A novel period mutation implicating nuclear export in temperature compensation of the Drosophila circadian clock.

Circadian clocks are self-sustained molecular oscillators controlling daily changes of behavioral activity and physiology. For functional reliability and precision, the frequency of these molecular oscillations must be stable at different environmental temperatures, known as "temperature compensation." Despite being an intrinsic property of all circadian clocks, this phenomenon is not well understood at the molecular level. Here, we use behavioral and molecular approaches to characterize a novel mutation in the period (per) clock gene of Drosophila melanogaster, which alters a predicted nuclear export signal (NES) of the PER protein and affects temperature compensation. We show that this new perI530A allele leads to progressively longer behavioral periods and clock oscillations with increasing temperature in both clock neurons and peripheral clock cells. While the mutant PERI530A protein shows normal circadian fluctuations and post-translational modifications at cool temperatures, increasing temperatures lead to both severe amplitude dampening and hypophosphorylation of PERI530A. We further show that PERI530A displays reduced repressor activity at warmer temperatures, presumably because it cannot inactivate the transcription factor CLOCK (CLK), indicated by temperature-dependent altered CLK post-translational modification in perI530A flies. With increasing temperatures, nuclear accumulation of PERI530A within clock neurons is increased, suggesting that wild-type PER is exported out of the nucleus at warm temperatures. Downregulating the nuclear export factor CRM1 also leads to temperature-dependent changes of behavioral rhythms, suggesting that the PER NES and the nuclear export of clock proteins play an important role in temperature compensation of the Drosophila circadian clock.

Impaired Pre-Motor Circuit Activity and Movement in a Drosophila Model of KCNMA1-Linked Dyskinesia.

BACKGROUND: Paroxysmal dyskinesias (PxDs) are characterized by involuntary movements and altered pre-motor circuit activity. Causative mutations provide a means to understand the molecular basis of PxDs. Yet in many cases, animal models harboring corresponding mutations are lacking. Here we utilize the fruit fly, Drosophila, to study a PxD linked to a gain-of-function (GOF) mutation in the KCNMA1/hSlo1 BK potassium channel. OBJECTIVES: We aimed to recreate the equivalent BK (big potassium) channel mutation in Drosophila. We sought to determine how this mutation altered action potentials (APs) and synaptic release in vivo; to test whether this mutation disrupted pre-motor circuit function and locomotion; and to define neural circuits involved in locomotor disruption. METHODS: We generated a knock-in Drosophila model using homologous recombination. We used electrophysiological recordings and calcium-imaging to assess AP shape, neurotransmission, and the activity of the larval pre-motor central pattern generator (CPG). We used video-tracking and automated systems to measure movement, and developed a genetic method to limit BK channel expression to defined circuits. RESULTS: Neuronal APs exhibited reduced width and an enhanced afterhyperpolarization in the PxD model. We identified calcium-dependent reductions in neurotransmitter release, dysfunction of the CPG, and corresponding alterations in movement, in model larvae. Finally, we observed aberrant locomotion and dyskinesia-like movements in adult model flies, and partially mapped the impact of GOF BK channels on movement to cholinergic neurons. CONCLUSION: Our model supports a link between BK channel GOF and hyperkinetic movements, and provides a platform to dissect the mechanistic basis of PxDs. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

CRYPTOCHROMES confer robustness, not rhythmicity, to circadian timekeeping.

Circadian rhythms are a pervasive property of mammalian cells, tissues and behaviour, ensuring physiological adaptation to solar time. Models of cellular timekeeping revolve around transcriptional feedback repression, whereby CLOCK and BMAL1 activate the expression of PERIOD (PER) and CRYPTOCHROME (CRY), which in turn repress CLOCK/BMAL1 activity. CRY proteins are therefore considered essential components of the cellular clock mechanism, supported by behavioural arrhythmicity of CRY-deficient (CKO) mice under constant conditions. Challenging this interpretation, we find locomotor rhythms in adult CKO mice under specific environmental conditions and circadian rhythms in cellular PER2 levels when CRY is absent. CRY-less oscillations are variable in their expression and have shorter periods than wild-type controls. Importantly, we find classic circadian hallmarks such as temperature compensation and period determination by CK1δ/ε activity to be maintained. In the absence of CRY-mediated feedback repression and rhythmic Per2 transcription, PER2 protein rhythms are sustained for several cycles, accompanied by circadian variation in protein stability. We suggest that, whereas circadian transcriptional feedback imparts robustness and functionality onto biological clocks, the core timekeeping mechanism is post-translational.

