Cryptic Genomic Rearrangements in Three Patients with 46,XY Disorders of Sex Development

Background

46,XY disorders of sex development (46,XY DSD) are genetically heterogeneous conditions. Recently, a few submicroscopic genomic rearrangements have been reported as novel genetic causes of 46,XY DSD.

Methodology/Principal Findings

To clarify the role of cryptic rearrangements in the development of 46,XY DSD, we performed array-based comparative genomic hybridization analysis for 24 genetic males with genital abnormalities. Heterozygous submicroscopic deletions were identified in three cases (cases 1–3). A ∼8.5 Mb terminal deletion at 9p24.1–24.3 was detected in case 1 that presented with complete female-type external genitalia and mental retardation; a ∼2.0 Mb interstitial deletion at 20p13 was identified in case 2 with ambiguous external genitalia and short stature; and a ∼18.0 Mb interstitial deletion at 2q31.1–32 was found in case 3 with ambiguous external genitalia, mental retardation and multiple anomalies. The genital abnormalities of case 1 could be ascribed to gonadal dysgenesis caused by haploinsufficiency of DMRT1, while those of case 3 were possibly associated with perturbed organogenesis due to a deletion of the HOXD cluster. The deletion in case 2 affected 36 genes, none of which have been previously implicated in sex development.

Conclusions/Significance

The results indicate that cryptic genomic rearrangements constitute an important part of the molecular bases of 46,XY DSD and that submicroscopic deletions can lead to various types of 46,XY DSD that occur as components of contiguous gene deletion syndromes. Most importantly, our data provide a novel candidate locus for 46,XY DSD at 20p13.     

Deletions in Xq26.3–q27.3 including FMR1 result in a severe phenotype in a male and variable phenotypes in females depending upon the X inactivation pattern

High resolution cytogenetics, microsatellite marker analyses, and fluorescence in situ hybridization were used to define Xq deletions encompassing the fragile X gene, FMR1, detected in individuals from two unrelated families. In Family 1, a 19-year-old male had facial features consistent with fragile X syndrome; however, his profound mental and growth retardation, small testes, and lover limb skeletal defects and contractures demonstrated a more severe phenotype, suggestive of a contiguous gene syndrome. A cytogenetic deletion including Xq26.3–q27.3 was observed in the proband, his phenotypically normal mother, and his learning-disabled non-dysmorphic sister. Methylation analyses at the FMR1 and androgen receptor loci indicated that the deleted X was inactive in > 95% of his mother’s white blood cells and 80–85% of the sister’s leukocytes. The proximal breakpoint for the deletion was approximately 10 Mb centromeric to FMR1, and the distal breakpoint mapped 1 Mb distal to FMR1. This deletion, encompassing ∼13 Mb of DNA, is the largest deletion including FMR1 reported to date. In the second family, a slightly smaller deletion was detected. A female with moderate to severe mental retardation, seizures, and hypothyroidism, had a de novo cytogenetic deletion extending from Xq26.3 to q27.3, which removed ∼12 Mb of DNA around the FMR1 gene. Cytogenetic and molecular data revealed that ∼50% of her white blood cells contained an active deleted X. These findings indicate that males with deletions including Xq26.3–q27.3 may exhibit a more severe phenotype than typical fragile X males, and females with similar deletions may have an abnormal phenotype if the deleted X remains active in a significant proportion of the cells. Thus, important genes for intellectual and neurological development, in addition to FMR1, may reside in Xq26.3–q27.3. One candidate gene in this region, SOX3, is thought to be involved in neuronal development and its loss may partly explain the more severe phenotypes of our patients. Received: 19 December 1996 / Accepted: 13 March 1997     

Delineation of the critical deletion region for congenital heart defects, on chromosome 8p23.1

