Prenatal diagnosis and molecular cytogenetic characterization of a de novo interstitial deletion of 7q (7q22.1 → q31.1)

We present prenatal diagnosis and molecular cytogenetic characterization of de novo interstitial deletion of 7q (7q22.1 → q31.1) by aCGH, FISH and QF-PCR in a fetus with an abnormal maternal serum screening result and ultrasound findings of facial cleft and hypogenitalism. We discuss the genotype–phenotype correlation and the consequence of haploinsufficiency of ZKSCAN5, ARPC1A, CYP3A43, RELN, LAMB1, IMMP2L and DOCK4 in this case.     

Prenatal diagnosis and molecular cytogenetic characterization of de novo partial trisomy 12q (12q24.21 → qter) and partial monosomy 6q (6q27 → qter) associated with coarctation of the aorta,ventriculomegaly and thickened nuchal fold

We present rapid aneuploidy diagnosis of de novo partial trisomy 12q (12q24.21 → qter) and partial monosomy 6q (6q27 → qter) by aCGH using uncultured amniocytes in a fetus with coarctation of the aorta, ventriculomegaly and thickened nuchal fold. We discuss the association of TBX3, TBX5 and MED13L gene duplication with coarctation of the aorta, and the association of RNASET2 gene haploinsufficiency with ventriculomegaly in this case.     

Interstitial 2q24.3 deletion including SCN2A and SCN3A genes in a patient with autistic features,psychomotor delay,microcephaly and no history of seizures

Mutations in neuronal voltage-gated sodium channel genes SCN1A, SCN2A, and SCN3A may play an important role in the etiology of neurological diseases and psychiatric disorders, besides various types of epilepsy. Here we describe a 3-year-old boy with autistic features, language delay, microcephaly and no history of seizures. Array-CGH analysis revealed an interstitial deletion of ~ 291.9 kB at band 2q24.3 disrupting the entire SCN2A gene and part of SCN3A. We discuss the effects of haploinsufficiency of SCN2A and SCN3A on the genetic basis of neurodevelopmental and neurobehavioral disorders and we propose that this haploinsufficiency may be associated not only with epilepsy, but also with autistic features.     

Testosterone treatment increases androgen receptor and aromatase gene expression in myotubes from patients with PCOS and controls,but does not induce insulin resistance

Polycystic ovary syndrome (PCOS) is associated with insulin resistance and increased risk of type 2 diabetes. Skeletal muscle is the major site of insulin mediated glucose disposal and the skeletal muscle tissue is capable to synthesize, convert and degrade androgens. Insulin sensitivity is conserved in cultured myotubes (in vitro) from patients with PCOS, but the effect of testosterone on this insulin sensitivity is unknown. We investigated the effect of 7 days testosterone treatment (100 nmol/l) on glucose transport and gene expression levels of hormone receptors and enzymes involved in the synthesis and conversion of testosterone (HSD17B1, HSD17B2, CYP19A1, SRD5A1-2, AR, ER-α, HSD17B6 and AKR1-3) in myotubes from ten patients with PCOS and ten matched controls.     

Prenatal diagnosis of ring chromosome 2 with lissencephaly and 2p25.3 and 2q37.3 microdeletions detected using array comparative genomic hybridization

We present rapid aneuploidy diagnosis of ring chromosome 2 with 2p25.3 and 2q37.3 microdeletions by aCGH using uncultured amniocytes in a fetus with IUGR, microcephaly, lissencephaly and ambiguous external genitalia. Our case adds lissencephaly to the list of CNS abnormalities in ring chromosome 2 with 2p25.3 and 2q37.3 microdeletions. We discuss the consequence of haploinsufficiency of HDAC4, KIF1A, PASK, HDLBP, FRAP2 and D2HGDH on 2q37.3, and haploinsufficiency of MYT1L, SNTG2 and TPO on 2p25.3 in this case.     

6p21.2–p12.3 deletion detected by aCGH in an 8-year-old girl with cleidocranial dysplasia and developmental delay

We present an 8-year-old girl with cleidocranial dysplasia, psychomotor developmental delay, poor wound healing and a 6p21.2–p12.3 deletion detected by aCGH. The patient was previously found to have a normal karyotype on conventional cytogenetic analysis and no RUNX2 mutation on sequence analysis. We discuss the genotype–phenotype correlation and the consequence of haploinsufficiency of CUL7, VEGFA, NFKBIE and RUNX2 in this case.     

