The clinical phenotype of Koolen-de Vries syndrome in Turkish patients and literature review.

Koolen-de Vries syndrome (KdVS) is a rare multisystemic disorder caused by a microdeletion on chromosome 17q21.31 including KANSL1 gene or intragenic pathogenic variants in KANSL1 gene. Here, we describe the clinical and genetic spectrum of eight Turkish children with KdVS due to a de novo 17q21.31 deletion, and report on several rare/new conditions. Eight patients from unrelated families aged between 17 months and 19 years enrolled in this study. All patients evaluated by a clinical geneticist, and the clinical diagnosis were confirmed by molecular karyotyping. KdVS patients had some common distinctive facial features. All patients had neuromotor retardation, and speech and language delay. Epilepsy, structural brain anomalies, ocular, ectodermal, and musculoskeletal findings, and friendly personality were remarkable in more than half of the patients. Hypertension, hypothyroidism, celiac disease, and postaxial polydactyly were among the rare/new conditions. Our study contributes to the clinical spectrum of patients with KdVS, while also provide a review by comparing them with previous cohort studies.

Detection of ploidy and chromosomal aberrations in commercial oil palm using high-throughput SNP markers.

MAIN CONCLUSION: Karyotyping using high-density genome-wide SNP markers identified various chromosomal aberrations in oil palm (Elaeis guineensis Jacq.) with supporting evidence from the 2C DNA content measurements (determined using FCM) and chromosome counts. Oil palm produces a quarter of the world's total vegetable oil. In line with its global importance, an initiative to sequence the oil palm genome was carried out successfully, producing huge amounts of sequence information, allowing SNP discovery. High-capacity SNP genotyping platforms have been widely used for marker-trait association studies in oil palm. Besides genotyping, a SNP array is also an attractive tool for understanding aberrations in chromosome inheritance. Exploiting this, the present study utilized chromosome-wide SNP allelic distributions to determine the ploidy composition of over 1,000 oil palms from a commercial F1 family, including 197 derived from twin-embryo seeds. Our method consisted of an inspection of the allelic intensity ratio using SNP markers. For palms with a shifted or abnormal distribution ratio, the SNP allelic frequencies were plotted along the pseudo-chromosomes. This method proved to be efficient in identifying whole genome duplication (triploids) and aneuploidy. We also detected several loss of heterozygosity regions which may indicate small chromosomal deletions and/or inheritance of identical by descent regions from both parents. The SNP analysis was validated by flow cytometry and chromosome counts. The triploids were all derived from twin-embryo seeds. This is the first report on the efficiency and reliability of SNP array data for karyotyping oil palm chromosomes, as an alternative to the conventional cytogenetic technique. Information on the ploidy composition and chromosomal structural variation can help to better understand the genetic makeup of samples and lead to a more robust interpretation of the genomic data in marker-trait association analyses.

Low-pass genome sequencing versus chromosomal microarray analysis: implementation in prenatal diagnosis.

PURPOSE: Emerging studies suggest that low-pass genome sequencing (GS) provides additional diagnostic yield of clinically significant copy-number variants (CNVs) compared with chromosomal microarray analysis (CMA). However, a prospective back-to-back comparison evaluating accuracy, efficacy, and incremental yield of low-pass GS compared with CMA is warranted. METHODS: A total of 1023 women undergoing prenatal diagnosis were enrolled. Each sample was subjected to low-pass GS and CMA for CNV analysis in parallel. CNVs were classified according to guidelines of the American College of Medical Genetics and Genomics. RESULTS: Low-pass GS not only identified all 124 numerical disorders or pathogenic or likely pathogenic (P/LP) CNVs detected by CMA in 121 cases (11.8%, 121/1023), but also defined 17 additional and clinically relevant P/LP CNVs in 17 cases (1.7%, 17/1023). In addition, low-pass GS significantly reduced the technical repeat rate from 4.6% (47/1023) for CMA to 0.5% (5/1023) and required less DNA (50 ng) as input. CONCLUSION: In the context of prenatal diagnosis, low-pass GS identified additional and clinically significant information with enhanced resolution and increased sensitivity of detecting mosaicism as compared with the CMA platform used. This study provides strong evidence for applying low-pass GS as an alternative prenatal diagnostic test.