Deletions in the distal region of chromosome 8p (del8p) are associated with congenital heart malformations. Other major manifestations include microcephaly, intrauterine growth retardation, mental retardation, and a characteristic hyperactive, impulsive behavior. We studied genotype-phenotype correlations in nine unrelated patients with a de novo del8p, by using the combination of classic cytogenetics, FISH, and the analysis of polymorphic DNA markers. With the exception of one large terminal deletion, all deletions were interstitial. In five patients, a commonly deleted region of approximately 6 Mb was present, with breakpoints clustering in the same regions. One patient without a heart defect or microcephaly but with mild mental retardation and characteristic behavior had a smaller deletion within this commonly deleted region. Two patients without a heart defect had a more proximal interstitial deletion that did not overlap with the commonly deleted region. Taken together, these data allowed us to define the critical deletion regions for the major features of a del8p.     

The generation of chromosomal deletions to provide extensive coverage and subdivision of the Drosophila melanogaster genome

Background

Chromosomal deletions are used extensively in Drosophila melanogaster genetics research. Deletion mapping is the primary method used for fine-scale gene localization. Effective and efficient deletion mapping requires both extensive genomic coverage and a high density of molecularly defined breakpoints across the genome.

Results

A large-scale resource development project at the Bloomington Drosophila Stock Center has improved the choice of deletions beyond that provided by previous projects. FLP-mediated recombination between FRT-bearing transposon insertions was used to generate deletions, because it is efficient and provides single-nucleotide resolution in planning deletion screens. The 793 deletions generated pushed coverage of the euchromatic genome to 98.4%. Gaps in coverage contain haplolethal and haplosterile genes, but the sizes of these gaps were minimized by flanking these genes as closely as possible with deletions. In improving coverage, a complete inventory of haplolethal and haplosterile genes was generated and extensive information on other haploinsufficient genes was compiled. To aid mapping experiments, a subset of deletions was organized into a Deficiency Kit to provide maximal coverage efficiently. To improve the resolution of deletion mapping, screens were planned to distribute deletion breakpoints evenly across the genome. The median chromosomal interval between breakpoints now contains only nine genes and 377 intervals contain only single genes.

Conclusions

Drosophila melanogaster now has the most extensive genomic deletion coverage and breakpoint subdivision as well as the most comprehensive inventory of haploinsufficient genes of any multicellular organism. The improved selection of chromosomal deletion strains will be useful to nearly all Drosophila researchers.     

A 1.1 Mb deletion in distal 13q deletion syndrome region with congenital heart defect and postaxial polydactyly: Additional support for a CHD locus at distal 13q34 region

13q deletion syndrome is a rare genetic disorder, especially for group 3 deletion (13q33–q34 deletion). Previously we described a patient with congenital heart defect and mental retardation and proposed that a distal 6 Mb region might contain the causative gene of congenital heart defect. Here we present a new patient with congenital heart defects (CHD), hand and foot anomalies and mild mental retardation. We identified a 1.1 Mb deletion at chromosome 13q34 with high resolution SNP-array BeadChips (HumanOmni1-Quad, Illumina, USA). This chromosome region contains ten annotated genes, including GRK1, TFDP1, RASA3 and GAS6. To our knowledge, this represents the smallest 13q34 deletion identified to date. Our study provides additional support that distal 13q34 deletion region might contain key gene(s) responsible for cardiac development.     

