MLPA analysis of 32 foetuses with a congenital heart defect and 1 foetus with renal defects - pilot study. The significant frequency rate of presented pathological CNV.

AIMS: The aim of this retrospective study was to determine the detection rate of the pathogenic copy number variants (CNVs) in a cohort of 33 foetuses - 32 with CHD (congenital heart defects) and 1 with kidney defect, after exclusion of common aneuploidies (trisomy 13, 18, 21, and monosomy X) by karyotyping, Multiplex ligation - dependent probe amplification (MLPA) and chromosomal microarray analysis (CMA). We also assess the effectivity of MLPA as a method of the first tier for quick and inexpensive detection of mutations, causing congenital malformations in foetuses. METHODS: MLPA with probe mixes P070, P036 - Telomere 3 and 5, P245 - microdeletions, P250 - DiGeorge syndrome, and P311 - CHD (Congenital heart defects) was performed in 33 samples of amniotic fluid and chorionic villi. CMA was performed in 10 relevant cases. RESULTS: Pathogenic CNVs were found in 5 samples: microdeletions in region 22q11.2 (≈2 Mb) in two foetuses, one distal microdeletion of the 22q11.2 region containing genes LZTR1, CRKL, AIFM3 and SNAP29 (≈416 kb) in the foetus with bilateral renal agenesis, 8p23.1 (3.8 Mb) microdeletion syndrome and microdeletion in area 9q34.3 (1.7 Mb, Kleefstra syndrome). MLPA as an initial screening method revealed unambiguously pathogenic CNVs in 15.2 % of samples. CONCLUSION: Our study suggests that MLPA and CMA are a reliable and high-resolution technology and should be used as the first-tier test for prenatal diagnosis of congenital heart disease. Determination of the cause of the abnormality is crucial for genetic counselling and further management of the pregnancy.

Duplication of 9p24.3 in three unrelated patients and their phenotypes, considering affected genes, and similar recurrent variants.

BACKGROUND: Recent studies suggest that duplication of the 9p24.3 chromosomal locus, which includes the DOCK8 and KANK1 genes, is associated with autism spectrum disorders (ASD), intellectual disability/developmental delay (ID/DD), learning problems, language disorders, hyperactivity, and epilepsy. Correlation between this duplication and the carrier phenotype needs further discussion. METHODS: In this study, three unrelated patients with ID/DD and ASD underwent SNP aCGH and MLPA testing. Similarities in the phenotypes of patients with 9p24.3, 15q11.2, and 16p11.2 duplications were also observed. RESULTS: All patients with ID/DD and ASD carried the 9p24.3 duplication and showed intragenic duplication of DOCK8. Additionally, two patients had ADHD, one was hearing impaired and obese, and one had macrocephaly. Inheritance of the 9p24.3 duplication was confirmed in one patient and his sibling. In one patient KANK1 was duplicated along with DOCK8. Carriers of 9p24.3, 15q11.2, and 16p11.2 duplications showed several phenotypic similarities, with ID/DD more strongly associated with duplication of 9p24.3 than of 15q11.2 and 16p11.2. CONCLUSION: We concluded that 9p24.3 is a likely cause of ASD and ID/DD, especially in cases of DOCK8 intragenic duplication. DOCK8 is a likely causative gene, and KANK1 aberrations a modulator, of the clinical phenotype observed. Other modulators were not excluded.

Short stature and SHOX (Short stature homeobox) variants-efficacy of screening using various strategies.