Fetal Cystic Hygroma Associated with Terminal 2p25.1 Duplication and Terminal 3p25.3 Deletion: Cytogenetic, Fluorescent in Situ Hybridization and Microarray Familial Characterization of Two Different Chromosomal Structural Rearrangements.

We report a prenatally diagnosed case of partial trisomy 2p and partial monosomy 3p, resulting from unbalanced translocation (2;3)(p25.1;p25.3) of paternal origin. Parents were non consanguineous Caucasians, with familial history of recurrent miscarriages on the father's side. Detailed sonographic examination of the fetus showed a septated cystic hygroma measuring 6 mm at 13 weeks' gestation. Karyotyping and fluorescent in situ hybridization (FISH) analysis of cultured amniotic fluid cells revealed an unbalanced translocation der(3)t(2;3)(p25.1; p25.3) and apparently balanced inv(3)(p13p25.3) in a fetus. Parental cytogenetic evaluation using karyotyping and FISH analysis showed the presence of both a balanced translocation and a paracentric inversion in father t(2;3) (p25.1;p25.3) inv(3)(p13p25.3). Microarray analysis showed a 11.6 Mb deletion at 3p26.3-p25.3 and duplication of 10.5 Mb at the 2p25.3-p25 region. The duplicated region at 2p25.1p25.3 contains 45 different genes, where 12 are reported as OMIM morbid genes with different phenotypical implications. The deleted region at 3p26.3-p25.3 contains 65 genes, out of which 27 are OMIM genes. Three of these (CNTN4, SETD5 and VHL) were curated by Clingene Dosage Gene Map and were given a high haplo-insufficiency score. Genes affected by the unbalanced translocation could have contributed to some specific phenotypic changes of the fetus in late pregnancy. The application of different cytogenetic methods was essential in our case, allowing the detection of different types of structural chromosomal aberrations and more thorough genetic counseling for future pregnancies.

Translocation of a parthenogenesis gene candidate to an alternate carrier chromosome in apomictic Brachiaria humidicola.

BACKGROUND: The apomictic reproductive mode of Brachiaria (syn. Urochloa) forage species allows breeders to faithfully propagate heterozygous genotypes through seed over multiple generations. In Brachiaria, reproductive mode segregates as single dominant locus, the apospory-specific genomic region (ASGR). The AGSR has been mapped to an area of reduced recombination on Brachiaria decumbens chromosome 5. A primer pair designed within ASGR-BABY BOOM-like (BBML), the candidate gene for the parthenogenesis component of apomixis in Pennisetum squamulatum, was diagnostic for reproductive mode in the closely related species B. ruziziensis, B. brizantha, and B. decumbens. In this study, we used a mapping population of the distantly related commercial species B. humidicola to map the ASGR and test for conservation of ASGR-BBML sequences across Brachiaria species. RESULTS: Dense genetic maps were constructed for the maternal and paternal genomes of a hexaploid (2n = 6x = 36) B. humidicola F1 mapping population (n = 102) using genotyping-by-sequencing, simple sequence repeat, amplified fragment length polymorphism, and transcriptome derived single nucleotide polymorphism markers. Comparative genomics with Setaria italica provided confirmation for x = 6 as the base chromosome number of B. humidicola. High resolution molecular karyotyping indicated that the six homologous chromosomes of the sexual female parent paired at random, whereas preferential pairing of subgenomes was observed in the apomictic male parent. Furthermore, evidence for compensated aneuploidy was found in the apomictic parent, with only five homologous linkage groups identified for chromosome 5 and seven homologous linkage groups of chromosome 6. The ASGR mapped to B. humidicola chromosome 1, a region syntenic with chromosomes 1 and 7 of S. italica. The ASGR-BBML specific PCR product cosegregated with the ASGR in the F1 mapping population, despite its location on a different carrier chromosome than B. decumbens. CONCLUSIONS: The first dense molecular maps of B. humidicola provide strong support for cytogenetic evidence indicating a base chromosome number of six in this species. Furthermore, these results show conservation of the ASGR across the Paniceae in different chromosomal backgrounds and support postulation of the ASGR-BBML as candidate genes for the parthenogenesis component of apomixis.