Disrupted Glutamate Signaling in Drosophila Generates Locomotor Rhythms in Constant Light.

We have used the Cambridge Protein Trap resource (CPTI) to screen for flies whose locomotor rhythms are rhythmic in constant light (LL) as a means of identifying circadian photoreception genes. From the screen of ∼150 CPTI lines, we obtained seven hits, two of which targeted the glutamate pathway, Got1 (Glutamate oxaloacetate transaminase 1) and Gs2 (Glutamine synthetase 2). We focused on these by employing available mutants and observed that variants of these genes also showed high levels of LL rhythmicity compared with controls. It was also clear that the genetic background was important with a strong interaction observed with the common and naturally occurring timeless (tim) polymorphisms, ls-tim and s-tim. The less circadian photosensitive ls-tim allele generated high levels of LL rhythmicity in combination with Got1 or Gs2, even though ls-tim and s-tim alleles do not, by themselves, generate the LL phenotype. The use of dsRNAi for both genes as well as for Gad (Glutamic acid decarboxylase) and the metabotropic glutamate receptor DmGluRA driven by clock gene promoters also revealed high levels of LL rhythmicity compared to controls. It is clear that the glutamate pathway is heavily implicated in circadian photoreception. TIM levels in Got1 and Gs2 mutants cycled and were more abundant than in controls under LL. Got1 but not Gs2 mutants showed diminished phase shifts to 10 min light pulses. Neurogenetic dissection of the LL rhythmic phenotype using the gal4/gal80 UAS bipartite system suggested that the more dorsal CRY-negative clock neurons, DNs and LNds were responsible for the LL phenotype. Immunocytochemistry using the CPTI YFP tagged insertions for the two genes revealed that the DN1s but not the DN2 and DN3s expressed Got1 and Gs2, but expression was also observed in the lateral neurons, the LNds and s-LNvs. Expression of both genes was also found in neuroglia. However, downregulation of glial Gs2 and Got1 using repo-gal4 did not generate high levels of LL rhythmicity, so it is unlikely that this phenotype is mediated by glial expression. Our results suggest a model whereby the DN1s and possibly CRY-negative LNds use glutamate signaling to supress the pacemaker s-LNvs in LL.

Neurocalcin regulates nighttime sleep and arousal in Drosophila.

Sleep-like states in diverse organisms can be separated into distinct stages, each with a characteristic arousal threshold. However, the molecular pathways underlying different sleep stages remain unclear. The fruit fly, Drosophila melanogaster, exhibits consolidated sleep during both day and night, with night sleep associated with higher arousal thresholds compared to day sleep. Here we identify a role for the neuronal calcium sensor protein Neurocalcin (NCA) in promoting sleep during the night but not the day by suppressing nocturnal arousal and hyperactivity. We show that both circadian and light-sensing pathways define the temporal window in which NCA promotes sleep. Furthermore, we find that NCA promotes sleep by suppressing synaptic release from a dispersed wake-promoting neural network and demonstrate that the mushroom bodies, a sleep-regulatory center, are a module within this network. Our results advance the understanding of how sleep stages are genetically defined.

A Wake-Promoting Circadian Output Circuit in Drosophila.

Circadian clocks play conserved roles in gating sleep and wake states throughout the day-night cycle [1-5]. In the fruit fly Drosophila melanogaster, DN1p clock neurons have been reported to play both wake- and sleep-promoting roles [6-11], suggesting a complex coupling of DN1p neurons to downstream sleep and arousal centers. However, the circuit logic by which DN1p neurons modulate sleep remains poorly understood. Here, we show that DN1p neurons can be divided into two morphologically distinct subsets. Projections from one subset surround the pars intercerebralis, a previously defined circadian output region [12]. In contrast, the second subset also sends presynaptic termini to a visual processing center, the anterior optic tubercle (AOTU) [13]. Within the AOTU, we find that DN1p neurons inhibit a class of tubercular-bulbar (TuBu) neurons that act to promote consolidated sleep. These TuBu neurons in turn form synaptic connections with R neurons of the ellipsoid body, a region linked to visual feature detection, locomotion, spatial memory, and sleep homeostasis [14-17]. Our results define a second output arm from DN1p neurons and suggest a role for TuBu neurons as regulators of sleep drive.