BACKGROUND: SHOX mutations have previously been described as causes of Léri-Weill dyschondrosteosis (LWD), Langer mesomelic dysplasia (LMD), and idiopathic short stature. The loss of X chromosome-Turner syndrome or mosaic 45,X/46,XX or 46,XY-also leads to the heterozygous loss of SHOX in patients with short stature only or with features similar to LWD. The aim of this study was to assess the efficacy of the targeted screening for SHOX variants, which involved different methods in the laboratory analysis of short stature. We determined the significance and positive predictive value of short stature for the detection of SHOX variants. METHODS: Targeted screening for variants in SHOX involving MLPA, sequencing, karyotyping and FISH was performed in the short stature cohort (N = 174) and control cohort (N = 91). The significance of short stature and particular characteristics for the detection of SHOX variants was determined by Fisher's exact test, and the probability of SHOX mutation occurrence was calculated using a forward/stepwise logistic regression model. RESULTS: In total, 27 and 15 variants influencing SHOX were detected in the short stature and control cohorts, respectively (p > 0.01). Sex chromosome aberrations and pathogenic CNV resulting in diagnosis were detected in eight (4.6%) and five (2.9%) patients of the short stature group and three (3.3%) and one (1.1%) individuals of the control group. VUS variants were discovered in 14 (8.0%) and 11 (12.1%) individuals of the short stature and control groups, respectively. MLPA demonstrated the detection rate of 13.22%, and it can be used as a frontline method for detection of aberrations involving SHOX. However, only mosaicism of monosomy X with a higher frequency of monosomic cells could be reliably discovered by this method. Karyotyping and FISH can compensate for this limitation; their detection rates in short stature group were 3.55% and 13.46% (N = 52), respectively. FISH proved to be more effective than karyotyping in the study as it could reveal cryptic mosaics in some cases where karyotyping initially failed to detect such a clone. We suggest adding FISH on different tissue than peripheral blood to verify sex-chromosome constitution, especially in cases with karyotypes: 45,X; mosaic 45,X/46,XX or 46,XY; 46,Xidic(Y) detected from blood; in children, where mosaic 45,X was detected prenatally but was not confirmed from peripheral blood. The correlation of short stature with the occurrence of SHOX mutations was insignificant and short stature demonstrates a low positive predictive value-15.5% as unique indicator for SHOX mutations. The typical skeletal signs of LWD, including Madelung deformity and disproportionate growth, positively correlate with the findings of pathogenic SHOX variants (p < 0.01) by Fisher's exact test but not with the findings of VUS variants in SHOX which are more prevalent in the individuals with idiopathic short stature or in the individuals with normal height.

Differences in the importance of microcephaly, dysmorphism, and epilepsy in the detection of pathogenic CNVs in ID and ASD patients.

BACKGROUND: Autism spectrum disorders (ASD) and intellectual disabilities (ID) are heterogeneous and complex developmental diseases with significant genetic backgrounds and overlaps of genetic susceptibility loci. Copy number variants (CNVs) are known to be frequent causes of these impairments. However, the clinical heterogeneity of both disorders causes the diagnostic efficacy of CNV analysis to be modest. This could be resolved by stratifying patients according to their clinical features. AIM: First, we sought to assess the significance of particular clinical features for the detection of pathogenic CNVs in separate groups of ID and ASD patients and determine whether and how these groups differ from each other in the significance of these variables. Second, we aimed to create a statistical model showing how particular clinical features affect the probability of pathogenic CNV findings. METHOD: We tested a cohort of 204 patients with ID (N = 90) and ASD (N = 114) for the presence of pathogenic CNVs. We stratified both groups according to their clinical features. Fisher's exact test was used to determine the significance of these variables for pathogenic CNV findings. Logistic regression was used to create a statistical model of pathogenic CNV findings. RESULTS: The frequency of pathogenic CNV was significantly higher in the ID group than in the ASD group: 18 (19.78%) versus 8 (7%) (p < 0.004). Microcephaly showed a significant association with pathogenic findings in ID patients (p < 0.01) according to Fisher's exact test, whereas epilepsy showed a significant association with pathogenic findings in ASD patients (p < 0.01). The probability of pathogenic CNV findings when epilepsy occurred in ASD patients was more than two times higher than if epilepsy co-occurred with ID (29.6%/14.0%). Facial dysmorphism was a significant variable for detecting pathogenic CNVs in both groups (ID p = 0.05, ASD p = 0.01). However, dysmorphism increased the probability of pathogenic CNV detection in the ID group nearly twofold compared to the ASD group (44.4%/23.7%). The presence of macrocephaly in the ASD group showed a 25% probability of pathogenic CNV findings by logistic regression, but this was insignificant according to Fisher's exact test. The probability of detecting pathogenic CNVs decreases up to 1% in the absence of dysmorphism, macrocephaly, and epilepsy in the ASD group. CONCLUSION: Dysmorphism, microcephaly, and epilepsy increase the probability of pathogenic CNV findings in ID and ASD patients. The significance of each feature as a predictor for pathogenic CNV detection differs depending on whether the patient has only ASD or ID. The probability of pathogenic CNV findings without dysmorphism, macrocephaly, or epilepsy in ASD patients is low. Therefore the efficacy of CNV analysis is limited in these patients.