BAP1-deficient tumor/nevus with germline aberration: A potential pitfall in assessing melanocytic neoplasms with single nucleotide polymorphism array.

BRCA1-associated protein 1 (BAP1) is a tumor suppressor gene, located on chromosome 3p21, encoding BAP1 nuclear protein, which is associated with a subset of melanocytic tumors with distinct cytologic features. Single nucleotide polymorphism array (SNP-array) is a molecular karyotyping technique that can detect copy number variations and loss of heterozygosity in various fresh and formalin-fixed paraffin-embedded tissues. Herein we present a 56-year-old female, who presented with a lesion on her left nose/cheek that was growing in size and changing in color. Histopathology was characteristic of a BAP1-deficient melanocytic neoplasm, with a biphasic population of cytologically bland conventional nevomelanocytes and a proliferation of large epithelioid melanocytes with abundant eosinophilic cytoplasm. Immunohistochemistry for BAP1 showed loss of nuclear labeling in the epithelioid melanocytes. SNP-array revealed a chromosome 21q22.1 monoaberration with no chromosome 3 abnormalities. The detection of this aberration prompted a discussion as to whether the lesion was best designated as a nevus or tumor. SNP-array on the patient's blood showed the same monoaberration of chromosome 21q22.1. This case emphasizes the importance of interpreting microarray results in the context of morphology, as germline aberrations can be a pitfall when assessing the genomic stability of a melanocytic proliferation by SNP array.

A Systematic Clinical Review of Prenatally Diagnosed Tetrasomy 9p.

Tetrasomy 9p was first described in 1973 and approximately 68 cases with a variable phenotype have been reported to date with 22 of them being detected prenatally. The objective of this study was to review prenatally-reported cases of tetrasomy 9p thus far and to identify ultrasound phenotypes that may be suggestive of this specific syndrome. A PubMed database search was done in February 2018 without any restriction of publication date orjournals, with the use of the following keywords: tetrasomy 9p, tetrasomy 9p prenatal, mosaic tetrasomy 9p, mosaic tetrasomy 9p prenatal, isochromosome 9p, duplication 9p prenatal, trisomy 9p prenatal. Reported cases were included if the clinical presentation and diagnostic approach of each case was clearly described. The most common characteristics of prenatally-detected tetrasomy 9p are intrauterine growth retardation (IUGR, 57.0%), central nervous system (CNS) abnormalities (59.0%), skeletal anomalies (29.0%), genitourinary and renal anomalies (29.0%) and cardiac defects (29.0%). The phenotypic spectrum of tetrasomy 9p is rather unspecific as these findings are commonly associated with other chromosome anomalies, as well as microdeletion/microduplication or monogenic syndromes. The combination of early fetal morphology and diagnostic genetic testing enables a definite tetrasomy 9p diagnosis and effective further pregnancy management.

Diagnosis of fetal submicroscopic chromosomal abnormalities in failed array CGH samples: copy number by sequencing as an alternative to microarrays for invasive fetal testing.

OBJECTIVES: Array comparative genomic hybridization (CGH) has become the technology of choice for high-resolution prenatal whole genome analysis. Limitations of microarrays are mainly related to the analog nature of the analysis, and poor-quality DNA can result in failed quality metrics with these platforms. We examined a cohort of abnormal fetuses with failed array CGH results using a next-generation sequencing algorithm, CNV-Seq. We assessed the ability of the platform to handle suboptimal prenatal samples and generate interpretable molecular karyotypes. METHODS: Nine samples obtained from abnormal fetuses and one from a normal control fetus were sequenced using an Illumina GAIIx. A segmentation algorithm for sequencing data was used to determine regional copy number data on the sequencing datasets. RESULTS: Phred quality scores were satisfactory for analysis of all samples. CNV-Seq identified both large- and small-scale abnormalities in the cohort, and normal results were obtained for fetuses for which microarray data were previously uninterpretable. No variants of uncertain significance were detected. Analysis of the digital sequencing datasets offered some advantages over array CGH output. CONCLUSIONS: Using next-generation sequencing for the detection of genomic copy number variants may be advantageous for poor-quality, invasively-acquired prenatal samples. CNV-Seq could become a potential alternative to array CGH in this setting.

