Molecular characterization of deletion breakpoints in adults with 22q11 deletion syndrome

22q11 Deletion syndrome (22q11DS) is a common microdeletion syndrome with variable expression, including congenital and later onset conditions such as schizophrenia. Most studies indicate that expression does not appear to be related to length of the deletion but there is limited information on the endpoints of even the common deletion breakpoint regions in adults. We used a real-time quantitative PCR (qPCR) approach to fine map 22q11.2 deletions in 44 adults with 22q11DS, 22 with schizophrenia (SZ; 12 M, 10 F; mean age 35.7 SD 8.0 years) and 22 with no history of psychosis (NP; 8 M, 14 F; mean age 27.1 SD 8.6 years). QPCR data were consistent with clinical FISH results using the TUPLE1 or N25 probes. Two subjects (one SZ, one NP) negative for clinical FISH had atypical 22q11.2 deletions confirmed by FISH using the RP11-138C22 probe. Most (n = 34; 18 SZ, 16 NP) subjects shared a common 3 Mb hemizygous 22q11.2 deletion. However, eight subjects showed breakpoint variability: a more telomeric proximal breakpoint (n = 2), or more centromeric (n = 3) or more telomeric distal breakpoint (n = 3). One NP subject had a proximal nested 1.4 Mb deletion. COMT and TBX1 were deleted in all 44 subjects, and PRODH in 40 subjects (19 SZ, 21 NP). The results delineate proximal and distal breakpoint variants in 22q11DS. Neither deletion extent nor PRODH haploinsufficiency appeared to explain the clinical expression of schizophrenia in the present study. Further studies are needed to elucidate the molecular basis of schizophrenia and clinical heterogeneity in 22q11DS.     

Molecular Characterization of a Deletion in the HPRT1 Gene in a Patient with Lesch–Nyhan Syndrome

Lesch–Nyhan syndrome is caused by a deficiency of hypoxanthine phosphoribosyltransferase (HPRT) encoded by HPRT1. About 20% of patients have a deletion of HPRT1 and large deletions of HPRT1 are not always fully characterized at the molecular level. Here, we report on a case of Lesch–Nyhan syndrome with a 33-kb deletion involving exon 1 of HPRT1. This novel mutation is caused by a nonhomologous recombination between different classes of interspersed repetitive DNA.     

Reorganization of inter‐chromosomal interactions in the 2q37‐deletion syndrome

Chromosomes occupy distinct interphase territories in the three‐dimensional nucleus. However, how these chromosome territories are arranged relative to one another is poorly understood. Here, we investigated the inter‐chromosomal interactions between chromosomes 2q, 12, and 17 in human mesenchymal stem cells (MSCs) and MSC‐derived cell types by DNA‐FISH. We compared our findings in normal karyotypes with a three‐generation family harboring a 2q37‐deletion syndrome, featuring a heterozygous partial deletion of histone deacetylase 4 (HDAC4) on chr2q37. In normal karyotypes, we detected stable, recurring arrangements and interactions between the three chromosomal territories with a tissue‐specific interaction bias at certain loci. These inter‐chromosomal interactions were confirmed by Hi‐C. Interestingly, the disease‐related HDAC4 deletion resulted in displaced inter‐chromosomal arrangements and altered interactions between the deletion‐affected chromosome 2 and chromosome 12 and/or 17 in 2q37‐deletion syndrome patients. Our findings provide evidence for a direct link between a structural chromosomal aberration and altered interphase architecture that results in a nuclear configuration, supporting a possible molecular pathogenesis.     

The RIN2 syndrome: a new autosomal recessive connective tissue disorder caused by deficiency of Ras and Rab interactor 2 (RIN2)

Defects leading to impaired intracellular trafficking have recently been shown to play an important role in the pathogenesis of genodermatoses, such as the Ehlers–Danlos and the cutis laxa syndromes. A new genodermatosis, termed macrocephaly, alopecia, cutis laxa and scoliosis (MACS) syndrome has been described, resulting from a homozygous 1-bp deletion in RIN2. RIN2 encodes the Ras and Rab interactor 2, involved in the regulation of Rab5-mediated early endocytosis. We performed a clinical, ultrastructural and molecular study in a consanguineous Algerian family with three siblings affected by a distinctive autosomal recessive genodermatosis, reported in 2005 by Verloes et al. The most striking clinical features include progressive facial coarsening, gingival hypertrophy, severe scoliosis, sparse hair and skin and joint hyperlaxity. Ultrastructural studies of the skin revealed important abnormalities in the collagen fibril morphology, and fibroblasts exhibited a dilated endoplasmic reticulum and an abnormal Golgi apparatus with rarefied and dilated cisternae. Molecular analysis of RIN2 revealed a novel homozygous 2-bp deletion in all affected individuals. The c.1914_1915delGC mutation introduces a frameshift and creates a premature termination codon, leading to nonsense-mediated mRNA decay. These findings confirm that RIN2 defects are associated with a distinct genodermatosis and underscore the involvement of RIN2 and its associated pathways in the pathogenesis of connective tissue disorders. The current family displays considerable phenotypic overlap with MACS syndrome. However, our family shows a dermatological and ultrastructural phenotype belonging to the Ehlers–Danlos rather than the cutis laxa spectrum. Therefore, the MACS acronym is not entirely appropriate for the current family.     

