A novel non-invasive prenatal sickle cell disease test for all at-risk pregnancies.

Sickle cell disease (SCD) is the most common genetic haematological disorder. The availability of non-invasive prenatal diagnosis (NIPD) is predicted to increase uptake of prenatal diagnosis for SCD, as it has no perceived procedure-related miscarriage risk. We report the development of a targeted massively parallel sequencing (MPS) assay for the NIPD of fetal SCD using fetal cell-free (cf)DNA from maternal plasma, with no requirement for paternal or proband samples. In all, 64 plasma samples from pregnant women were analysed: 42 from SCD carriers, 15 from women with homozygous (Hb SS) SCD and seven from women with compound heterozygous (Hb SC) SCD. Our assay incorporated a relative mutation dosage assay for maternal carriers and a wild type allele detection assay for affected women (Hb SS/Hb SC). Selective analysis of only smaller cfDNA fragments and modifications to DNA fragment hybridisation capture improved diagnostic accuracy. Clinical sensitivity was 100% and clinical specificity was 100%. One sample with a fetal fraction of <4% was correctly called as 'unaffected', but with a discordant genotype (Hb AA rather than Hb AS). Six samples gave inconclusive results, of which two had a fetal fraction of <4%. This study demonstrates that NIPD for SCD is approaching clinical utility.

Autopsy findings in EPG5-related Vici syndrome with antenatal onset.

Vici syndrome is one of the most extensive inherited human multisystem disorders and due to recessive mutations in EPG5 encoding a key autophagy regulator with a crucial role in autophagosome-lysosome fusion. The condition presents usually early in life, with features of severe global developmental delay, profound failure to thrive, (acquired) microcephaly, callosal agenesis, cataracts, cardiomyopathy, hypopigmentation, and combined immunodeficiency. Clinical course is variable but usually progressive and associated with high mortality. Here, we present a fetus, offspring of consanguineous parents, in whom callosal agenesis and other developmental brain abnormalities were detected on fetal ultrasound scan (US) and subsequent MRI scan in the second trimester. Postmortem examination performed after medically indicated termination of pregnancy confirmed CNS abnormalities and provided additional evidence for skin hypopigmentation, nascent cataracts, and hypertrophic cardiomyopathy. Genetic testing prompted by a suggestive combination of features revealed a homozygous EPG5 mutation (c.5870-1G>A) predicted to cause aberrant splicing of the EPG5 transcript. Our findings expand the phenotypical spectrum of EPG5-related Vici syndrome and suggest that this severe condition may already present in utero. While callosal agenesis is not an uncommon finding in fetal medicine, additional presence of hypopigmentation, cataracts and cardiomyopathy is rare and should prompt EPG5 testing.

Mutations in FBXL4 cause mitochondrial encephalopathy and a disorder of mitochondrial DNA maintenance.

Nuclear genetic disorders causing mitochondrial DNA (mtDNA) depletion are clinically and genetically heterogeneous, and the molecular etiology remains undiagnosed in the majority of cases. Through whole-exome sequencing, we identified recessive nonsense and splicing mutations in FBXL4 segregating in three unrelated consanguineous kindreds in which affected children present with a fatal encephalopathy, lactic acidosis, and severe mtDNA depletion in muscle. We show that FBXL4 is an F-box protein that colocalizes with mitochondria and that loss-of-function and splice mutations in this protein result in a severe respiratory chain deficiency, loss of mitochondrial membrane potential, and a disturbance of the dynamic mitochondrial network and nucleoid distribution in fibroblasts from affected individuals. Expression of the wild-type FBXL4 transcript in cell lines from two subjects fully rescued the levels of mtDNA copy number, leading to a correction of the mitochondrial biochemical deficit. Together our data demonstrate that mutations in FBXL4 are disease causing and establish FBXL4 as a mitochondrial protein with a possible role in maintaining mtDNA integrity and stability.

Mutations in B3GALNT2 cause congenital muscular dystrophy and hypoglycosylation of α-dystroglycan.

Mutations in several known or putative glycosyltransferases cause glycosylation defects in α-dystroglycan (α-DG), an integral component of the dystrophin glycoprotein complex. The hypoglycosylation reduces the ability of α-DG to bind laminin and other extracellular matrix ligands and is responsible for the pathogenesis of an inherited subset of muscular dystrophies known as the dystroglycanopathies. By exome and Sanger sequencing we identified two individuals affected by a dystroglycanopathy with mutations in ß-1,3-N-acetylgalactosaminyltransferase 2 (B3GALNT2). B3GALNT2 transfers N-acetyl galactosamine (GalNAc) in a ß-1,3 linkage to N-acetyl glucosamine (GlcNAc). A subsequent study of a separate cohort of individuals identified recessive mutations in four additional cases that were all affected by dystroglycanopathy with structural brain involvement. We show that functional dystroglycan glycosylation was reduced in the fibroblasts and muscle (when available) of these individuals via flow cytometry, immunoblotting, and immunocytochemistry. B3GALNT2 localized to the endoplasmic reticulum, and this localization was perturbed by some of the missense mutations identified. Moreover, knockdown of b3galnt2 in zebrafish recapitulated the human congenital muscular dystrophy phenotype with reduced motility, brain abnormalities, and disordered muscle fibers with evidence of damage to both the myosepta and the sarcolemma. Functional dystroglycan glycosylation was also reduced in the b3galnt2 knockdown zebrafish embryos. Together these results demonstrate a role for B3GALNT2 in the glycosylation of α-DG and show that B3GALNT2 mutations can cause dystroglycanopathy with muscle and brain involvement.

