Silhouette scores for assessment of SNP genotype clusters

BACKGROUND: High-throughput genotyping of single nucleotide polymorphisms (SNPs) generates large amounts of data. In many SNP genotyping assays, the genotype assignment is based on scatter plots of signals corresponding to the two SNP alleles. In a robust assay the three clusters that define the genotypes are well separated and the distances between the data points within a cluster are short. "Silhouettes" is a graphical aid for interpretation and validation of data clusters that provides a measure of how well a data point was classified when it was assigned to a cluster. Thus "Silhouettes" can potentially be used as a quality measure for SNP genotyping results and for objective comparison of the performance of SNP assays at different circumstances. RESULTS: We created a program (ClusterA) for calculating "Silhouette scores", and applied it to assess the quality of SNP genotype clusters obtained by single nucleotide primer extension ("minisequencing") in the Tag-microarray format. A Silhouette score condenses the quality of the genotype assignment for each SNP assay into a single numeric value, which ranges from 1.0, when the genotype assignment is unequivocal, down to -1.0, when the genotype assignment has been arbitrary. In the present study we applied Silhouette scores to compare the performance of four DNA polymerases in our minisequencing system by analyzing 26 SNPs in both DNA polarities in 16 DNA samples. We found Silhouettes to provide a relevant measure for the quality of SNP assays at different reaction conditions, illustrated by the four DNA polymerases here. According to our result, the genotypes can be unequivocally assigned without manual inspection when the Silhouette score for a SNP assay is > 0.65. All four DNA polymerases performed satisfactorily in our Tag-array minisequencing system. CONCLUSION: "Silhouette scores" for assessing the quality of SNP genotyping clusters is convenient for evaluating the quality of SNP genotype assignment, and provides an objective, numeric measure for comparing the performance of SNP assays. The program we created for calculating Silhouette scores is freely available, and can be used for quality assessment of the results from all genotyping systems, where the genotypes are assigned by cluster analysis using scatter plots

Hyperestrogenism Affects Adult Height Outcome in Growth Hormone Treated Boys With Silver-Russell Syndrome

Background: Intrauterine growth retardation and short stature are common features in Silver-Russell syndrome (SRS). Despite recombinant growth hormone (rGH) treatment, poor pubertal height gain, affecting adult height (AH), is common. This study investigated whether growth patterns and estrogen concentrations are associated with AH outcome in rGH treated SRS males.Methods: In this retrospective longitudinal single-center study, 11 males with SRS were classified as non-responders (NR = 6) or responders (R = 5), depending on AH adjusted for midparental height. Epigenetic analysis and longitudinal growth measures, including bone age, rGH related parameters, pubertal development, gonadotropins and estrogen concentrations, were analyzed until AH.Results: Pubarche before 9 years was only observed in one NR. At 10 years of age, there was no difference in gonadotropins between NR and R. However, estradiol (E2) concentrations at 10 years of age showed a strong association to AH adjusted for MPH (r = −0.78, p < 0.001). Serum E2 (pmol/L) was significantly higher in NR at ages 10 years [median (range) 2 (<2–5) vs. <2 (<2)], 12 years [23 (10–57) vs. 2 (<2–2)] and 14 years [77 (54–87) vs. 24 (<2–38)] but not at 16 years. Birth weight standard deviation score (SDS) was lower in NR [−4.1 (−4.7 to −2.1) vs. −2.7 (−3.3 to −1.7)]. Weight gain (SDS) until pubertal onset was greater in NR [2.4 (1.4–3.5) vs. 0.8 (−0.4 to 1.7)] and pubertal height gain (SDS) was lower in NR [−1.0 (−2.7–0.4) vs. 0.1 (−0.1 to 1.1)]. At AH, a number of NR and R had high E2 concentrations and small testes.Conclusion: Increased E2 concentrations at age 10, 12, and 14 years were associated to less pubertal height gain, thus affecting AH. Due to the small number of patients, the results need to be confirmed in larger cohorts. The finding of impaired testicular development stresses the need of hormonal evaluation as a complement to clinical and radiological assessment when predicting AH in males with SRS