High-resolution molecular karyotyping uncovers pairing between ancestrally related Brassica chromosomes.

How do chromosomal regions with differing degrees of homology and homeology interact at meiosis? We provide a novel analytical method based on simple genetics principles which can help to answer this important question. This method interrogates high-throughput molecular marker data in order to infer chromosome behavior at meiosis in interspecific hybrids. We validated this method using high-resolution molecular marker karyotyping in two experimental Brassica populations derived from interspecific crosses among B. juncea, B. napus and B. carinata, using a single nucleotide polymorphism chip. This method of analysis successfully identified meiotic interactions between chromosomes sharing different degrees of similarity: full-length homologs; full-length homeologs; large sections of primary homeologs; and small sections of secondary homeologs. This analytical method can be applied to any allopolyploid species or fertile interspecific hybrid in order to detect meiotic associations. This genetic information can then be used to identify which genomic regions share functional homeology (i.e., retain enough similarity to allow pairing and segregation at meiosis). When applied to interspecific hybrids for which reference genome sequences are available, the question of how differing degrees of homology and homeology affect meiotic interactions may finally be resolved.

Chromosomal-array analysis reveals partial 11q duplication and partial 12p deletion in a mildly affected case.

Partial trisomy 11q is a rare syndrome and may be observed due to an intra-chromosomal duplication or an inter-chromosomal insertion. The deletions of the short arm of chromosome 12 are also uncommon structural aberrations. Only a small fraction of structural chromosome anomalies are related to the unbalanced progeny of balanced translocation carrier parents. We here report on a 10-month-old baby boy who shows a very mild phenotype related to unique chromosomal abnormality, partial trisomy of 11q, and partial monosomy of 12p, due to the maternal balanced reciprocal translocation (11;12). The proband showed a 49.64 Mb duplication of 11q14.1-q25 and 0.44 Mb deletion of 12p13.33 in chromosomal array analysis. Since it is known that the duplications may cause a milder phenotype than deletions. Dysmorphic facial features, minor cardiac anomalies, respiratory distress, central nervous system anomalies, and psychomotor delay observed in the patient was similar to the reported pure 11q duplication cases, while behavioral problems observed in pure monosomy 12p cases could not be evaluated due to the young age of the patient. Phenotype-genotype correlation will be discussed in view of all the reported pure partial 11q trisomies and pure partial 12p deletion cases.

High-resolution microarray in the assessment of fetal anomalies detected by ultrasound.

AIMS: The main aim of this study was to determine the feasibility of using high-resolution microarray to assist with prenatal diagnosis of ultrasound-detected fetal abnormality and to describe the frequency of abnormal results in different categories of fetal anomalies. METHODS: Prospective cross-sectional study was conducted on women diagnosed with a fetal anomaly (ies) between February 2009 and December 2011 who were offered testing by microarray analysis (Affymetrix 2.7M SNP) and fluorescent in situ hybridisation (FISH) instead of standard karyotyping. Fetal anomalies were categorised according to organ system involvement. RESULTS: One hundred and eighteen women consented to testing with microarray. Eleven of one hundred eighteen (9.3%) cases had aneuploidy detected by FISH. Of the remaining 107, 23 (21.5%) had an abnormality detected on microarray, only three of which would have been detected using the combination of six-probe FISH and banded karyotype. The maximum expected yield for six-probe FISH and karyotype was thus 14/118 (11.8%), compared to 34/118 (28.8%), P < 0.0001. Of the 23 abnormalities detected with microarray, 10 (43%) were pathogenic, six (26%) were long continuous stretches of homozygosity and seven (30%) were of uncertain significance. The maximum yield was in cases with cardiovascular (100%); multiple (40%); central nervous system (CNS) (25%) and skeletal (9%) abnormalities. CONCLUSION: This study has confirmed the feasibility of translation of microarray into clinical practice. 11.8% (14/118) of the cases would have a genetic basis of an abnormality with a FISH and banded karyotype. This figure is approximately tripled to 28.8% (34/118) if we offer FISH and microarray. High yield for imbalances are multiple, cardiovascular, CNS and skeletal abnormalities.