Prion protein stabilizes amyloid-ß (Aß) oligomers and enhances Aß neurotoxicity in a Drosophila model of Alzheimer's disease.

The cellular prion protein (PrPC) can act as a cell-surface receptor for ß-amyloid (Aß) peptide; however, a role for PrPC in the pathogenesis of Alzheimer's disease (AD) is contested. Here, we expressed a range of Aß isoforms and PrPC in the Drosophila brain. We found that co-expression of Aß and PrPC significantly reduces the lifespan, disrupts circadian rhythms, and increases Aß deposition in the fly brain. In contrast, under the same conditions, expression of Aß or PrPC individually did not lead to these phenotypic changes. In vitro studies revealed that substoichiometric amounts of PrPC trap Aß as oligomeric assemblies and fragment-preformed Aß fibers. The ability of membrane-anchored PrPC to trap Aß as cytotoxic oligomers at the membrane surface and fragment inert Aß fibers suggests a mechanism by which PrPC exacerbates Aß deposition and pathogenic phenotypes in the fly, supporting a role for PrPC in AD. This study provides a second animal model linking PrPC expression with Aß toxicity and supports a role for PrPC in AD pathogenesis. Blocking the interaction of Aß and PrPC represents a potential therapeutic strategy.

Central and peripheral circadian clocks and their role in Alzheimer's disease.

Molecular and cellular oscillations constitute an internal clock that tracks the time of day and permits organisms to optimize their behaviour and metabolism to suit the daily demands they face. The workings of this internal clock become impaired with age. In this review, we discuss whether such age-related impairments in the circadian clock interact with age-related neurodegenerative disorders, such as Alzheimer's disease. Findings from mouse and fly models of Alzheimer's disease have accelerated our understanding of the interaction between neurodegeneration and circadian biology. These models show that neurodegeneration likely impairs circadian rhythms either by damaging the central clock or by blocking its communication with other brain areas and with peripheral tissues. The consequent sleep and metabolic deficits could enhance the susceptibility of the brain to further degenerative processes. Thus, circadian dysfunction might be both a cause and an effect of neurodegeneration. We also discuss the primary role of light in the entrainment of the central clock and describe important, alternative time signals, such as food, that play a role in entraining central and peripheral circadian clocks. Finally, we propose how these recent insights could inform efforts to develop novel therapeutic approaches to re-entrain arrhythmic individuals with neurodegenerative disease.

Quasimodo mediates daily and acute light effects on Drosophila clock neuron excitability.

We have characterized a light-input pathway regulating Drosophila clock neuron excitability. The molecular clock drives rhythmic electrical excitability of clock neurons, and we show that the recently discovered light-input factor Quasimodo (Qsm) regulates this variation, presumably via an Na+, K+, Cl- cotransporter (NKCC) and the Shaw K+ channel (dKV3.1). Because of light-dependent degradation of the clock protein Timeless (Tim), constant illumination (LL) leads to a breakdown of molecular and behavioral rhythms. Both overexpression (OX) and knockdown (RNAi) of qsm, NKCC, or Shaw led to robust LL rhythmicity. Whole-cell recordings of the large ventral lateral neurons (l-LNv) showed that altering Qsm levels reduced the daily variation in neuronal activity: qsmOX led to a constitutive less active, night-like state, and qsmRNAi led to a more active, day-like state. Qsm also affected daily changes in K+ currents and the GABA reversal potential, suggesting a role in modifying membrane currents and GABA responses in a daily fashion, potentially modulating light arousal and input to the clock. When directly challenged with blue light, wild-type l-LNvs responded with increased firing at night and no net response during the day, whereas altering Qsm, NKKC, or Shaw levels abolished these day/night differences. Finally, coexpression of ShawOX and NKCCRNAi in a qsm mutant background restored LL-induced behavioral arrhythmicity and wild-type neuronal activity patterns, suggesting that the three genes operate in the same pathway. We propose that Qsm affects both daily and acute light effects in l-LNvs probably acting on Shaw and NKCC.

Flyglow: Single-fly observations of simultaneous molecular and behavioural circadian oscillations in controls and an Alzheimer's model.

