Saliva DNA: An alternative biospecimen for single nucleotide polymorphism chromosomal microarray analysis in autism.

Chromosomal microarray analysis (CMA) is typically performed for investigation of autism using blood DNA. However, blood collection poses significant challenges for autistic children with repetitive behaviors and sensory and communication issues, often necessitating physical restraint or sedation. Noninvasive saliva collection offers an alternative, however, no published studies to date have evaluated saliva DNA for CMA in autism. Furthermore, previous reports suggest that saliva is suboptimal for detecting copy number variation. We therefore aimed to evaluate saliva DNA for single nucleotide polymorphism (SNP) CMA in autistic children. Saliva DNA from 48 probands and parents (n = 133) was obtained with a mean concentration of 141.7 ng/µL. SNP CMA was successful in 131/133 (98.5%) patients from which we correlated the size and accuracy of a copy number variant(s) called between a proband and carrier parent, and for a subgroup (n = 17 probands) who had a previous CMA using blood sample. There were no discordant copy number variant results between the proband and carrier parent, or the subgroup, however, there was an acceptable mean size difference of 0.009 and 0.07 Mb, respectively. Our findings demonstrate that saliva DNA can be an alternative for SNP CMA in autism, which avoids blood collection with significant implications for clinical practice guidelines.

SNP chromosome microarray genotyping for detection of uniparental disomy in the clinical diagnostic laboratory.

Single nucleotide polymorphism (SNP) chromosome microarray is well established for investigation of children with intellectual deficit/development delay and prenatal diagnosis of fetal malformation but has also emerged for uniparental disomy (UPD) genotyping. Despite published guidelines on clinical indications for testing there are no laboratory guidelines published for performing SNP microarray UPD genotyping. We evaluated SNP microarray UPD genotyping using Illumina beadchips on family trios/duos within a clinical cohort (n=98) and then explored our findings in a post-study audit (n=123). UPD occurred in 18.6% and 19.5% cases, respectively, with chromosome 15 most frequent (62.5% and 25.0%). UPD was predominantly maternal in origin (87.5% and 79.2%), highest in suspected genomic imprinting disorder cases (56.3% and 41.7%) but absent amongst children of translocation carriers. We assessed regions of homozygosity among UPD cases. The smallest interstitial and terminal regions were 2.5 Mb and 9.3 Mb, respectively. We found regions of homozygosity confounded genotyping in a consanguineous case with UPD15 and another with segmental UPD due to non-informative probes. In a unique case with chromosome 15q UPD mosaicism, we established the detection limit of mosaicism as ∼5%. From the benefits and pitfalls identified in this study, we propose a testing model and recommendations for UPD genotyping by SNP microarray.

Neuroblastoma Molecular Risk-Stratification of DNA Copy Number and ALK Genotyping via Cell-Free Circulating Tumor DNA Profiling.

BACKGROUND: MYCN amplification (MNA), segmental chromosomal aberrations (SCA) and ALK activating mutations are biomarkers for risk-group stratification and for targeted therapeutics for neuroblastoma, both of which are currently assessed on tissue biopsy. Increase in demand for tumor genetic testing for neuroblastoma diagnosis is posing a challenge to current practice, as the small size of the core needle biopsies obtained are required for multiple molecular tests. We evaluated the utility of detecting these biomarkers in the circulation. METHODS: Various pre-analytical conditions tested to optimize circulating-tumor DNA (ctDNA) copy number changes evaluations. Plasma samples from 10 patients diagnosed with neuroblastoma assessed for SCA and MNA using single nucleotide polymorphism (SNP) array approach currently used for neuroblastoma diagnosis, with MNA status assessed independently using digital-droplet PCR (ddPCR). Three patients (one in common with the previous 10) tested for ALK activating mutations p.F1174L and p.F1245I using ddPCR. RESULTS: Copy number detection is highly affected by physical perturbations of the blood sample (mimicking suboptimal sample shipment), which could be overcome using specialized preservative collection tubes. Pre-analytical DNA repair procedures on ctDNA before SNP chromosome microarray processing improved the lower limit of detection for SCA and MNA, defined as 20% and 10%, respectively. We detected SCA in 10/10 (100%) patients using SNP array, 7 of which also presented MNA. Circulating-free DNA (cfDNA) and matched tumor DNA profiles were generally identical. MNA was detected using ddPCR in 7/7 (100%) of MNA and 0/12 (0%) non-MNA cases. MNA and ALK mutation dynamic change was assessed in longitudinal samples from 4 and 3 patients (one patient with both), respectively, accurately reflected response to treatment in 6/6 (100%) and disease recurrence in 5/6 (83%) of cases. Samples taken prior to targeted treatment with the ALK inhibitor Lorlatinib and 6-8 weeks on treatment showed reduction/increase in ALK variants according to response to treatment. CONCLUSIONS: These results demonstrate the feasibility of ctDNA profiling for molecular risk-stratification, and treatment monitoring in a clinically relevant time frame and the potential to reduce fresh tissue requirements currently embedded in the management of neuroblastoma.