Chromosome 18p deletion syndrome presenting holoprosencephaly and premaxillary agenesis: Prenatal diagnosis and aCGH characterization using uncultured amniocytes

We present prenatal diagnosis of a de novo distal 18p deletion involving 14.06 Mb at 18p11.32–p11.21 by aCGH using uncultured amniocytes in a pregnancy with fetal holoprosencephaly and premaxillary agenesis. QF-PCR analysis showed that distal 18p deletion was from maternal origin. Metaphase FISH analysis confirmed haploinsufficiency of TGIF. We discuss the functions of the genes that are deleted within this region. The present case shows the usefulness of applying aCGH on uncultured amniocytes for rapid aneuploidy diagnosis in cases with prenatally detected fetal structural abnormalities.     

Prenatal diagnosis of de novo interstitial deletions involving 5q23.1–q23.3 and 18q12.1–q12.3 by array CGH using uncultured amniocytes in a pregnancy with fetal interrupted aortic arch and atrial septal defect

We present prenatal diagnosis of de novo interstitial deletions involving 5q23.1–q23.3 and 18q12.1–q12.3 by aCGH using uncultured amniocytes in pregnancy with interrupted aortic arch and atrial septal defect in a fetus. The fetus postnatally manifested facial dysmorphisms and long slender fingers. We discuss the genotype–phenotype correlation and the consequence of haploinsufficiency of FBN2, DTNA and CELF4 in this case.     

Partial monosomy 21 (q11.2→q21.3) combined with 3p25.3→pter monosomy due to an unbalanced translocation in a patient presenting dysmorphic features and developmental delay

We describe a female patient of 1 year and 5 months-old, referred for genetic evaluation due to neuropsychomotor delay, hearing impairment and dysmorphic features. The patient presents a partial chromosome 21 monosomy (q11.2→q21.3) in combination with a chromosome 3p terminal monosomy (p25.3→pter) due to an unbalanced de novo translocation. The translocation was confirmed by fluorescence in situ hybridization (FISH) and the breakpoints were mapped with high resolution array. After the combined analyses with these techniques the final karyotype was defined as 45,XX,der(3)t(3;21)(p25.3;q21.3)dn,-21.ish der(3)t(3;21)(RP11-329A2-,RP11-439F4-,RP11-95E11-,CTB-63H24 +).arr 3p26.3p25.3(35,333-10,888,738)) × 1,21q11.2q21.3(13,354,643-27,357,765) × 1. Analysis of microsatellite DNA markers pointed to a paternal origin for the chromosome rearrangement. This is the first case described with a partial proximal monosomy 21 combined with a 3p terminal monosomy due to a de novo unbalanced translocation.     

A 1.5 Mb terminal deletion of 12p associated with autism spectrum disorder

We report a patient with a terminal 12p deletion associated with autism spectrum disorder (ASD). This 12p13.33 deletion is 1.5 Mb in size and encompasses 13 genes (B4GALNT3, CCDC77, ERC1, FBXL14, IQSEC3, KDM5A, LINC00942, LOC574538, NINJ2, RAD52, SLC6A12, SLC6A13 and WNK1). All previous cases reported with partial monosomy of 12p13.33 are associated with neurodevelopmental delay, and we suggest that ERC1, which encodes a regulator of neurotransmitter release, is the best gene candidate contributing to this phenotype as well as to the ASD of our patient.     

Sample-to-SNP kit: A reliable,easy and fast tool for the detection of HFE p.H63D and p.C282Y variations associated to hereditary hemochromatosis

Classical hereditary hemochromatosis involves the HFE-gene and diagnostic analysis of the DNA variants HFE p.C282Y (c.845G > A; rs1800562) and HFE p.H63D (c.187C > G; rs1799945). The affected protein alters the iron homeostasis resulting in iron overload in various tissues. The aim of this study was to validate the TaqMan-based Sample-to-SNP protocol for the analysis of the HFE-p.C282Y and p.H63D variants with regard to accuracy, usefulness and reproducibility compared to an existing SNP protocol. The Sample-to-SNP protocol uses an approach where the DNA template is made accessible from a cell lysate followed by TaqMan analysis. Besides the HFE-SNPs other eight SNPs were used as well. These SNPs were: Coagulation factor II-gene F2 c.20210G > A, Coagulation factor V-gene F5 p.R506Q (c.1517G > A; rs121917732), Mitochondria SNP: mt7028 G > A, Mitochondria SNP: mt12308 A > G, Proprotein convertase subtilisin/kexin type 9-gene PCSK9 p.R46L (c.137G > T), Plutathione S-transferase pi 1-gene GSTP1 p.I105V (c313A > G; rs1695), LXR g.-171 A > G, ZNF202 g.-118 G > T. In conclusion the Sample-to-SNP kit proved to be an accurate, reliable, robust, easy to use and rapid TaqMan-based SNP detection protocol, which could be quickly implemented in a routine diagnostic or research facility.     