The value of genome-wide analysis in craniosynostosis

Background: This study assessed the diagnostic yield of high-throughput sequencing methods in a cohort of craniosynostosis (CS) patients not presenting causal variants identified through previous targeted analysis.Methods: Whole-genome or whole-exome sequencing (WGS/WES) was performed in a cohort of 59 patients (from 57 families) assessed by retrospective phenotyping as having syndromic or nonsyndromic CS.Results: A syndromic form was identified in 51% of the unrelated cases. A genetic cause was identified in 38% of syndromic cases, with novel variants detected in FGFR2 (a rare Alu insertion), TWIST1, TCF12, KIAA0586, HDAC9, FOXP1, and NSD2. Additionally, we report two patients with rare recurrent variants in KAT6A and YY1 as well as two patients with structural genomic aberrations: one with a 22q13 duplication and one with a complex rearrangement involving chromosome 2 (2p25 duplication including SOX11 and deletion of 2q22). Moreover, we identified potentially relevant variants in 87% of the remaining families with no previously detected causal variants, including novel variants in ADAMTSL4, ASH1L, ATRX, C2CD3, CHD5, ERF, H4C5, IFT122, IFT140, KDM6B, KMT2D, LTBP1, MAP3K7, NOTCH2, NSD1, SOS1, SPRY1, POLR2A, PRRX1, RECQL4, TAB2, TAOK1, TET3, TGFBR1, TCF20, and ZBTB20.Conclusion: These results confirm WGS/WES as a powerful diagnostic tool capable of either targeted in silico or broad genomic analysis depending on phenotypic presentation (e.g., classical or unusual forms of syndromic CS)

Methods for Analysis of Disease Associated Genomic Sequence Variation

In Molecular Medicine a wide range of methods are applied to analyze the genome to find genetic predictors of human disease. Apart from predisposing disease, genetic variations may also serve as genetic markers in the search for factors underlying complex diseases. Additionally, they provide a means to distinguish between species, analyze evolutionary relationships and subdivide species into strains. The development and improvement of laboratory techniques and computational methods was a spin-off effect of the Human Genome Project. The same techniques for analyzing genomic sequence variations may be used independent of organism or source of DNA or RNA. In this thesis, methods for high-throughput analysis of sequence variations were developed, evaluated and applied. The performance of several genotyping assays were investigated prior to genotyping 4000 samples in a co-operative genetic epidemiological study. Sequence variations in the estrogen receptor alpha gene were found to be associated with an increased risk of breast and endometrial cancer in Swedish women. Whole genome amplification (WGA) enables large scale genetic analysis of sparse amounts of biobanked DNA samples. The performance of two WGA methods was evaluated using four-color minisequencing on tag-arrays. Our in-house developed assay and “array of arrays” format allow up to 80 samples to be analyzed in parallel on a single microscope slide. Multiple displacement amplification by the Φ29 DNA polymerase gave essentially identical genotyping results as genomic DNA. To facilitate accurate method comparisons, a cluster quality assessment approach was established and applied to assess the performance of four commercially available DNA polymerases in the tag-array minisequencing assay. A microarray method for genotyping human group A rotavirus (HRV) was developed and applied to an epidemiological survey of infectious HRV strains in Nicaragua. The method combines specific capture of amplified viral sequences on microarrays with genotype-specific DNA-polymerase mediated extension of capture oligonucleotides with fluorescent dNTPs

Microarrays for Genotyping Human Group A Rotavirus by Multiplex Capture and Type-Specific Primer Extension

Human group A rotavirus (HRV) is the major cause of severe gastroenteritis in infants worldwide. HRV shares the feature of a high degree of genetic diversity with many other RNA viruses, and therefore, genotyping of this organism is more complicated than genotyping of more stable DNA viruses. We describe a novel microarray-based method that allows high-throughput genotyping of RNA viruses with a high degree of polymorphism by multiplex capture and type-specific extension on microarrays. Denatured reverse transcription (RT)-PCR products derived from two outer capsid genes of clinical isolates of HRV were hybridized to immobilized capture oligonucleotides representing the most commonly occurring P and G genotypes on a microarray. Specific primer extension of the type-specific capture oligonucleotides was applied to incorporate the fluorescent nucleotide analogue cyanine 5-labeled dUTP as a detectable label. Laser scanning and fluorescence detection of the microarrays was followed by visual or computer-assisted interpretation of the fluorescence patterns generated on the microarrays. Initially, the method detected HRV in all 40 samples and correctly determined both the G and the P genotypes of 35 of the 40 strains analyzed. After modification by inclusion of additional capture oligonucleotides specific for the initially unassigned genotypes, all genotypes could be correctly defined. The results of genotyping with the microarray fully agreed with the results obtained by nucleotide sequence analysis and sequence-specific multiplex RT-PCR. Owing to its robustness, simplicity, and general utility, the microarray-based method may gain wide applicability for the genotyping of microorganisms, including highly variable RNA and DNA viruses

Hyperestrogenism Affects Adult Height Outcome in Growth Hormone Treated Boys With Silver-Russell Syndrome