Validation of a Chromosomal Microarray for Prenatal Diagnosis Using a Prospective Cohort of Pregnancies with Increased Risk for Chromosome Abnormalities.

AIM: Validation of a chromosomal microarray for improved prenatal diagnosis for chromosomal abnormalities among high-risk pregnancies. METHODS: A cohort of 213 pregnancies was investigated by chromosomal microarray and the results were compared with quantitative fluorescent polymerase chain reaction (QF-PCR), karyotype, and 850K single-nucleotide polymorphism microarray results. The detection limit of mosaicism was determined by assaying different trisomy mosaic constructs down to ∼12%. Imprecision estimates from replicates of mean log2 ratio values for a 200 kb deletion and 400 kb duplication were determined by evaluating the coefficient of variation (CV%). RESULTS: Excluding pregnancies with aneuploidy, the chromosomal microarray detected 19/213 (8.9%) pregnancies with copy number abnormalities. These were classified as pathogenic in 11/213 (5.2%) cases, as variants of uncertain significance in 4/213 (1.9%) cases, and as likely benign in 4/213 (1.9%) cases. In 15/213 (7.0%) pregnancies, these abnormalities were not detectable by karyotype. Importantly, 8/11 (72.7%) of the pathogenic abnormalities detected by chromosomal microarray were only detectable by this modality. There were no false-positive results and only eight false-negative results. The chromosomal microarray showed excellent sensitivity (96.2%) and specificity (100.0%). The lower detection limit for mosaicism was ∼12%. Imprecision for the 0.2 Mb deletion (11.6 CV%) and 0.4 Mb duplication (5.9 CV%) was very low. CONCLUSION: This chromosomal microarray showed excellent diagnostic performance with improved detection rates compared to karyotyping for prenatal diagnosis of clinically relevant fetal chromosomal abnormalities.

14q12 microdeletions excluding FOXG1 give rise to a congenital variant Rett syndrome-like phenotype.

Rett syndrome is a clinically defined neurodevelopmental disorder almost exclusively affecting females. Usually sporadic, Rett syndrome is caused by mutations in the X-linked MECP2 gene in ∼90-95% of classic cases and 40-60% of individuals with atypical Rett syndrome. Mutations in the CDKL5 gene have been associated with the early-onset seizure variant of Rett syndrome and mutations in FOXG1 have been associated with the congenital Rett syndrome variant. We report the clinical features and array CGH findings of three atypical Rett syndrome patients who had severe intellectual impairment, early-onset developmental delay, postnatal microcephaly and hypotonia. In addition, the females had a seizure disorder, agenesis of the corpus callosum and subtle dysmorphism. All three were found to have an interstitial deletion of 14q12. The deleted region in common included the PRKD1 gene but not the FOXG1 gene. Gene expression analysis suggested a decrease in FOXG1 levels in two of the patients. Screening of 32 atypical Rett syndrome patients did not identify any pathogenic mutations in the PRKD1 gene, although a previously reported frameshift mutation affecting FOXG1 (c.256dupC, p.Gln86ProfsX35) was identified in a patient with the congenital Rett syndrome variant. There is phenotypic overlap between congenital Rett syndrome variants with FOXG1 mutations and the clinical presentation of our three patients with this 14q12 microdeletion, not encompassing the FOXG1 gene. We propose that the primary defect in these patients is misregulation of the FOXG1 gene rather than a primary abnormality of PRKD1.

Cyclin-dependent kinase-like 5 (CDKL5) mutation screening in Rett syndrome and related disorders.

Rett syndrome (RTT) is a severe neurodevelopmental disorder affecting females almost exclusively and is characterized by a wide spectrum of clinical manifestations. Mutations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene have been found in up to 95% of classical RTT cases and a lesser proportion of atypical cases. Recently, mutations in another X-linked gene, CDKL5 (cyclin-dependent kinase-like 5) have been found to cause atypical RTT, in particular the early onset seizure (Hanefeld variant) and one female with autism. In this study we screened several cohorts of children for CDKL5 mutations, totaling 316 patients, including individuals with a clinical diagnosis of RTT but who were negative for MECP2 mutations (n=102), males with X-linked mental retardation (n=9), patients with West syndrome (n=52), patients with autism (n=59), patients with epileptic encephalopathy (n=33), patients with Aicardi syndrome (n=7) and other patients with intellectual disability with or without seizures (n=54). In all, seven polymorphic variations and four de novo mutations (c.586C>T [p.S196L]; c.58G>C [p.G20R]; c.2504delC [p.P835fs]; deletion of exons 1-3) were identified, and in all instances of the latter the clinical phenotype was that of an epileptic encephalopathy. These results suggest that pathogenic CDKL5 mutations are unlikely to be identified in the absence of severe early-onset seizures and highlight the importance of screening for large intragenic and whole gene deletions.

