compare molecular combing to Southern blot in the analysis of the facioscapulohumeral muscular dystrophy type 1 locus (FSHD1) on chromosome 4q35-qter (chr 4q) in genomic DNA specimens sent to a clinical laboratory for FSHD testing. A de-identified set of 87 genomic DNA specimens determined by Southern blot as normal (n = 71), abnormal with D4Z4 macrosatellite repeat array contractions (n = 7), indeterminate (n = 6), borderline (n = 2), or mosaic (n = 1) was independently re-analyzed by molecular combing in a blinded fashion. The molecular combing results were identical to the Southern blot results in 75 (86%) of cases. All contractions (n = 7) and mosaics (n = 1) detected by Southern blot were confirmed by molecular combing. Of the 71 samples with normal Southern blot results, 67 (94%) had concordant molecular combing results. The four discrepancies were either mosaic (n = 2), rearranged (n = 1), or borderline by molecular combing (n = 1). All indeterminate Southern blot results (n = 6) were resolved by molecular combing as either normal (n = 4), borderline (n = 1), or rearranged (n = 1). The two borderline Southern blot results showed a D4Z4 contraction on the chr 4qA allele and a normal result by molecular combing. Molecular combing overcomes a number of technical limitations of Southern blot by providing direct visualization of D4Z4 macrosatellite repeat arrays on specific chr 4q and chr 10q alleles and more precise D4Z4 repeat sizing. This study suggests that molecular combing has superior analytical validity compared to Southern blot for determining D4Z4 contraction size, detecting mosaicism, and resolving borderline and indeterminate Southern blot results. Further studies are needed to establish the clinical validity and diagnostic accuracy of these findings in FSHD.

Improving the Positive Predictive Value of Non-Invasive Prenatal Screening (NIPS)

We evaluated performance characteristics of a laboratory-developed, non-invasive prenatal screening (NIPS) assay for fetal aneuploidies. This assay employs massively parallel shotgun sequencing with full automation. GC sequencing bias correction and statistical smoothing were performed to enhance discrimination of affected and unaffected pregnancies. Maternal plasma samples from pregnancies with known aneuploidy status were used for assay development, verification, and validation. Assay verification studies using 2,085 known samples (1873 unaffected, 69 trisomy 21, 20 trisomy 18, 17 trisomy 13) demonstrated complete discrimination between autosomal trisomy (Z scores >8) and unaffected (Z scores <4) singleton pregnancies. A validation study using 552 known samples (21 trisomy 21, 10 trisomy 18, 1 trisomy 13) confirmed complete discrimination. Twin pregnancies showed similar results. Follow-up of abnormal results from the first 10,000 clinical samples demonstrated PPVs of 98% (41/42) for trisomy 21, 92% (23/25) for trisomy 18, and 69% (9/13) for trisomy 13. Adjustment for causes of false-positive results identified during clinical testing (eg, maternal duplications) improved PPVs to 100% for trisomy 21 and 96% for trisomy 18. This NIPS test demonstrates excellent discrimination between trisomic and unaffected pregnancies. The PPVs obtained in initial clinical testing are substantially higher than previously reported NIPS methods.

First Report of a Deletion Encompassing an Entire Exon in the Homogentisate 1,2-Dioxygenase Gene Causing Alkaptonuria

Alkaptonuria is often diagnosed clinically with episodes of dark urine, biochemically by the accumulation of peripheral homogentisic acid and molecularly by the presence of mutations in the homogentisate 1,2-dioxygenase gene (HGD). Alkaptonuria is invariably associated with HGD mutations, which consist of single nucleotide variants and small insertions/deletions. Surprisingly, the presence of deletions beyond a few nucleotides among over 150 reported deleterious mutations has not been described, raising the suspicion that this gene might be protected against the detrimental mechanisms of gene rearrangements. The quest for an HGD mutation in a proband with AKU revealed with a SNP array five large regions of homozygosity (5–16 Mb), one of which includes the HGD gene. A homozygous deletion of 649 bp deletion that encompasses the 72 nucleotides of exon 2 and surrounding DNA sequences in flanking introns of the HGD gene was unveiled in a proband with AKU. The nature of this deletion suggests that this in-frame deletion could generate a protein without exon 2. Thus, we modeled the tertiary structure of the mutant protein structure to determine the effect of exon 2 deletion. While the two β-pleated sheets encoded by exon 2 were missing in the mutant structure, other β-pleated sheets are largely unaffected by the deletion. However, nine novel α-helical coils substituted the eight coils present in the native HGD crystal structure. Thus, this deletion results in a deleterious enzyme, which is consistent with the proband’s phenotype. Screening for mutations in the HGD gene, particularly in the Middle East, ought to include this exon 2 deletion in order to determine its frequency and uncover its origin.

Mosaic isochromosome 15q and maternal uniparental isodisomy for chromosome 15 in a patient with morbid obesity and variant PWS-like phenotype

Angelman and Prader–Willi syndromes are reciprocal imprinting disorders caused by loss of maternally or paternally expressed genes, respectively, within 15q11.2–q13. Angelman syndrome (AS; OMIM 105830) is a neurodevelopmental disorder and is due to the loss of maternally expressed UBE3A gene. Prader–Willi syndrome (PWS; OMIM 176270) is a clinically distinct disorder caused by the loss of paternally expressed genes in the human chromosome region 15q11.2–q13. Recently published data strongly suggest a role for the paternally expressed small nucleolar RNA (snoRNA) cluster, SNORD116, in PWS etiology. Uniparental disomy (UPD) 15 is one of the important causes of PWS and AS. Interestingly, balanced and unbalanced chromosomal aberrations in the form of Robertsonian translocation, isochromosomes, supernumerary marker chromosomes and copy number variations have been strongly linked with the occurrence of UPD. Here we report on a very unique case with a mosaic isochromosome for the entire long arm of 15, that is, i(15)(q10), resulting in mosaic uniparental isodisomy for 15q and with no copy number alterations. This is the first report of UPD15 constituted by a mosaic, but copy number neutral chromosomal rearrangement in a patient with a variant PWS‐like phenotype.