Mutations in GDP-mannose pyrophosphorylase B cause congenital and limb-girdle muscular dystrophies associated with hypoglycosylation of α-dystroglycan.

Congenital muscular dystrophies with hypoglycosylation of α-dystroglycan (α-DG) are a heterogeneous group of disorders often associated with brain and eye defects in addition to muscular dystrophy. Causative variants in 14 genes thought to be involved in the glycosylation of α-DG have been identified thus far. Allelic mutations in these genes might also cause milder limb-girdle muscular dystrophy phenotypes. Using a combination of exome and Sanger sequencing in eight unrelated individuals, we present evidence that mutations in guanosine diphosphate mannose (GDP-mannose) pyrophosphorylase B (GMPPB) can result in muscular dystrophy variants with hypoglycosylated α-DG. GMPPB catalyzes the formation of GDP-mannose from GTP and mannose-1-phosphate. GDP-mannose is required for O-mannosylation of proteins, including α-DG, and it is the substrate of cytosolic mannosyltransferases. We found reduced α-DG glycosylation in the muscle biopsies of affected individuals and in available fibroblasts. Overexpression of wild-type GMPPB in fibroblasts from an affected individual partially restored glycosylation of α-DG. Whereas wild-type GMPPB localized to the cytoplasm, five of the identified missense mutations caused formation of aggregates in the cytoplasm or near membrane protrusions. Additionally, knockdown of the GMPPB ortholog in zebrafish caused structural muscle defects with decreased motility, eye abnormalities, and reduced glycosylation of α-DG. Together, these data indicate that GMPPB mutations are responsible for congenital and limb-girdle muscular dystrophies with hypoglycosylation of α-DG.

The relationship between spoken language and speech and nonspeech processing in children with autism: a magnetic event-related field study.

It has been proposed that language impairments in children with Autism Spectrum Disorders (ASD) stem from atypical neural processing of speech and/or nonspeech sounds. However, the strength of this proposal is compromised by the unreliable outcomes of previous studies of speech and nonspeech processing in ASD. The aim of this study was to determine whether there was an association between poor spoken language and atypical event-related field (ERF) responses to speech and nonspeech sounds in children with ASD (n = 14) and controls (n = 18). Data from this developmental population (ages 6-14) were analysed using a novel combination of methods to maximize the reliability of our findings while taking into consideration the heterogeneity of the ASD population. The results showed that poor spoken language scores were associated with atypical left hemisphere brain responses (200 to 400 ms) to both speech and nonspeech in the ASD group. These data support the idea that some children with ASD may have an immature auditory cortex that affects their ability to process both speech and nonspeech sounds. Their poor speech processing may impair their ability to process the speech of other people, and hence reduce their ability to learn the phonology, syntax, and semantics of their native language.

Evidence of digenic inheritance in Alport syndrome.

BACKGROUND: Alport syndrome is a clinically heterogeneous, progressive nephropathy caused by mutations in collagen IV genes, namely COL4A3 and COL4A4 on chromosome 2 and COL4A5 on chromosome X. The wide phenotypic variability and the presence of incomplete penetrance suggest that a simple Mendelian model cannot completely explain the genetic control of this disease. Therefore, we explored the possibility that Alport syndrome is under digenic control. METHODS: Using massively parallel sequencing, we identified 11 patients who had pathogenic mutations in two collagen IV genes. For each proband, we ascertained the presence of the same mutations in up to 12 members of the extended family for a total of 56 persons studied. RESULTS: Overall, 23 mutations were found. Individuals with two pathogenic mutations in different genes had a mean age of renal function deterioration intermediate with respect to the autosomal-dominant form and the autosomal-recessive one, in line with molecule stoichiometry of the disruption of the type IV collagen triple helix. CONCLUSIONS: Segregation analysis indicated three possible digenic segregation models: (i) autosomal inheritance with mutations on different chromosomes, resembling recessive inheritance (five families); (ii) autosomal inheritance with mutations on the same chromosome resembling dominant inheritance (two families) and (iii) unlinked autosomal and X-linked inheritance having a peculiar segregation (four families). This pedigree analysis provides evidence for digenic inheritance of Alport syndrome. Clinical geneticists and nephrologists should be aware of this possibility in order to more accurately assess inheritance probabilities, predict prognosis and identify other family members at risk.

ISPD gene mutations are a common cause of congenital and limb-girdle muscular dystrophies.