A novel tetra-primer ARMS-PCR approach for the molecular karyotyping of chromosomal inversion 2Ru in the main malaria vectors Anopheles gambiae and Anopheles coluzzii.

BACKGROUND: Chromosomal inversion polymorphisms have been associated with adaptive behavioral, physiological, morphological and life history traits in the two main Afrotropical malaria vectors, Anopheles coluzzii and Anopheles gambiae. The understanding of the adaptive value of chromosomal inversion systems is constrained by the feasibility of cytological karyotyping. In recent years in silico and molecular approaches have been developed for the genotyping of most widespread inversions (2La, 2Rb and 2Rc). The 2Ru inversion, spanning roughly 8% of chromosome 2R, is commonly polymorphic in West African populations of An. coluzzii and An. gambiae and shows clear increases in frequency with increasing rainfall seasonally and geographically. The aim of this work was to overcome the constraints of currently available cytological and high-throughput molecular assays by developing a simple PCR assay for genotyping the 2Ru inversion in individual specimens of both mosquito species. METHODS: We designed tetra-primer amplification refractory mutation system (ARMS)-PCR assays based on five tag single-nucleotide polymorphisms (SNPs) previously shown to be strongly correlated with 2Ru inversion orientation. The most promising assay was validated against laboratory and field samples of An. coluzzii and An. gambiae karyotyped either cytogenetically or molecularly using a genotyping-in-thousands by sequencing (GT-seq) high-throughput approach that employs targeted sequencing of multiplexed PCR amplicons. RESULTS: A successful assay was designed based on the tag SNP at position 2R, 31710303, which is highly predictive of the 2Ru genotype. The assay, which requires only one PCR, and no additional post-PCR processing other than electrophoresis, produced a clear banding pattern for 98.5% of the 454 specimens tested, which is a 96.7% agreement with established karyotyping methods. Sequences were obtained for nine of the An. coluzzii specimens manifesting 2Ru genotype discrepancies with GT-seq. Possible sources of these discordances are discussed. CONCLUSIONS: The tetra-primer ARMS-PCR assay represents an accurate, streamlined and cost-effective method for the molecular karyotyping of the 2Ru inversion in An. coluzzii and An. gambiae. Together with approaches already available for the other common polymorphic inversions, 2La, 2Rb and 2Rc, this assay will allow investigations of the adaptive value of the complex set of inversion systems observed in the two major malaria vectors in the Afrotropical region.

Microduplication 3p26.3p24.3 and 4q34.3q35.2 Microdeletion Identified in a Patient with Developmental Delay Associated with Brain Malformation.

Microdeletions and microduplications are involved in many of prenatal and postnatal cases of multiple congenital malformations (MCM), developmental delay/intellectual disability (DD/ID), and autism spectrum disorders (ASD). Molecular karyotyping analysis (MCA), performed by DNA microarray technology, is a valuable method used to elucidate the ethology of these clinical expressions, essentially contributing to the diagnosis of rare genetic diseases produced by DNA copy number variations (CNVs). MCA is frequently used as the first-tier cytogenetic diagnostic test for patients with MCM, DD/ID, or ASD due to its much higher resolution (≥10×) for detecting microdeletions and microduplications than classic cytogenetic analysis by G-banded karyotyping. Therefore, MCA can detect about 10% pathogenic genomic imbalances more than G-banded karyotyping alone. In addition, MCA using the Single Nucleotide Polymorphism-array (SNP-array) method also allows highlighting the regions of loss of heterozygosity and uniparental disomy, which are the basis of some genetic syndromes. We presented a case of a five-year-old patient, with global development delay, bilateral fronto-parietal lysencephaly, and pachygyria, for which MCA through SNP-Array led to the detection of the genetic changes, such as 3p26.3p24.3 microduplication and 4q34.3q35.2 microdeletion, which were the basis of the patient's phenotype and to the precise establishment of the diagnosis.