INDEL variation in the regulatory region of the major flowering time gene LanFTc1 is associated with vernalization response and flowering time in narrow‐leafed lupin (Lupinus angustifolius L.)

Narrow‐leafed lupin (Lupinus angustifolius L.) cultivation was transformed by 2 dominant vernalization‐insensitive, early flowering time loci known as Ku and Julius (Jul), which allowed expansion into shorter season environments. However, reliance on these loci has limited genetic and phenotypic diversity for environmental adaptation in cultivated lupin. We recently predicted that a 1,423‐bp deletion in the cis‐regulatory region of LanFTc1, a FLOWERING LOCUS T (FT) homologue, derepressed expression of LanFTc1 and was the underlying cause of the Ku phenotype. Here, we surveyed diverse germplasm for LanFTc1 cis‐regulatory variation and identified 2 further deletions of 1,208 and 5,162 bp in the 5' regulatory region, which overlap the 1,423‐bp deletion. Additionally, we confirmed that no other polymorphisms were perfectly associated with vernalization responsiveness. Phenotyping and gene expression analyses revealed that Jul accessions possessed the 5,162‐bp deletion and that the Jul and Ku deletions were equally capable of removing vernalization requirement and up‐regulating gene expression. The 1,208‐bp deletion was associated with intermediate phenology, vernalization responsiveness, and gene expression and therefore may be useful for expanding agronomic adaptation of lupin. This insertion/deletion series may also help resolve how the vernalization response is mediated at the molecular level in legumes.     

Microdeletion 11q13.1.q13.2 in a patient presenting with developmental delay,facial dysmorphism,and esophageal atresia: Possible role of the GSTP1 gene in esophagus malformation

BACKGROUND: Esophageal atresia is a major congenital malformation characterized by a complete interruption of the esophageal continuity. It is frequently observed in associations and syndromes. As an isolated finding, it has a multifactorial etiology whose genetic factors are poorly known. Recently, the GST family, especially the GSTM1 null genotype (but not the GSTP1 polymorphism I105V), has been associated with esophageal atresia. These enzymes play a role in phase II detoxification of xenobiotics. Here we present the clinical and molecular findings observed in a patient suggesting that the loss of the GSTP1 allele might predispose to this malformation. CASE: We describe a patient presenting with esophageal atresia associated with developmental delay and facial dysmorphism, whose mother used tobacco and alcohol during the first 2 months of her pregnancy. Microdeletion/microduplication analysis was performed using comparative genomic hybridization and a 180K Agilent array. It detected a de novo 2 Mb chromosome 11q13.1.q13.2 deletion. CONCLUSION: The deleted chromosomal segment includes the GSTP1 gene. We hypothesize that the deletion of one GSTP1 allele (an isoform highly expressed in embryonic tissues), associated with specific environmental factors, such as tobacco and alcohol, could cause the esophageal atresia observed in our patient. Birth Defects Research (Part A) 97:463–466, 2013. © 2013 Wiley Periodicals, Inc.     

Deletion of chromosome 21 and normal intelligence: molecular definition of the lesion

Summary Application of a method for the fine structure analysis of unbalanced chromosomal rearrangements using quantitative Southern blot analysis has established that an individual of normal intelligence and largely normal appearance has a significant interstitial deletion of chromosome 21. Using high resolution cytogenetic analysis and molecular analysis with five single copy DNA sequences unique to chromosome 21 and a probe for human SOD1 (CuZn, Superoxide dismutase), we find that the deletion extends from the border of bands 21q11.1–11.2 and extends to the border of bands 21q21.2–q21.3. The latter border is established molecularly by the presence of two copies of SOD1, previously mapped to band 21q22.1, and of four single copy sequences known to be located distal to this region. The presence of SOD1 was confirmed by enzyme dosage analysis. These findings demonstrate that deletion of close to 20,000kb of autosomal material is compatible with normal intelligence. Further, they suggest that chromosome 21 may include a large region of relative developmental neutrality whose molecular basis may now be investigated. Because of the limits of even high resolution cytogenetic analysis, fine structure molecular analyses of this type will be necessary to reliably detect and define similar small chromosomal deletions or insertions. The molecular definition of such aneuploidy provides the basis for increasing the resolution of the human physical genetic map.     