Mouse and Human CRKL Is Dosage Sensitive for Cardiac Outflow Tract Formation

The human chromosome 22q11.2 region is susceptible to rearrangements during meiosis leading to velo-cardio-facial/DiGeorge/22q11.2 deletion syndrome (22q11DS) characterized by conotruncal heart defects (CTDs) and other congenital anomalies. The majority of individuals have a 3 Mb deletion whose proximal region contains the presumed disease-associated gene TBX1 (T-box 1). Although a small subset have proximal nested deletions including TBX1, individuals with distal deletions that exclude TBX1 have also been identified. The deletions are flanked by low-copy repeats (LCR22A, B, C, D). We describe cardiac phenotypes in 25 individuals with atypical distal nested deletions within the 3 Mb region that do not include TBX1 including 20 with LCR22B to LCR22D deletions and 5 with nested LCR22C to LCR22D deletions. Together with previous reports, 12 of 37 (32%) with LCR22B–D deletions and 5 of 34 (15%) individuals with LCR22C–D deletions had CTDs including tetralogy of Fallot. In the absence of TBX1, we hypothesized that CRKL (Crk-like), mapping to the LCR22C–D region, might contribute to the cardiac phenotype in these individuals. We created an allelic series in mice of Crkl, including a hypomorphic allele, to test for gene expression effects on phenotype. We found that the spectrum of heart defects depends on Crkl expression, occurring with analogous malformations to that in human individuals, suggesting that haploinsufficiency of CRKL could be responsible for the etiology of CTDs in individuals with nested distal deletions and might act as a genetic modifier of individuals with the typical 3 Mb deletion.     

Analysis of severely affected patients with dihydropyrimidine dehydrogenase deficiency reveals large intragenic rearrangements of DPYD and a de novo interstitial deletion del(1)(p13.3p21.3)

Dihydropyrimidine dehydrogenase (DPD) deficiency is an infrequently described autosomal recessive disorder of the pyrimidine degradation pathway and can lead to mental and motor retardation and convulsions. DPD deficiency is also known to cause a potentially lethal toxicity following administration of the antineoplastic agent 5-fluorouracil. In an ongoing study of 72 DPD deficient patients, we analysed the molecular background of 5 patients in more detail in whom initial sequence analysis did not reveal pathogenic mutations. In three patients, a 13.8 kb deletion of exon 12 was found and in one patient a 122 kb deletion of exon 14–16 of DPYD. In the fifth patient, a c.299_302delTCAT mutation in exon 4 was found and also loss of heterozygosity of the entire DPD gene. Further analysis demonstrated a de novo deletion of approximately 14 Mb of chromosome 1p13.3–1p21.3, which includes DPYD. Haploinsufficiency of NTNG1, LPPR4, GPSM2, COL11A1 and VAV3 might have contributed to the severe psychomotor retardation and unusual craniofacial features in this patient. Our study showed for the first time the presence of genomic deletions affecting DPYD in 7% (5/72) of all DPD deficient patients. Therefore, screening of DPD deficient patients for genomic deletions should be considered.     

Disruption of Contactin 4 in two subjects with autism in Chinese population

Autism is a heterogeneous childhood neurodevelopmental disorder that is characterised by deficits in verbal communication, impaired social interactions, restricted interests and repetitive behaviours. Using an Illumina HumanCNV370-Quad BeadChip, we identified two Han Chinese individuals with autism and large duplications (~1.6 Mb and ~2.4 Mb) disrupting the same CNTN4 gene. CNTN4 encodes a protein that functions as a cell-adhesion molecule and may play an essential role in the formation of axon connections in the developing nervous system. The disruption of this gene has been reported to be the cause of the 3p deletion syndrome and also a possible susceptibility factor for autism spectrum disorders (ASDs). Our results suggest that rare copy number variations (CNVs) in CNTN4 may also influence autism susceptibility in Asian populations. Interestingly, a comparison of the clinical phenotypes between the two subjects revealed that the subject with the 2.4 Mb CNV (involving several other genes) presented with a more severe phenotype than the subject with the 1.6 Mb CNV (disrupting only CNTN4 and CNTN6). This suggests that other genes in the nearby region may contribute to the pathogenesis.     