MLPA is a practical and complementary alternative to CMA for diagnostic testing in patients with autism spectrum disorders and identifying new candidate CNVs associated with autism.

BACKGROUND: Autism spectrum disorder (ASD) is a complex heterogeneous developmental disease with a significant genetic background that is frequently caused by rare copy number variants (CNVs). Microarray-based whole-genome approaches for CNV detection are widely accepted. However, the clinical significance of most CNV is poorly understood, so results obtained using such methods are sometimes ambiguous. We therefore evaluated a targeted approach based on multiplex ligation-dependent probe amplification (MLPA) using selected probemixes to detect clinically relevant variants for diagnostic testing of ASD patients. We compare the reliability and efficiency of this test to those of chromosomal microarray analysis (CMA) and other tests available to our laboratory. In addition, we identify new candidate genes for ASD identified in a cohort of ASD-diagnosed patients. METHOD: We describe the use of MLPA, CMA, and karyotyping to detect CNV in 92 ASD patients and evaluate their clinical significance. RESULT: Pathogenic and likely pathogenic mutations were identified by CMA in eight (8.07% of the studied cohort) and 12 (13.04%) ASD patients, respectively, and in eight (8.07%) and four (4.35%) patients, respectively, by MLPA. The detected mutations include the 22q13.3 deletion, which was attributed to ring chromosome 22 formation based on karyotyping. CMA revealed a total of 91 rare CNV in 55 patients: eight pathogenic, 15 designated variants of unknown significance (VOUS)-likely pathogenic, 10 VOUS-uncertain, and 58 VOUS-likely benign or benign. MLPA revealed 18 CNV in 18 individuals: eight pathogenic, four designated as VOUS-likely pathogenic, and six designated as VOUS-likely benign/benign. Rare CNVs were detected in 17 (58.62%) out of 29 females and 38 (60.32%) out of 63 males in the cohort. Two genes, DOCK8 and PARK2, were found to be overlapped by CNV designated pathogenic, VOUS-likely pathogenic, or VOUS-uncertain in multiple patients. Moreover, the studied ASD cohort exhibited significant (p < 0.05) enrichment of duplications encompassing DOCK8. CONCLUSION: Multiplex ligation-dependent probe amplification and CMA yielded concordant results for 12 patients bearing CNV designated pathogenic or VOUS-likely pathogenic. Unambiguous diagnoses were achieved for eight patients (corresponding to 8.7% of the total studied population) by both MLPA and CMA, for one (1.09%) patient by karyotyping, and for one (1.09%) patient by FRAXA testing. MLPA and CMA thus achieved identical reliability with respect to clinically relevant findings. As such, MLPA could be useful as a fast and inexpensive test in patients with syndromic autism. The detection rate of potentially pathogenic variants (VOUS-likely pathogenic) achieved by CMA was higher than that for MLPA (13.04% vs. 4.35%). However, there was no corresponding difference in the rate of unambiguous diagnoses of ASD patients. In addition, the results obtained suggest that DOCK8 may play a role in the etiology of ASD.

Individual human odor fallout as detected by trained canines.

We tested the hypothesis that if odor fallout (the release of a human's odor onto an untouched object) in human subjects exists, then holding a hand above an absorbent will produce a detectable scent which will be subsequently matched in a detection test by trained canines. Scents were collected from seven males to sterile cotton absorbent squares. The left hand was used to get the control scent and the right hand served as the target scent. Each experimental subject was sitting; his left hand was laid down on a cotton square for 3 min. The right hand was held 5 cm above another cotton square for 3 min. The scent identification was done by two specially trained police German shepherds. These canines had routinely performed scent identification line-ups as part of criminal investigation procedures. Both canines performed 14 line-ups and correctly matched the collected scents of all test subjects. The results suggest the existence of human odor fallout, whereby a human scent trace is left by humans even if they do not touch an object.