Evaluation of muscle strength and motor abilities in children with type II and III spinal muscle atrophy treated with valproic acid

Background  

Spinal muscular atrophy (SMA) is an autosomal recessive disorder that affects the motoneurons of the spinal anterior horn, resulting in hypotonia and muscle weakness. The disease is caused by deletion or mutation in the telomeric copy of SMN gene (SMN1) and clinical severity is in part determined by the copy number of the centromeric copy of the SMN gene (SMN2). The SMN2 mRNA lacks exon 7, resulting in a production of lower amounts of the full-length SMN protein. Knowledge of the molecular mechanism of diseases has led to the discovery of drugs capable of increasing SMN protein level through activation of SMN2 gene. One of these drugs is the valproic acid (VPA), a histone deacetylase inhibitor.     

Evidence for a new contiguous gene syndrome, the chromosome 16p13.3 deletion syndrome alias severe Rubinstein–Taybi syndrome

Rubinstein–Taybi syndrome (RSTS) is a well-known autosomal dominant mental retardation syndrome with typical facial and skeletal abnormalities. Previously, we have reported two patients presenting with RSTS and additional clinical features including failure to thrive, seizures, and intractable infections (Bartsch et al. in Eur J Hum Genet 7:748–756, 1999). Recently we identified a third patient with this condition, termed here severe RSTS, or chromosome 16p13.3 deletion syndrome. The three patients died in infancy, and all displayed a specific mutation, a chromosomal microdeletion including the 3′-end of the CREBBP gene. Using fluorescence in situ hybridization and closely spaced DNA probes, we characterized the deletion intervals in these patients and in three individuals with a deletion of CREBBP and typical RSTS. The deleted DNA segments were found to greatly vary in size, spanning from ∼40 kb to >3 Mb. Four individuals, including the patients with severe RSTS, exhibited deletions containing gene/s in addition to CREBBP. The patients with severe RSTS all had deletions comprising telomeric neighbor genes of CREBBP, including DNASE1, a dominant gene encoding a nuclease that has been associated with systemic lupus erythematodes. Our findings suggest that severe RSTS is distinct from RSTS and represents a novel true contiguous gene syndrome (chromosome 16p13.3 deletion syndrome). Because of the risk of critical infections and high mortality rate, we recommend that the size of the deletion interval should be determined in CREBBP deletion-positive patients with RSTS, especially in young children. Further studies are needed to delineate the clinical spectrum of the new disorder and to clarify the role of DNASE1.     

Location of antibiotic resistance determinants, copy control, and replication functions on the double-selective streptococcal cloning vector pGB301

Summary The physical map of the streptococcal cloning vector pGB301 (9.8 kb) has been extended by localizing the cleavage sites of restriction endonucleases AvaII, AvaI, BclI, BstEII, and PvuII. The latter four enzymes cleaved pGB301 at unique sites. Insertion of chromosomal DNA from Staphylococcus aureus strain 3Ar^–m^– into the single BstEII site of pGB301 led to inactivation of the plasmid's chloramphenicol resistance determinant. Twelve deletion derivatives of pGB301 were isolated either by in vitro manipulation of pGB301 or as spontaneous deletion mutants following transformation of Challis by mixtures of recombinant plasmids. The overlapping deletions which spanned a continuous sequence of 7.7 kb ranged in size from 0.3 kb to 4.1 kb and allowed to localize the chloramphenicol and MLS-resistance determinants, the copy control function, and the replication region on the physical map of pGB301. Plasmid pGB301 together with its deletion mutants constitutes a valuable tool for further molecular cloning experiments in streptococci.     

Subunit dissociation and stability alteration of <Emphasis Type="SmallCaps">d</Emphasis>-hydantoinase deleted at the terminal amino acid residue

Two variants of d-hydantoinase (HYD), created by deletion of one amino acid residue of at either the N- or C-terminus, were expressed in Escherichia coli and purified by two-step chromatography. Compared with HYD, HYDc1 with the C-terminal Arg deletion retained 43% activity, while HYDn1 with the N-terminal Ser deletion had no activity using dl-Hydantoin as substrate. Based on HYD dimer with a molecular weight of 103 kDa, HYDc1 is a monomer of 52 kDa and HYDn1 is a mixture of dimer and monomer. Moreover, HYDc1 displayed higher pH stability and lower thermal stability compared to HYD. In addition, the secondary and tertiary structures of HYDc1 were not significantly changed in contrast to the ones of HYDn1. All data imply that the C-terminal Arg of the HYD is crucial for homodimeric architecture of the enzyme, but non-essential for catalysis, while the N-terminal Ser is required for both conformation and catalysis of the enzyme.     