High-resolution mapping of genotype-phenotype relationships in cri du chat syndrome using array comparative genomic hybridization

We have used array comparative genomic hybridization to map DNA copy-number changes in 94 patients with cri du chat syndrome who had been carefully evaluated for the presence of the characteristic cry, speech delay, facial dysmorphology, and level of mental retardation (MR). Most subjects had simple deletions involving 5p (67 terminal and 12 interstitial). Genotype-phenotype correlations localized the region associated with the cry to 1.5 Mb in distal 5p15.31, between bacterial artificial chromosomes (BACs) containing markers D5S2054 and D5S676; speech delay to 3.2 Mb in 5p15.32-15.33, between BACs containing D5S417 and D5S635; and the region associated with facial dysmorphology to 2.4 Mb in 5p15.2-15.31, between BACs containing D5S208 and D5S2887. These results overlap and refine those reported in previous publications. MR depended approximately on the 5p deletion size and location, but there were many cases in which the retardation was disproportionately severe, given the 5p deletion. All 15 of these cases, approximately two-thirds of the severely retarded patients, were found to have copy-number aberrations in addition to the 5p deletion. Restriction of consideration to patients with only 5p deletions clarified the effect of such deletions and suggested the presence of three regions, MRI-III, with differing effect on retardation. Deletions including MRI, a 1.2-Mb region overlapping the previously defined cri du chat critical region but not including MRII and MRIII, produced a moderate level of retardation. Deletions restricted to MRII, located just proximal to MRI, produced a milder level of retardation, whereas deletions restricted to the still-more proximal MRIII produced no discernible phenotype. However, MR increased as deletions that included MRI extended progressively into MRII and MRIII, and MR became profound when all three regions were deleted.     

8q12.1q12.3 de novo microdeletion involving the CHD7 gene in a patient without the major features of CHARGE syndrome: Case report and critical review of the literature

CHARGE syndrome is an autosomal dominant inherited disorder characterized by a specific and recognizable pattern of anomalies. De novo mutations or deletions of the gene encoding chromodomain helicase DNA binding protein 7 (CHD7) are the major cause of CHARGE syndrome. In this report, we describe a patient with a typical phenotype characterized by psychomotor retardation, hypertrichosis, facial asymmetry, synophria, failure to thrive, developmental delay and gastro-esophageal reflux, carrying a de novo 6.04 Mb interstitial deletion in 8q12.1q12.3 detected by single nucleotide polymorphism (SNP) array analysis. Despite the deletion includes CHD7 and although the patient shares some of the clinical features of the CHARGE syndrome, she does not fulfill the clinical criteria for this syndrome. To the best of our knowledge, this is the second case with an entire deletion of the CHD7 gene not leading to CHARGE syndrome and, for this reason, useful to expand and further delineate the clinical features associated with the 8q12.1q12.3 deletion. Furthermore, the literature review revealed that the phenotype secondary to duplications of the same region partially overlaps with the phenotype reported in this study. Selected genes that are present in the hemizygous state and which might be important for the phenotype of this patient, are discussed in context of the clinical features.     

Contribution of SNP arrays in diagnosis of deletion 2p11.2-p12

Deletions of the short arm of chromosome 2 are exceedingly rare, having been reported in few patients. Furthermore most cases with deletion in 2p11.2-p12 have been studied using standard karyotype and so it is not possible to delineate the precise size of deletions.Here, we describe a 9-year-old girl with a 9.4 Mb de novo interstitial deletion of region 2p11.2-p12 identified by SNP array analysis.The deleted region encompasses over 40 known genes, including LRRTM1, CTNNA2 and REEP1, haploinsufficiency of which could explain some clinical features of this patient such as mental retardation, speech delay and gait abnormalities.A comparison of our case with previously reported patients who present deletions in 2p11.2-p12 was carried out.Our case adds new information to the deletion of 2p11.2-p12, improving the knowledge on this rearrangement.     

Induction of diploid gynogenesis in an evolutionary model organism, the three-spined stickleback (Gasterosteus aculeatus)

Background

X-linked alpha thalassemia, mental retardation syndrome in humans is a rare recessive disorder caused by mutations in the ATRX gene. The disease is characterised by severe mental retardation, mild alpha-thalassemia, microcephaly, short stature, facial, skeletal, genital and gonadal abnormalities.