Circadian rhythms are essential for health and are frequently disturbed in disease. A full understanding of the causal relationships between behavioural and molecular circadian rhythms requires simultaneous longitudinal observations over time in individual organisms. Current experimental paradigms require the measurement of each rhythm separately across distinct populations of experimental organisms, rendering the comparability of the resulting datasets uncertain. We therefore developed FLYGLOW, an assay using clock gene controlled luciferase expression detected by exquisitely sensitive EM-CCD imaging, to enable simultaneous quantification of parameters including locomotor, sleep consolidation and molecular rhythms in single flies over days/weeks. FLYGLOW combines all the strengths of existing techniques, and also allows powerful multiparametric paired statistics. We found the age-related transition from rhythmicity to arrhythmicity for each parameter occurs unpredictably, with some flies showing loss of one or more rhythms during middle-age. Using single-fly correlation analysis of rhythm robustness and period we demonstrated the independence of the peripheral clock from circadian behaviours in wild type flies as well as in an Alzheimer's model. FLYGLOW is a useful tool for investigating the deterioration of behavioural and molecular rhythms in ageing and neurodegeneration. This approach may be applied more broadly within behavioural neurogenetics research.

Insights into amyloid disease from fly models.

The formation of amyloid aggregates is a feature of most, if not all, polypeptide chains. In vivo modelling of this process has been undertaken in the fruitfly Drosophila melanogaster with remarkable success. Models of both neurological and systemic amyloid diseases have been generated and have informed our understanding of disease pathogenesis in two main ways. First, the toxic amyloid species have been at least partially characterized, for example in the case of the Aß (amyloid ß-peptide) associated with Alzheimer's disease. Secondly, the genetic underpinning of model disease-linked phenotypes has been characterized for a number of neurodegenerative disorders. The current challenge is to integrate our understanding of disease-linked processes in the fly with our growing knowledge of human disease, for the benefit of patients.

The central molecular clock is robust in the face of behavioural arrhythmia in a Drosophila model of Alzheimer's disease.

Circadian behavioural deficits, including sleep irregularity and restlessness in the evening, are a distressing early feature of Alzheimer's disease (AD). We have investigated these phenomena by studying the circadian behaviour of transgenic Drosophila expressing the amyloid beta peptide (Aß). We find that Aß expression results in an age-related loss of circadian behavioural rhythms despite ongoing normal molecular oscillations in the central clock neurons. Even in the absence of any behavioural correlate, the synchronised activity of the central clock remains protective, prolonging lifespan, in Aß flies just as it does in control flies. Confocal microscopy and bioluminescence measurements point to processes downstream of the molecular clock as the main site of Aß toxicity. In addition, there seems to be significant non-cell-autonomous Aß toxicity resulting in morphological and probably functional signalling deficits in central clock neurons.

Functional genomics in Drosophila models of human disease.

It is occasionally observed that common sporadic diseases have rare familial counterparts in which mutations at a single locus result in a similar disorder exhibiting simple Mendelian inheritance. Such an observation is often sufficient justification for the creation of a disease model in the fly. Whether the system is based on the over-expression of a toxic variant of a human protein or requires the loss of function of an orthologous fly gene, the consequent phenotypes can be used to understand pathogenesis through the discovery of genetic modifiers. Such genetic screening can be completed rapidly in the fly and in this review we outline how libraries of mutants are generated and how consequent changes in disease-related phenotypes are assessed. The bioinformatic approaches to processing the copious amounts of data so generated are also presented. The next phase of fly modelling will tackle the challenges of complex diseases in which many genes are associated with risk in the human. There is growing interest in the use of interactomics and epigenetics to provide proteome- and genome-scale descriptions of the regulatory dysfunction that results in disease.

QUASIMODO, a Novel GPI-anchored zona pellucida protein involved in light input to the Drosophila circadian clock.