An innocuous duplication of 11.2 Mb at 13q21 is gene poor: sub-bands of gene paucity and pervasive CNV characterize the chromosome anomalies.

A boy with autistic spectrum disorder without dysmorphisms was found to have a chromosome duplication of part of band 13q21. His mother and grandfather both of normal intellect had the same chromosomal duplication. Comparison was made with the Chromosome anomaly database www.som.soton.ac.uk/research/geneticsdiv/anomaly%20register which revealed similar cases. Mapping on DNA microarray for the proband and mother showed the duplication to be of length 11.2 Mb, encompassing the 13q21.1-13q21.32 region. The duplicated region is profoundly gene poor, with a mean gene density of 0.45 genes/Mb. We estimate, that the mean gene density in the sub-bands of the chromosome anomalies is 2.4-2.5 genes/Mb. In addition the percentages of the sub-bands reported as copy number variants (CNV) was estimated from the Database of Chromosome Genomic Variants (http://projects.tcag.ca/variation/). It was found that for some of these sub-bands, gene paucity was likely to be a major contributor to their innocuous phenotypic effect, for example, the gene densities were for: 1p31.2 (1.25 genes/Mb); 2p12 (1.7); 4p15.31 (1.3); 5p14.1 (0.22); 5p14.3 (0.8); 5q21.2 (0.6); 5q21.3 (1.2); 8p23.2 (0.25); 13q21.1 (0.9); 14q31.1 (1.4); 18q22.1 (1.4); 21q21.1 (1.2); and 21q21.2 (0.7). For other sub-bands the percentage of the band in which CNV have been reported was found to be markedly increased, for example, 8p23.2 (94.7% of the band is defined by reported CNV); 3p26.3 (81.6); 5p14.3 (59.3); 8p22 (48.8); 2p12 (44.0); 5q21.1 (43.6); 6q24.2 (41.4); 9p23 (38.8); 10q21.1 (36.5); 5q21.2 (35.4), and 11q14.3 (33.8). We argue that both gene paucity and pervasive CNV are major indicators of bands conforming to the Chromosome Anomaly phenomenon.

Paternally inherited submicroscopic duplication at 11p15.5 implicates insulin-like growth factor II in overgrowth and Wilms' tumorigenesis.

Loss of imprinting at insulin-like growth factor II (IGFII), in association with H19 silencing, has been described previously in a subgroup of Beckwith-Wiedemann syndrome (BWS) patients who have an elevated risk for Wilms' tumor. An equivalent somatic mutation occurs in sporadic Wilms' tumor. We describe a family with overgrowth in three generations and Wilms' tumor in two generations, with paternal inheritance of a cis-duplication at 11p15.5 spanning the BWS IC1 region and including H19, IGFII, INS, and TH. The duplicated region was below the limit of detection by high-resolution karyotyping and fluorescence in situ hybridization, has a predicted minimum size of 400 kb, and was confirmed by genotyping and gene-dosage analysis on a CytoChip comparative genomic hybridization bacterial artificial chromosome array. IGFII is the only known paternally expressed oncogene mapping within the duplicated region and our findings directly implicate IGFII in Wilms' tumorigenesis and add to the mutation spectrum that increases the effective dose of IGFII. Furthermore, this study raises the possibility that sporadic cases of overgrowth and Wilms' tumor, presenting with apparent gain of methylation at IC1, may be explained by submicroscopic paternal duplications. This finding has important implications for determining the transmission risk in these disorders.

Issues arising from the prenatal diagnosis of some rare trisomy mosaics--the importance of cryptic fetal mosaicism.