Impact of cytokine and cytokine receptor gene polymorphisms on cellular immunity after smallpox vaccination

We explored associations between SNPs in cytokine/cytokine receptor genes and cellular immunity in subjects following primary smallpox vaccination. We also analyzed the genotype–phenotype associations discovered in the Caucasian subjects among a cohort of African-Americans. In Caucasians we found 277 associations (p < 0.05) between gene SNPs and inter-individual variations in IFN-α, IL-12p40, IL-1β, IL-2, and TNF-α secretion levels. A collection of SNPs in the IL1RN, IL2RB, IL4R, IL6, IL10RB, IL12A, and IL12RB2 genes had consistent associations among both Caucasians and African-Americans. A regulatory SNP (rs452204) in the IL1RN gene was significantly associated with higher levels of IL-2 secretion in an allele dose-dependent manner in both race groups (p = 0.05 for Caucasians and p = 0.002 for African-Americans). IL12RB2 polymorphism rs3790567 was associated with a dose-related decrease in IL-1β secretion (p = 0.009 for Caucasians and p = 0.01 for African-Americans). Our results demonstrate that variations in smallpox vaccine-induced cytokine responses are modulated by genetic polymorphisms in cytokine and cytokine receptor genes.     

Prenatal diagnosis and molecular cytogenetic characterization of de novo pure partial trisomy 6p associated with microcephaly,craniosynostosis and abnormal maternal serum biochemistry

We present prenatal diagnosis and molecular cytogenetic characterization of de novo pure trisomy 6p22.3 → p25.3 encompassing BMP6 in a fetus associated with microcephaly and craniosynostosis on prenatal ultrasound, abnormal maternal serum biochemistry of a low PAPP-A level in the first-trimester combined test, and a karyotype of 46,XX,der(22)t(6;22)(p22.3;p13)dn. The present case demonstrates the usefulness of rapid prenatal identification of the origin of the extra chromosome material on the short arm of an acrocentric chromosome by spectral karyotyping, fluorescence in situ hybridization and array comparative genomic hybridization. We review the phenotypic abnormality of craniosynostosis in previously reported patients with partial trisomy 6p. We discuss the genotype–phenotype correlation of the involved gene of BMP6 in this case.     

Novel nucleotide insertions in two unrelated Indian patients with 5α reductase 2 deficiency leading to premature termination of SRD5A2 enzyme

T is converted to a more potent androgen, DHT by the action of microsomal membrane enzyme 5α reductase 2. Defects in 5α reductase 2 isozyme results in incomplete virilisation of external male genitalia. Mutations in SRD5A2 gene leads to diminished enzyme activity, thus hampering DHT synthesis from T. We describe two unrelated patients from India with 5αRD2 due to novel insertion of nucleotides in the exon 1 of SRD5A2 gene that lead to premature termination of protein. Master S (case 1; III.8) was 3 years old at initial evaluation, had perineoscrotal hypospadias, microphallus and both testes were palpable in the inguinal region. Master P (case 2; III.9) was born as normal full term baby. He had primary complaint of microphallus, penoscrotal hypospadias and gonads in the inguinal region. Diagnosis of 5αRD2 was made, as T/DHT ratio in the two cases was 41 and 131.2 respectively. Sequence analysis of SRD5A2 gene showed an insertion of nucleotides TA in exon 1 (c.188_189). This resulted in premature termination of the protein due to stop codon at amino acid position 7. The protein formed is drastically truncated and inadequate protein synthesized explains the phenotypic characteristics of our patients.     