15q11.2 microdeletion and FMR1 premutation in a family with intellectual disabilities and autism

Genomic rearrangements of chromosome 15q11–q13 are responsible for diverse phenotypes including intellectual disabilities and autism. 15q11.2 deletion, implicating common PWS/AS breakpoints BP1–BP2, has been described in patients with delayed motor and speech development and behavioural problems. Here we report the clinical and molecular characterisation of a maternally inherited BP1–BP2 deletion in two siblings with intellectual, motor and speech delay, autistic syndrome disorder and several dysmorphic features. One of the patients was also a carrier of an FMR1 allele in the low premutation range. The four genes within the deletion were under-expressed in all deletion carriers but FMR1 mRNA levels remained normal. Our results suggest that BP1-BP2 deletion could be considered as a risk factor for neuropsychological phenotypes and that it presents with variable clinical expressivity.     

Extensive length variation in the cpDNA trnT-trnF region of hemiparasitic Pedicularis and its phylogenetic implications

The cpDNA trnT-trnF region, a molecular marker widely used in the phylogenetic reconstruction at lower taxonomic levels, is relatively conserved in size and structure. In this region single length variation over 100 bp is much less common than small deletion for congeneric species of angiosperms. Here we examined evolutionary patterns of the trnT-trnF region in 43 species of Pedicularis, a species-rich genus with adaptive radiation. Four independent large deletions, varying from 203 to 297 bp in length, were detected from nine species of the genus, which might result from slipped-strand mispairing. These deletions occurred in different locations of the cpDNA region and in different clades of the phylogenetic tree, indicating that the deletion of large cpDNA fragments may be very frequent in the hemiparasitic lineage of the family Orobanchaceae. Parsimony analyses showed that section Cyathophora of Pedicularis, endemic to the Sino-Himalayan region, was a strongly supported monophyletic group. This section could have a recent origin followed by rapid radiation, considering that it is characterized by a large deletion in the trnT-trnF region and a relatively low interspecific sequence divergence.     

Exclusion of the C/D box snoRNA gene cluster HBII-52 from a major role in Prader–Willi syndrome

Prader–Willi syndrome (PWS) and Angelman syndrome (AS) are distinct neurogenetic disorders caused by the loss of function of imprinted genes in 15q11–q13. The maternally expressed UBE3A gene is affected in AS. Four protein-encoding genes (MKRN3, MAGEL2, NDN and SNURF-SNRPN) and several small nucleolar (sno) RNA genes (HBII-13, HBII-436, HBII-85, HBII-438A, HBII-438B and HBII-52) are expressed from the paternal chromosome only but their contribution to PWS is unclear. To examine the role of the HBII-52 snoRNA genes, we have reinvestigated an AS family with a submicroscopic deletion spanning UBE3A and flanking sequences. By fine mapping of the centromeric deletion breakpoint in this family, we have found that the deletion affects all of the 47 HBII-52 genes. Since the complete loss of the HBII-52 genes in family members who carry the deletion on their paternal chromosome is not associated with an obvious clinical phenotype, we conclude that HBII-52 snoRNA genes do not play a major role in PWS. However, we cannot exclude the possibility that the loss of HBII-52 has a phenotypic effect when accompanied by the loss of function of other genes in 15q11–q13.Electronic Database Information: accession numbers and URLs for data presented herein are as follows: for PAR-4 (accession number AF019617), deletion junction fragment (L15422): GenBank, ; for Angelman syndrome [MIM105830]: Online Mendelian Inheritance in Man (OMIM),     

The lytA Gene and the DNA Region Located Downstream of This Gene Are Not Involved in the Formation of the Type 3 Capsular Polysaccharide of Streptococcus pneumoniae

Streptococcus pneumoniae strain M31, an unencapsulated, serotype 2 (S2⁻) mutant having a deletion of at least 5.5 kb containing the gene lytA that encodes the main pneumococcal autolysin, was transformed to the encapsulated serotype 3 (S3⁺) with DNA from the clinical pneumococcal strain 406. Hybridization analysis revealed that the S3⁺ transformants also have the deletion demonstrating that lytA and the DNA region located downstream of this gene are not involved in the encapsulation of S. pneumoniae. Received: 29 February 1996 / Accepted: 1 March 1996     

A single-base deletion in soybean flavonol synthase gene is associated with magenta flower color