Results

We examined the expression of ATRX and ATRY during early development and gonadogenesis in two distantly related mammals: the tammar wallaby (a marsupial) and the mouse (a eutherian). This is the first examination of ATRX and ATRY in the developing mammalian gonad and fetus. ATRX and ATRY were strongly expressed in the developing male and female gonad respectively, of both species. In testes, ATRY expression was detected in the Sertoli cells, germ cells and some interstitial cells. In the developing ovaries, ATRX was initially restricted to the germ cells, but was present in the granulosa cells of mature ovaries from the primary follicle stage onwards and in the corpus luteum. ATRX mRNA expression was also examined outside the gonad in both mouse and tammar wallaby whole embryos. ATRX was detected in the developing limbs, craniofacial elements, neural tissues, tail and phallus. These sites correspond with developmental deficiencies displayed by ATR-X patients.

Conclusions

There is a complex expression pattern throughout development in both mammals, consistent with many of the observed ATR-X syndrome phenotypes in humans. The distribution of ATRX mRNA and protein in the gonads was highly conserved between the tammar and the mouse. The expression profile within the germ cells and somatic cells strikingly overlaps with that of DMRT1, suggesting a possible link between these two genes in gonadal development. Taken together, these data suggest that ATRX has a critical and conserved role in normal development of the testis and ovary in both the somatic and germ cells, and that its broad roles in early mammalian development and gonadal function have remained unchanged for over 148 million years of mammalian evolution.     

Efficient auto-excision of a selectable marker gene from transgenic citrus by combining the Cre/loxP system and ipt selection

Key message

A highly efficient Cre-mediated deletion system, offering a good alternative for producing marker-free transgenic plants that will relieve public concerns regarding GMOs, was first developed in citrus.

Abstract

The presence of marker genes in genetically modified crops raises public concerns regarding their safety. The removal of marker genes can prevent the risk of their flow into the environment and hasten the public’s acceptance of transgenic products. In this study, a new construct based on the Cre/loxP site-recombination system was designed to delete marker genes from transgenic citrus. In the construct, the selectable marker gene isopentenyltransferase gene (ipt) from Agrobacterium tumefaciens and the Cre recombinase gene were flanked by two loxP recognition sites in the direct orientation. The green fluorescent protein (gfp) reporter gene for monitoring the transformation of foreign genes was located outside of the loxP sequences. Transformation and deletion efficiencies of the vector were investigated using nopaline synthase gene (NosP) and CaMV 35S promoters to drive expression of Cre. Analysis of GFP activity showed that 28.1 and 13.6 % transformation efficiencies could be obtained by NosP- and CaMV 35S-driven deletions, respectively. Molecular analysis demonstrated that 100 % deletion efficiency was observed in the transgenic plants. The complete excision of the marker gene was found in all deletion events driven by NosP and in 81.8 % of deletion events driven by CaMV 35S. The results showed that Cre/loxP-mediated excision was highly efficient and precise in citrus. This approach provides a reliable strategy for auto-deletion of selectable marker genes from transgenic citrus to produce marker-free transgenic plants.     

Progress in lactic acid bacterial phage research

Background

The cyanobacterium Synechocystis sp. PCC 6803 is widely used for research on photosynthesis and circadian rhythms, and also finds application in sustainable biotechnologies. Synechocystis is naturally transformable and undergoes homologous recombination, which enables the development of a variety of tools for genetic and genomic manipulations. To generate multiple gene deletions and/or replacements, marker-less manipulation methods based on counter-selection are generally employed. Currently available methods require two transformation steps with different DNA plasmids.

Results

In this study, we present a marker-less gene deletion and replacement strategy in Synechocystis sp. PCC 6803 which needs only a single transformation step. The method utilizes an nptI-sacB double selection cassette and exploits the ability of the cyanobacterium to undergo two successive genomic recombination events via double and single crossing-over upon application of appropriate selective procedures.