OBJECTIVES: To add to the knowledge of fetal mosaicism, confined placental mosaicism (CPM), and uniparental disomy (UPD), in rare trisomies detected at prenatal diagnosis. METHODS: The origin of rare trisomy mosaics, mostly (8/11) seen in amniocytes, was examined in 11 cases by follow-up karyotyping and the study of microsatellite inheritance. RESULTS: Of the rare trisomies presented, three were mosaic trisomy 16 (two of which were CPM), and the remainder comprised single cases of mosaic trisomies of 8, 9, 10, 11, 12, 14, 5 and 15--the last two being CPM. Cases varied in parental derivation and meiotic versus post-zygotic origin but no case involved UPD. There was evidence for cryptic fetal mosaicism in three cases (5, 7, 11)--involving chromosomes 11, 14 and 16. CONCLUSIONS: These cases contribute further data to phenotypes associated with rare trisomies and the relative influences on the phenotype of CPM, UPD and fetal mosaicism. From sparse published data, we estimate that approximately 10% of apparent CPM cases for a rare trisomy (i.e. aneuploid CVS, normal amniocytes) may actually be cryptic fetal mosaics undetected in cultured amniocytes. In many cases, this cryptic mosaicism may be of limited clinical significance, but in others, the associated phenotypic effects may be obvious. There is no general approach to resolve this issue; the finding of even a few similar aneuploid cells in different amniocyte culture vessels may be clinically significant. It may be useful to study such an amniocyte culture with FISH with the relevant centromeric probe. Careful follow-up is recommended, particularly for infants where apparent correction of autosomal trisomy has occurred.

Three different origins for apparent triploid/diploid mosaics.

Four apparent triploid/diploid mosaic cases were studied. Three of the cases were detected at prenatal diagnosis and the other was of an intellectually handicapped, dysmorphic boy. Karyotypes were performed in multiple tissues if possible, and the inheritance of microsatellites was studied with DNA from fetal tissues and parental blood. Non-mosaic triploids have a different origin from these mosaics with simple digyny or diandry documented in many cases. Three different mechanisms of origin for these apparent mosaics were detected: (1) chimaerism with karyotypes from two separate zygotes developing into a single individual, (2) delayed digyny, by incorporation of a pronucleus from a second polar body into one embryonic blastomere, and (3) delayed dispermy, similarly, by incorporation of a second sperm pronucleus into one embryonic blastomere. In three of the four cases, there was segregation within the embryos of triploid and diploid cell lines into different tissues from which DNA could be isolated. In case 2 originating by digyny, the same sperm allele at each locus could be detected in both triploid and diploid tissues, which is supportive evidence for the involvement of a single sperm and for true mosaicism rather than chimaerism. Similarly, in case 4 originating by dispermy, the same single ovum allele at each locus could be detected in diploid and triploid tissues, confirming mosaicism. In the chimaeric case (case 3), the diploid line had the karyotype 47,XY,+16 while the triploid line was 69,XXY. This suggests a chimaera, since, in a true mosaic, the triploid line should also contain the additional chromosome 16. Supporting the interpretation of a chimaeric origin for this case, the DNA data showed that the triploidy was consistent with MII non-disjunction (i.e. involving a diploid ovum). In the mosaic cases (1, 2, 4), there was no evidence of the involvement of a diploid sperm or a diploid ova, and in triploid/diploid mosaicism, an origin from a diploid gamete is excluded, since all such conceptuses would be simple triploids. In one of these triploid/diploid mosaics detected at prenatal diagnosis by CVS, the triploid line seemed to be sequestered into the extra-fetal tissues (confined placental mosaicism). This fetus developed normally and a normal infant was born with no evidence of triploidy in newborn blood or cord blood at three months of age.

Recombinants of intrachromosomal transposition of subtelomeres in chromosomes 1 and 2: a cause of minute terminal chromosomal imbalances.

Two cases of submicroscopic recombinants of intrachromosomal transposition of telomeres, one each from chromosome 1 and 2 are described. Meiotic crossing-over would generate the recombinants from these reciprocal rearrangements. In both cases, which were detected by FISH with subtelomeric probes, there is a minute deletion of the qter region and a second presence of the pter subtelomeric region on the respective qter, i.e., a duplication of 1pter or 2pter respectively. The deletion on 2qter (case 2) was confirmed by microsatellite inheritance and was of paternal origin, but in case 1 there was no detectable 1q deletion other than of the subtelomeric probe, and parental origin could not be determined. The present case 2 with del(2qter)/dup(2pter) shares many features with reported cases of simple deletion (2qter) but did not have features of Albright hereditary osteodystrophy, which are seen in half of such deletion patients. The clinical features present in case 1 were similar to those of the previously reported case of a submicroscopic 1qter deletion but also to cases with microscopically visible 1qter deletions, presumably because of gene enrichment in subtelomeric regions. Recombinants of such intrachromosomal subtelomere transpositions detected by subtelomeric probes may comprise up to 10% of submicroscopic pter or qter deletion cases. Other cases of this unusual mechanism may be detected with more common use of subtelomeric probes. It is suggested the bouquet associations of telomeres in early meiosis may facilitate such unusual rearrangements.