The Wm locus of soybean [Glycine max (L.) Merr.] controls flower color. Dominant Wm and recessive wm allele of the locus produce purple and magenta flower, respectively. A putative full-length cDNA of flavonol synthase (FLS), gmfls1 was isolated by 5′ RACE and end-to-end PCR from a cultivar Harosoy with purple flower (WmWm). Sequence analysis revealed that gmfls1 consisted of 1,208 nucleotides encoding 334 amino acids. It had 59–72% homology with FLS proteins of other plant species. Conserved dioxygenase domains A and B were found in the deduced polypeptide. Sequence comparison between Harosoy and Harosoy-wm (magenta flower mutant of Harosoy; wmwm) revealed that they differed by a single G deletion in the coding region of Harosoy-wm. The deletion changed the subsequent reading frame resulting in a truncated polypeptide consisting of 37 amino acids that lacked the dioxygenase domains A and B. Extracts of E. coli cells expressing gmfls1 of Harosoy catalyzed the formation of quercetin from dihydroquercetin, whereas cell extracts expressing gmfls1 of Harosoy-wm had no FLS activity. Genomic Southern analysis suggested the existence of three to four copies of the FLS gene in the soybean genome. CAPS analysis was performed to detect the single-base deletion. Harosoy and Clark (WmWm) exhibited longer fragments, while Harosoy-wm had shorter fragments due to the single-base deletion. The CAPS marker co-segregated with genotypes at Wm locus in a F₂ population segregating for the locus. Linkage mapping using SSR markers revealed that the Wm and gmfls1 were mapped at similar position in the molecular linkage group F. The above results strongly suggest that gmfls1 represents the Wm gene and that the single-base deletion may be responsible for magenta flower color.

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Development of a simple molecular marker specific for detecting the self-compatible<Emphasis Type="Italic">S4′</Emphasis> haplotype in sweet cherry (<Emphasis Type="Italic">Prunus avium</Emphasis> L.)

In sweet cherry (Prunus avium L.), theS4′ haplotype, characterized by a self-incompatibility (SI) defect in pollen, is self-compatible and is derived from the self-incompatibleS4 haplotype by x-ray mutagenesis.SFBs (S haplotype-specific F-box protein genes) have been found to associate with pollen determinant of SI. This report identified theSFB4′ of the self-compatibleS4′ haplotype. The alignment of the sequences ofSFB4′ andSFB4 by the BLAST program revealed a 4-bp deletion inSFB4′, which is TTTA. The sequence polymorphism generated by the TTTA deletion inSFB4′ was exploited to develop a simple molecular marker specific for detecting theS4′ but not theS4 haplotype. The simple marker specific to theS4′ haplotype can be visualized directly on an agarose gel, so it can be immediately applied to a marker-assistant cherry-breeding program. Thus, this work provides a practical molecular marker for cherry breeding. Principal author. An erratum to this article is available at .     

Distribution of puroindoline alleles in bread wheat cultivars of the Yellow and Huai valley of China and discovery of a novel puroindoline a allele without PINA protein

Kernel hardness is one of the most important factors determining the milling and processing quality of bread wheat (Triticum aestivum L.). In the present study, 267 wheat cultivars and advanced lines from the Yellow and Huai Valley of China, CIMMYT, Russia and Ukraine were used for identification of SKCS (Single Kernel Characterization System) hardness and puroindoline alleles. Results indicated that Pinb-D1b is the most popular genotype in wheat cultivars from the Yellow and Huai Valley, Russia and Ukraine, whereas PINA null is a predominant genotype in wheat cultivars and advanced lines from CIMMYT. Molecular characterization of PINA-null alleles indicated that one Chinese landrace Chiyacao had the allele Pina-D1l with a single nucleotide C deletion at position 265 in Pina coding region based on sequencing results, and 35 of 39 PINA-null alleles belonged to Pina-D1b according to PCR amplification with the sequence-tagged site (STS) marker Pina-N developed previously. The remaining three cultivars (Jiangdongmen, Heshangtou and Hongquanmang from China) with PINA-null alleles were characterized at the DNA level by a primer walking strategy, and the results showed that all three cultivars with PINA-null alleles possessed a uniform 10,415-bp deletion from −5,117 bp to +5,298 bp (ATG codon references zero), designated as Pina-D1r. Correspondingly, an STS marker Pina-N2 with an expected fragment size of 436-bp spanning the 10,415-bp deletion was developed for detection of the Pina-D1r allele. This study provided a useful molecular marker for straightforward detection of one of the PINA-null alleles and would also be helpful to further understand the molecular and genetic basis of kernel hardness in bread wheat.