Microdeletions in 1q21 and 8q12.1 depict two additional molecular subgroups of Silver-Russell syndrome like phenotypes.

BACKGROUND: Silver-Russell syndrome (SRS) is a genetic disorder characterized by intrauterine and postnatal growth restriction, relative macrocephaly at birth, body asymmetry and typical facial features. Clinical and molecular heterogeneity is described in SRS. Common causes are loss of methylation of the imprinting center 1 in 11p15 and maternal uniparental disomy of chromosome 7. Other genetic alterations include disturbances of imprinted regions in 14q32, 7q32 and 11p15 as well as submicroscopic deletions and duplications. Single nucleotide variants in genes like IGF2, HMGA2, PLAG1, CDKN1C have also been identified in patients with SRS phenotypes. However, routine molecular diagnostics usually focus on 11p15 and chromosome 7, while less frequent causes are not systematically addressed. RESULTS: Here we report two patients with SRS features in which molecular karyotyping revealed microdeletions in 1q21 and 8q12.1 respectively. In a 3.5-year-old girl with postnatal growth restriction, feeding difficulties, relative macrocephaly and distinct SRS features a 2 Mb deletion in 1q21.1q21.2 was identified. Our second case is a 1.5-year-old boy with intrauterine and postnatal growth restriction, feeding difficulties and distinct facial features with a 77 kb deletion in 8q12.1 affecting PLAG1 as the only protein-encoding gene with known function. CONCLUSIONS: The 1q21 region has not yet been assigned as an SRS region, although six patients with the same deletion and SRS features including relative macrocephaly have been described before. This new case adds to the evidence that distal 1q21 should be annotated as an SRS candidate region. The PLAGL1 alteration is the smallest deletion in 8q12.1 ever reported in a patient with SRS phenotype and it finally confirms that PLAG1 is the SRS causing gene in 8q12.1. To increase the diagnostic yield in patients with suspected SRS, we recommend both molecular karyotyping and next generation sequencing-based approaches.

Distribution of segmental chromosomal alterations in neuroblastoma.

BACKGROUND: Neuroblastoma (NB) is a heterogeneous tumor with extremely diverse prognosis according to clinical and genetic factors such as specific combinations of chromosomal imbalances. METHODS: Molecular karyotyping data from a national neuroblastic tumor database of 155 NB samples were analyzed and related to clinical data. RESULTS: Segmental chromosomal alterations (SCA) were detected in 102 NB, whereas 45 only displayed numerical alterations. Incidence of SCA was higher in stage M (92%) and MYCN amplified (MNA) NB (96%). Presence of SCA was associated with older age, especially 1q gain and 3p deletion. 96% of the deaths were observed in the SCA group and 85% of the relapsed NB contained SCA. The alteration most commonly associated with a higher number of other segmental rearrangements was 11q deletion, followed by 4p deletion. Whole-chromosome 19 gain was associated with lower stages, absence of SCA and better outcome. CONCLUSIONS: SCA are not randomly distributed and are concentrated on recurrent chromosomes. The most frequently affected chromosomes identify prognostic factors in specific risk groups. SCA are associated with older age and MNA. We have identified a small subset of patients with better outcome that share whole-chromosome 19 numeric gain, suggesting its use as a prognostic biomarker in NB.