BACKGROUND: Circadian clocks are synchronized to the solar day via visual and specialized photoreceptors. In Drosophila, CRYPTOCHROME (CRY) is a major photoreceptor that mediates resetting of the circadian clock via light-dependent degradation of the clock protein TIMELESS (TIM). However, in the absence of CRY, this TIM-mediated resetting still occurs in some pacemaker neurons, resulting in synchronized behavioral rhythms when flies are exposed to light-dark cycles. Even in the additional absence of visual photoreception, partial molecular and behavioral light synchronization persists. Therefore, other important clock-related photoreceptive and synchronization mechanisms must exist. RESULTS: We identified a novel clock-controlled gene (quasimodo) that encodes a light-responsive and membrane-anchored Zona Pellucida domain protein that supports light-dependent TIM degradation. Whereas wild-type flies become arrhythmic in constant light (LL), quasimodo mutants elicit rhythmic expression of clock proteins and behavior in LL. QUASIMODO (QSM) can function independently of CRY and is predominantly expressed within CRY-negative clock neurons. Interestingly, downregulation of qsm in the clock circuit restores LL clock protein rhythms in qsm-negative neurons, indicating that qsm-mediated light input is not entirely cell autonomous and can be accessed by the clock circuit. CONCLUSIONS: Our findings indicate that QSM constitutes part of a novel and CRY-independent light input to the circadian clock. Like CRY, this pathway targets the clock protein TIM. QSM's light-responsive character in conjunction with the predicted localization at the outer neuronal membrane suggests that its function is linked to a yet unidentified membrane-bound photoreceptor.

A role for the PERIOD:PERIOD homodimer in the Drosophila circadian clock.

Circadian clocks in eukaryotes rely on transcriptional feedback loops, in which clock genes repress their own transcription resulting in molecular oscillations with a period of approximately 24 h. In Drosophila, the clock proteins Period (PER) and Timeless (TIM) operate in such a feedback loop, whereby they first accumulate in the cytoplasm of clock cells as a heterodimer. Nuclear translocation of the complex or the individual PER and TIM proteins is followed by repression of per and tim transcription, whereby PER seems to act as the prime repressor. We found that in addition to PER:TIM complexes, functional PER:PER homodimers exist in flies. Specific disruption of PER homodimers results in drastically impaired behavioral and molecular rhythmicity, pointing the biological importance of this clock protein complex. Analysis of PER subcellular distribution and repressor competence in the PER dimer mutant revealed defects in PER nuclear translocation and a disruption of rhythmic period transcription. The striking similarity of these phenotypes with that of reduced CKII activity suggests that the formation or function of the PER dimer is closely linked to this kinase. Our results confirm a previous structural model for PER and provide strong evidence that PER homodimers are important for circadian clock function.

Light-dependent interactions between the Drosophila circadian clock factors cryptochrome, jetlag, and timeless.

Circadian clocks regulate daily fluctuations of many physiological and behavioral aspects in life. They are synchronized with the environment via light or temperature cycles [1]. Natural fluctuations of the day length (photoperiod) and temperature necessitate a daily reset of the circadian clock on the molecular level. In Drosophila, the blue-light photoreceptor Cryptochrome (Cry) mediates a rapid light-dependent degradation of the clock protein Timeless (Tim) via the F box protein Jetlag (Jet) and the proteasome, which initiates the resetting of the molecular clock [2, 3]. Cry is also degraded in the light but whereas the degradation of Tim is well characterized [4-8], the mechanism for light-dependent degradation of Cry is mostly unknown. Until now it was believed that these two degradation pathways are distinct [4, 9]. Here we reveal that Jetlag also interacts with Cry in a light-dependent manner. After illumination, Jetlag induces massive degradation of Cry, which can be prevented in vitro and in vivo by adding Tim as an antagonist. We show that the affinity of Tim for Cry and Jetlag determines the sequential order of Tim and Cry degradation and thus reveal an intimate connection between the light-dependent degradation of these two proteins by the same proteasomal pathway.

Induction of pyruvate dehydrogenase kinase-3 by hypoxia-inducible factor-1 promotes metabolic switch and drug resistance.

The switch of cellular metabolism from mitochondrial respiration to glycolysis is the hallmark of cancer cells and associated with tumor malignancy. However, the mechanism of this metabolic switch remains largely unknown. Herein, we reported that hypoxia-inducible factor-1 (HIF-1) induced pyruvate dehydrogenase kinase-3 (PDK3) expression leading to inhibition of mitochondrial respiration. Promoter activity assay, small interference RNA knockdown assay, and chromatin immunoprecipitation assay demonstrated that hypoxia-induced PDK3 gene activity was regulated by HIF-1 at the transcriptional level. Forced expression of PDK3 in cancer cells resulted in increased lactic acid accumulation and drugs resistance, whereas knocking down PDK3 inhibited hypoxia-induced cytoplasmic glycolysis and cell survival. These data demonstrated that increased PDK3 expression due to elevated HIF-1alpha in cancer cells may play critical roles in metabolic switch during cancer progression and chemoresistance in cancer therapy.