Distribution of repetitive DNA sequences in chromosomes of five opisthorchid species (Trematoda, Opisthorchiidae)

Genomes of opisthorchid species are characterized by small size, suggesting a reduced amount of repetitive DNA in their genomes. Distribution of repetitive DNA sequences in the chromosomes of five species of the family Opisthorchiidae (Opisthorchis felineus 2n = 14 (Rivolta, 1884), Opisthorchis viverrini 2n = 12 (Poirier, 1886), Metorchis xanthosomus 2n = 14 (Creplin, 1846), Metorchis bilis 2n = 14 (Braun, 1890), Clonorchis sinensis 2n = 14 (Cobbold, 1875)) was studied with C- and AgNOR-banding, generation of microdissected DNA probes from individual chromosomes and fluorescent in situ hybridization on mitotic and meiotic chromosomes. Small-sized C-bands were discovered in pericentric regions of chromosomes. Ag-NOR staining of opisthorchid chromosomes and FISH with ribosomal DNA probe showed that karyotypes of all studied species were characterized by the only nucleolus organizer region in one of small chromosomes. The generation of DNA probes from chromosomes 1 and 2 of O. felineus and M. xanthosomus was performed with chromosome microdissection followed by DOP-PCR. FISH of obtained microdissected DNA probes on chromosomes of these species revealed chromosome specific DNA repeats in pericentric C-bands. It was also shown that microdissected DNA probes generated from chromosomes could be used as the Whole Chromosome Painting Probes without suppression of repetitive DNA hybridization. Chromosome painting using microdissected chromosome specific DNA probes showed the overall repeat distribution in opisthorchid chromosomes.     

Structure and characterization of amidase from Rhodococcus sp. N-771: Insight into the molecular mechanism of substrate recognition

In this study, we have structurally characterized the amidase of a nitrile-degrading bacterium, Rhodococcus sp. N-771 (RhAmidase). RhAmidase belongs to amidase signature (AS) family, a group of amidase families, and is responsible for the degradation of amides produced from nitriles by nitrile hydratase. Recombinant RhAmidase exists as a dimer of about 107 kDa. RhAmidase can hydrolyze acetamide, propionamide, acrylamide and benzamide with kcat/Km values of 1.14 ± 0.23 mM^− 1s^− 1, 4.54 ± 0.09 mM^− 1s^− 1, 0.087 ± 0.02 mM^− 1s^− 1 and 153.5 ± 7.1 mM^− 1s^− 1, respectively. The crystal structures of RhAmidase and its inactive mutant complex with benzamide (S195A/benzamide) were determined at resolutions of 2.17 Å and 2.32 Å, respectively. RhAmidase has three domains: an N-terminal α-helical domain, a small domain and a large domain. The N-terminal α-helical domain is not found in other AS family enzymes. This domain is involved in the formation of the dimer structure and, together with the small domain, forms a narrow substrate-binding tunnel. The large domain showed high structural similarities to those of other AS family enzymes. The Ser-cis Ser-Lys catalytic triad is located in the large domain. But the substrate-binding pocket of RhAmidase is relatively narrow, due to the presence of the helix α13 in the small domain. The hydrophobic residues from the small domain are involved in recognizing the substrate. The small domain likely participates in substrate recognition and is related to the difference of substrate specificities among the AS family amidases.     

Comparative cytogenetics of opisthorchid species (Trematoda, Opisthorchiidae)

In the present study karyotypes and chromosomes of five species of the family Opisthorchiidae (Opisthorchis felineus (Rivolta, 1884), O. viverrini (Poirier, 1886), Metorchis xanthosomus (Creplin, 1846), M. bilis (Braun, 1893), and Clonorchis sinensis (Cobbold, 1875)) were compared. Karyotypes of O. felineus, M. xanthosomus, M. bilis and C. sinensis consist of two pairs of large meta- and submetacentrics and five pairs of small chromosomes (2n = 14). The karyotype of O. viverrini is 2n = 12, which indicates a fusion of two chromosomes of opisthorchid ancestral karyotype. Analysis of mitotic and meiotic chromosomes was performed by heterologous in situ hybridization of microdissected DNA probes obtained from chromosomes 1 and 2 of O. felineus and chromosomes 1 and 2 of M. xanthosomus. Results of chromosome staining (C- and AgNOR-banding) and FISH of telomeric probes and ribosomal DNA probe on opisthorchid chromosomes were used for chromosome comparison. Data on chromosome number in opisthorchid species were also discussed.