The Clinical Utility of Optical Genome Mapping for the Assessment of Genomic Aberrations in Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) is the most prevalent type of cancer occurring in children. ALL is characterized by structural and numeric genomic aberrations that strongly correlate with prognosis and clinical outcome. Usually, a combination of cyto- and molecular genetic methods (karyotyping, array-CGH, FISH, RT-PCR, RNA-Seq) is needed to identify all aberrations relevant for risk stratification. We investigated the feasibility of optical genome mapping (OGM), a DNA-based method, to detect these aberrations in an all-in-one approach. As proof of principle, twelve pediatric ALL samples were analyzed by OGM, and results were validated by comparing OGM data to results obtained from routine diagnostics. All genomic aberrations including translocations (e.g., dic(9;12)), aneuploidies (e.g., high hyperdiploidy) and copy number variations (e.g., IKZF1, PAX5) known from other techniques were also detected by OGM. Moreover, OGM was superior to well-established techniques for resolution of the more complex structure of a translocation t(12;21) and had a higher sensitivity for detection of copy number alterations. Importantly, a new and unknown gene fusion of JAK2 and NPAT due to a translocation t(9;11) was detected. We demonstrate the feasibility of OGM to detect well-established as well as new putative prognostic markers in an all-in-one approach in ALL. We hope that these limited results will be confirmed with testing of more samples in the future.

A PCR-RFLP method for genotyping of inversion 2Rc in Anopheles coluzzii.

BACKGROUND: Genotyping of polymorphic chromosomal inversions in malaria vectors such as An. coluzzii Coetzee & Wilkerson is important, both because they cause cryptic population structure that can mislead vector analysis and control and because they influence epidemiologically relevant eco-phenotypes. The conventional cytogenetic method of genotyping is an impediment because it is labor intensive, requires specialized training, and can be applied only to one gender and developmental stage. Here, we circumvent these limitations by developing a simple and rapid molecular method of genotyping inversion 2Rc in An. coluzzii that is both economical and field-friendly. This inversion is strongly implicated in temporal and spatial adaptations to climatic and ecological variation, particularly aridity. METHODS: Using a set of tag single-nucleotide polymorphisms (SNPs) strongly correlated with inversion orientation, we identified those that overlapped restriction enzyme recognition sites and developed four polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) assays that distinguish alternative allelic states at the tag SNPs. We assessed the performance of these assays using mosquito population samples from Burkina Faso that had been cytogenetically karyotyped as well as genotyped, using two complementary high-throughput molecular methods based on tag SNPs. Further validation was performed using mosquito population samples from additional West African (Benin, Mali, Senegal) and Central African (Cameroon) countries. RESULTS: Of four assays tested, two were concordant with the 2Rc cytogenetic karyotype > 90% of the time in all samples. We recommend that these two assays be employed in tandem for reliable genotyping. By accepting only those genotypic assignments where both assays agree, > 99% of assignments are expected to be accurate. CONCLUSIONS: We have developed tandem PCR-RFLP assays for the accurate genotyping of inversion 2Rc in An. coluzzii. Because this approach is simple, inexpensive, and requires only basic molecular biology equipment, it is widely accessible. These provide a crucial tool for probing the molecular basis of eco-phenotypes relevant to malaria epidemiology and vector control.

Genome-wide identification of Chiari malformation type I associated candidate genes and chromosomal variations.

Chiari malformation type I (CMI) is a brain malformation that is characterized by herniation of the cerebellum into the spinal canal. Chiari malformation type I is highly heterogeneous; therefore, an accurate explanation of the pathogenesis of the disease is often not possible. Although some studies showed the role of genetics in CMI, the involvement of genetic variations in CMI pathogenesis has not been thoroughly elucidated. Therefore, in the current study we aim to reveal CMI-associated genomic variations in familial cases.Four CMI patients and 7 unaffected healthy members of two distinct families were analyzed. A microarray analysis of the affected and unaffected individuals from two Turkish families with CMI was conducted. Analyses of single nucleotide variations (SNVs) and copy number variations (CNVs) were performed by calculation of B allele frequency (BAF) and log R ratio (LRR) values from whole genome SNV data. Two missense variations, OLFML2A (rs7874348) and SLC4A9 (rs6860077), and a 5'UTR variation of COL4A1 (rs9521687) were significantly associated with CMI. Moreover, 12 SNVs in the intronic regions of FAM155A, NR3C1, TRPC7, ASTN2, and TRAF1 were determined to be associated with CMI. The CNV analysis showed that the 11p15.4 chromosome region is inherited in one of the families. The use of familial studies to explain the molecular pathogenesis of complex diseases such as CMI is crucial. It has been suggested that variations in OLFML2A, SLC4A9, and COL4A1 play a role in CMI molecular pathogenesis. The CNV analysis of individuals in both families revealed a potential chromosomal region, 11p15.4, and risk regions that are associated with CMI.