Conclusions

By reducing the number of cloning steps, this strategy will facilitate gene manipulation, gain-of-function studies, and automated screening of mutants.     

FISH-mapping of a 100-kb terminal 22q13 deletion

Both cytogenetically visible and cryptic deletions of the terminal region of chromosome 22q are associated with a clinical phenotype including mental retardation, delay in expressive speech development, hypotonia, normal to accelerated growth and minor facial dysmorphic features. The genes responsible for the development of the phenotype have not yet been identified, but a distal localization is probable, since the cytogenetically visible and the cryptic deletions show a similar pattern of symptoms. We report a 33-year-old woman with a submicroscopic 22q13 deletion, mild mental retardation, speech delay, autistic symptoms and mild facial dysmorphic features. The deletion was mapped by FISH using cosmid probes from terminal 22q13, and the size of the deletion was estimated to be 100 kb. Three genes are affected by the deletion in this patient. ACR and RABL2B are deleted and proSAP2 is disrupted. This observation, together with recently published data, supports the notion that proSAP2 is the most important contributor to the 22q13 deletion phenotype.     

Hotspots of Large Rare Deletions in the Human Genome

Background

We have examined the genomic distribution of large rare autosomal deletions in a sample of 440 parent-parent-child trios from the Quebec founder population (QFP) which was recruited for a study of Attention Deficit Hyperactivity Disorder.

Methodology/Principal Findings

DNA isolated from blood was genotyped on Illumina Hap300 arrays. PennCNV combined with visual evaluation of images generated by the Beadstudio program was used to determine deletion boundary definition of sufficient precision to discern independent events, with near-perfect concordance between parent and child in about 98% of the 399 events detected in the offspring; the remaining 7 deletions were considered de novo. We defined several genomic regions of very high deletion frequency (‘hotspots’), usually of 0.4–0.6 Mb in length where independent rare deletions were found at frequencies of up to 100 fold higher than the average for the genome as a whole. Five of the 7 de novo deletions were in these hotspots. The same hotspots were also observed in three other studies on members of the QFP, those with schizophrenia, with endometriosis and those from a longevity cohort.

Conclusions/Significance

Nine of the 13 hotspots carry one gene (7 of which are very long), while the rest contain no known genes. All nine genes have been implicated in disease. The patterns of exon deletions support the proposed roles for some of these genes in human disease, such as NRXN1 and PARKIN, and suggest limited roles or no role at all, for others, including MACROD2 and CTNNA3. Our results also offer an alternative interpretation for the observations of deletions in tumors which have been proposed as reflecting tumor-suppressive activity of genes in these hotspots.     

Hemizygosity of delta-catenin (CTNND2) is associated with severe mental retardation in cri-du-chat syndrome

Delta-catenin is an adherens junction protein involved in cell motility and expressed early in neuronal development. It was discovered as an interactor with presenilin-1. The genomic structure of the human delta-catenin gene (Human Gene Nomenclature Committee-approved symbol CTNND2) was determined and mapped to 5p15.2. A deletion of this chromosomal region has been associated with the cri-du-chat syndrome (CDCS), a segmental aneusomy syndrome of 5p that is associated with an unusual high-pitched cry at birth, facial dysmorphology, poor growth, and severe mental retardation. delta-catenin maps to a specific region in 5p15.2 that has been implicated in the mental retardation phenotype. The breakpoints in patients with 5p terminal deletions were characterized with respect to the severity of mental retardation and the physical location of the delta-catenin gene. A strong correlation was found between the hemizygous loss of delta-catenin and severe mental retardation. These findings and the properties of delta-catenin as a neuronal-specific protein, expressed early in development and involved in cell motility, support its role in the mental retardation of CDCS when present in only one copy.