Expression of sex-determining genes in the scalp of men with androgenetic alopecia.

BACKGROUND: The regulation of the cutaneous steroidogenesis in patients with androgenetic alopecia remains largely unclear. OBJECTIVE: The purpose of this study was to quantify the expression of the sex-determining genes in different scalp areas. METHODS: Paired scalp specimens from frontal and occipital scalp areas of 10 patients were examined by real-time RT-PCR for mRNA expression and of 40 patients (mean age 34.9 years, range 22-58) by Western blotting for protein analysis. RESULTS: The SOX-9 mRNA was most abundant in the skin, while SF-1 mRNA was sparsely detected. The protein levels of DAX-1, SRY and WT-1 were significantly higher in the bald scalp (p=0.003, 0.004 and 0.03, respectively). Only the SRY expression showed a positive correlation with the baldness severity in Norwood-Hamilton classification (p=0.024). There was no association between patient's age and the protein levels. Immunostaining of SOX-9 was detected in the outer root sheath keratinocytes of hair follicles but not in the dermal papillae. CONCLUSION: Further study on a larger population, including normal subjects and female patients, is needed to confirm the pathogenic role of sex-determining genes in androgenetic alopecia.

Aberrant expression of leptin in human endometriotic stromal cells is induced by elevated levels of hypoxia inducible factor-1alpha.

Elevated expression of leptin in endometriotic tissue results in an increase in stromal cell proliferation and may contribute to the development of endometriosis. However, the underlying mechanism responsible for aberrant expression of leptin is not known. We hypothesize that aberrant expression of leptin in endometriotic stroma may be regulated by increased levels of hypoxia-inducible factor-1alpha (HIF-1alpha), the master transcription factor that controls gene expression in response to hypoxia. Herein we show that the mRNA and protein levels of HIF-1alpha were greater in ectopic endometriotic tissue compared with its eutopic counterpart. Exposure of eutopic endometrial stromal cells under hypoxic conditions or treated with desferrioxamine (DFO, chemical hypoxia) resulted in a time-dependent increase in leptin gene expression. A promoter activity assay demonstrated that HIF-1alpha induced leptin promoter activity after DFO treatment. Chromatin immunoprecipitation assay further demonstrated that binding of HIF-1alpha to leptin promoter was evident after DFO treatment. Finally, depletion of HIF-1alpha by short interference RNA abolished leptin expression in ectopic endometriotic stromal cells. Taken together, our data demonstrate that aberrant expression of leptin in ectopic endometriotic stromal cells is induced, at least in part, by an elevated level of HIF-1alpha in these cells, providing new insights into the etiology of endometriosis.

Computational analysis and refinement of sequence structure on chromosome 22q11.2 region: application to the development of quantitative real-time PCR assay for clinical diagnosis.

The low-copy repeat (LCR) is a new class of repetitive DNA element and has been implicated in many human disorders, including DiGeorge/velocardiofacial syndrome (DGS/VCFS). It is now recognized that nonallelic homologous recombination (NAHR) through LCRs flanking the chromosome 22q11.2 region leads to genome rearrangements and results in the DGS/VCFS. To refine the structure and content of chromosome 22q11.2 LCRs, we applied computational analysis to dissect region-specific LCRs using publicly available sequences. Nine distinct duplicons between 1.6 and 65 kb long and sharing >95% sequence identity were identified. The presence of these sequence motifs supports the NAHR mechanism. Further sequence analysis suggested that the previously defined 3-Mb deletion may actually comprise two deletion intervals of similar size close to each other and thus indistinguishable when using fluorescence in situ hybridization (FISH) analysis. The differentially deleted regions contain several hypothetical proteins and UniGene clusters and may partially explain the clinical heterogeneity observed in DGS/VCFS patients with the 3-Mb common deletion. To implement further sequence information in molecular medicine, we designed a real-time quantitative PCR assay and validated the method in 122 patients with suspected DGS/VCFS. The assay detected 28 patients with chromosome 22q11.2 deletion later confirmed using FISH. Our results indicated that the developed assay is reliable as well as time and cost effective for clinical diagnosis of chromosome 22q11.2 deletion. They also suggest that this methodology can be applied to develop a molecular approach for clinical detection and diagnosis of other genomic disorders.