Molecular cytogenetic pilot study on pleomorphic adenomas of salivary glands.

Pleomorphic adenomas (PAs) of salivary glands are the most frequent entity of solid parotid tumors. Nonetheless, their genetics is not yet well understood. Thus, the current study characterized 14 PAs using a unique combination of cytogenetic, molecular cytogenetic and/or molecular karyotyping based approaches. The current study applied G-banding based on trypsin treatment and Giemsa-staining in peripheral blood and tumor tissue. Additionally, fluorescence in situ hybridization was performed using whole chromosome painting or centromeric probes. Array-based comparative genomic hybridization was also conducted. In 5 of 14 cases, chromosomal and/or submicroscopic alterations were characterized. Balanced and unbalanced translocations, loss or gain of whole chromosomes and submicroscopic copy number alterations were detected. Furthermore, the first case of a so-called 'jumping translocation' in a PA was reported. The genes twist-related protein 1 and distal-less homeobox 5 were also involved in copy number variations in two PAs. In conclusion, approaches utilized in the current study are highly suited to characterize the genetic constitution of PAs.

Highly specific PCR-RFLP assays for karyotyping the widespread 2Rb inversion in malaria vectors of the Anopheles gambiae complex.

BACKGROUND: Chromosomal inversion polymorphisms play a role in adaptation to heterogeneous environments. Inversion polymorphisms are implicated in the very high ecological flexibility of the three main malaria vector species of the Afrotropical Anopheles gambiae complex, facilitating the exploitation of anthropogenic environmental modifications and promoting a strong association with humans. In addition to extending the species' spatial and temporal distribution, inversions are associated with epidemiologically relevant mosquito behavior and physiology, underscoring their medical importance. We here present novel PCR-RFLP based assays strongly predictive of genotype for the cosmopolitan 2Rb inversion in An. coluzzii and An. gambiae, a development which overcomes the numerous constraints inherent to traditional cytological karyotyping. METHODS: We designed PCR-RFLP genotyping assays based on tag SNPs previously computationally identified as strongly predictive (> 95%) of 2Rb genotype. We targeted those tags whose alternative allelic states destroyed or created the recognition site of a commercially available restriction enzyme, and designed assays with distinctive cleavage profiles for each inversion genotype. The assays were validated on 251 An. coluzzii and 451 An. gambiae cytologically karyotyped specimens from nine countries across Africa and one An. coluzzii laboratory colony. RESULTS: For three tag SNPs, PCR-RFLP assays (denoted DraIII, MspAI, and TatI) reliably produced robust amplicons and clearly distinguishable electrophoretic profiles for all three inversion genotypes. Results obtained with the DraIII assay are ≥ 95% concordant with cytogenetic assignments in both species, while MspAI and TatI assays produce patterns highly concordant with cytogenetic assignments only in An. coluzzii or An. gambiae, respectively. Joint application of species-appropriate pairs of assays increased the concordance levels to > 99% in An. coluzzii and 98% in An. gambiae. Potential sources of discordance (e.g. imperfect association between tag and inversion, allelic dropout, additional polymorphisms in the restriction target site, incomplete or failed restriction digestion) are discussed. CONCLUSIONS: The availability of highly specific, cost effective and accessible molecular assays for genotyping 2Rb in An. gambiae and An. coluzzii allows karyotyping of both sexes and all developmental stages. These novel tools will accelerate deeper investigations into the role of this ecologically and epidemiologically important chromosomal inversion in vector biology.