Background: Intrauterine growth retardation and short stature are common features in Silver-Russell syndrome (SRS). Despite recombinant growth hormone (rGH) treatment, poor pubertal height gain, affecting adult height (AH), is common. This study investigated whether growth patterns and estrogen concentrations are associated with AH outcome in rGH treated SRS males. Methods: In this retrospective longitudinal single-center study, 11 males with SRS were classified as non-responders (NR = 6) or responders (R = 5), depending on AH adjusted for midparental height. Epigenetic analysis and longitudinal growth measures, including bone age, rGH related parameters, pubertal development, gonadotropins and estrogen concentrations, were analyzed until AH. Results: Pubarche before 9 years was only observed in one NR. At 10 years of age, there was no difference in gonadotropins between NR and R. However, estradiol (E2) concentrations at 10 years of age showed a strong association to AH adjusted for MPH (r = -0.78, p amp;lt; 0.001). Serum E2 (pmol/L) was significantly higher in NR at ages 10 years [median (range) 2 (amp;lt;2-5) vs. amp;lt;2 (amp;lt;2)], 12 years [23 (10-57) vs. 2 (amp;lt;2-2)] and 14 years [77 (54-87) vs. 24 (amp;lt;2-38)] but not at 16 years. Birth weight standard deviation score (SDS) was lower in NR [-4.1 (-4.7 to -2.1) vs. -2.7 (-3.3 to -1.7)]. Weight gain (SDS) until pubertal onset was greater in NR [2.4 (1.4-3.5) vs. 0.8 (-0.4 to 1.7)] and pubertal height gain (SDS) was lower in NR [-1.0 (-2.7-0.4) vs. 0.1 (-0.1 to 1.1)]. At AH, a number of NR and R had high E2 concentrations and small testes. Conclusion: Increased E2 concentrations at age 10, 12, and 14 years were associated to less pubertal height gain, thus affecting AH. Due to the small number of patients, the results need to be confirmed in larger cohorts. The finding of impaired testicular development stresses the need of hormonal evaluation as a complement to clinical and radiological assessment when predicting AH in males with SRS.Funding Agencies|Futurum - the Academy for Health and Care, Region Jonkoping County [FUTURUM-575601, FUTURUM-765281, FUTURUM-578061, FUTURUM-709351]; Pfizer [WI 182827]; IngaBritt and Arne Lundberg Research Foundation [2015-103]; Swedish government [ALFGBG-507931]; county councils, the ALF [ALFGBG-507931]</p

Estrogen receptor alpha gene polymorphism and endometrial cancer risk – a case-control study

Abstract Background Estrogen is an established endometrial carcinogen. One of the most important mediators of estrogenic action is the estrogen receptor alpha. We have investigated whether polymorphic variation in the estrogen receptor alpha gene (ESR1) is associated with endometrial cancer risk. Methods In 702 cases with invasive endometrial cancer and 1563 controls, we genotyped five markers in ESR1 and used logistic regression models to estimate odds ratios (OR) and 95 percent confidence intervals (CI). Results We found an association between rs2234670, rs2234693, as well as rs9340799, markers in strong linkage disequilibrium (LD), and endometrial cancer risk. The association with rs9340799 was the strongest, OR 0.75 (CI 0.60–0.93) for heterozygous and OR 0.53 (CI 0.37–0.77) for homozygous rare compared to those homozygous for the most common allele. Haplotype models did not fit better to the data than single marker models. Conclusion We found that intronic variation in ESR1 was associated with endometrial cancer risk.</p

Replicative and non-replicative mechanisms in the formation of clustered CNVs are indicated by whole genome characterization

Clustered copy number variants (CNVs) as detected by chromosomal microarray analysis (CMA) are often reported as germline chromothripsis. However, such cases might need further investigations by massive parallel whole genome sequencing (WGS) in order to accurately define the underlying complex rearrangement, predict the occurrence mechanisms and identify additional complexities. Here, we utilized WGS to delineate the rearrangement structure of 21 clustered CNV carriers first investigated by CMA and identified a total of 83 breakpoint junctions (BPJs). The rearrangements were further sub-classified depending on the patterns observed: I) Cases with only deletions (n = 8) often had additional structural rearrangements, such as insertions and inversions typical to chromothripsis; II) cases with only duplications (n = 7) or III) combinations of deletions and duplications (n = 6) demonstrated mostly interspersed duplications and BPJs enriched with microhomology. In two cases the rearrangement mutational signatures indicated both a breakage-fusion-bridge cycle process and haltered formation of a ring chromosome. Finally, we observed two cases with Alu- and LINE-mediated rearrangements as well as two unrelated individuals with seemingly identical clustered CNVs on 2p25.3, possibly a rare European founder rearrangement. In conclusion, through detailed characterization of the derivative chromosomes we show that multiple mechanisms are likely involved in the formation of clustered CNVs and add further evidence for chromoanagenesis mechanisms in both "simple" and highly complex chromosomal rearrangements. Finally, WGS characterization adds positional information, important for a correct clinical interpretation and deciphering mechanisms involved in the formation of these rearrangements.Peer reviewe