Dystroglycanopathies are a clinically and genetically diverse group of recessively inherited conditions ranging from the most severe of the congenital muscular dystrophies, Walker-Warburg syndrome, to mild forms of adult-onset limb-girdle muscular dystrophy. Their hallmark is a reduction in the functional glycosylation of α-dystroglycan, which can be detected in muscle biopsies. An important part of this glycosylation is a unique O-mannosylation, essential for the interaction of α-dystroglycan with extracellular matrix proteins such as laminin-α2. Mutations in eight genes coding for proteins in the glycosylation pathway are responsible for ∼50% of dystroglycanopathy cases. Despite multiple efforts using traditional positional cloning, the causative genes for unsolved dystroglycanopathy cases have escaped discovery for several years. In a recent collaborative study, we discovered that loss-of-function recessive mutations in a novel gene, called isoprenoid synthase domain containing (ISPD), are a relatively common cause of Walker-Warburg syndrome. In this article, we report the involvement of the ISPD gene in milder dystroglycanopathy phenotypes ranging from congenital muscular dystrophy to limb-girdle muscular dystrophy and identified allelic ISPD variants in nine cases belonging to seven families. In two ambulant cases, there was evidence of structural brain involvement, whereas in seven, the clinical manifestation was restricted to a dystrophic skeletal muscle phenotype. Although the function of ISPD in mammals is not yet known, mutations in this gene clearly lead to a reduction in the functional glycosylation of α-dystroglycan, which not only causes the severe Walker-Warburg syndrome but is also a common cause of the milder forms of dystroglycanopathy.

Comparing and contrasting barriers in augmentative alternative communication use in nonspeaking autism and complex communication needs: multi-stakeholder perspectives.

Augmentative alternative communication (AAC) devices or systems are often prescribed to minimally verbal or nonspeaking autistic individuals and other individuals with complex communication needs to facilitate communication or as an alternative to spoken language. AAC use can result in communication gains and improved quality of life for minimally verbal or nonspeaking individuals. Despite this, AAC abandonment is high, limiting societal participation of the individual on the autism spectrum with complex communication needs. Our study is a novel exploration of the barriers of AAC use from a multi-stakeholder perspective, and a qualitative analysis of similarities and differences between stakeholders. We conducted semi-structured interviews and focus groups with 30 parent-carers, educators and clinicians currently supporting AAC users in Western Australia and analysed the data using reflexive thematic analysis. Barriers from each stakeholder group were coded, resulting in 17 subthemes forming five main themes common to all stakeholders: Stakeholder Knowledge, Stakeholder Attitudes and Stigma, Resources, AAC User Engagement, and Device Fit. Contrasting perspectives included actual and perceived stigma associated with AAC use (parent-carers vs clinicians); different struggles with resources and knowledge (parent-carers vs clinicians and educators); and a lack of clinician communication in the processes that determined AAC-fit for school environments (educators only). Findings are discussed in the context of improving inter-stakeholder collaboration and capacity building in Australian health service and practice to better support minimally verbal or nonspeaking autistic individuals and individuals with complex communication needs. Suggestions are also offered for communication partner training.

The efficacy of mindfulness-based therapy for anxiety, social skills, and aggressive behaviors in children and young people with Autism Spectrum Disorder: A systematic review.

Introduction: The purpose of this systematic review was to examine the efficacy of mindfulness-based interventions for improving anxiety, social skills, and aggressive behaviors in children and young people (CYP) with Autism Spectrum Disorder (ASD); summarize the results across clinic, home, and school contexts; and evaluate the quality of these interventions for clinical practice. Methods: A search of the PsycINFO, Medline (Ovid), Web of Science, and Scopus databases was conducted in June 2021, and no date restrictions were applied. Inclusion criteria were quantitative or qualitative research implementing a mindfulness-based intervention for CYP aged 6-25 years with a diagnosis of ASD, Pervasive Development Disorder, or Asperger's Syndrome. Results: We identified 23 articles for inclusion including within subject pre- and post-testing, multiple baselines, and randomized control trials, among other research designs. Of these, a quality analysis conducted using an ASD research-specific risk of bias tool found over half (14) were of weak methodological quality, whereas only four and five were found to be of strong and adequate quality, respectively. Discussion: While the results of this systematic review suggest promising evidence for the use of mindfulness-based interventions to improve anxiety, social skills, and aggressive behaviors in CYP with ASD, results should be interpreted with caution due to the limitations resulting from the overall weak quality of the studies.The review protocol was pre-registered on PROSPERO (CRD42021259125) and can be viewed at https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=259125.

A hemizygous mutation in the FOXP3 gene (IPEX syndrome) resulting in recurrent X-linked fetal hydrops: a case report.

BACKGROUND: Fetal hydrops is excessive extravasation of fluid into the third space in a fetus, which could be due to a wide differential of underlying pathology. IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome primarily affects males. It is a monogenic primary immunodeficiency syndrome of X-linked recessive inheritance due to FOXP3 gene variants. It is characterised by the development of multiple autoimmune disorders in affected individuals. CASE PRESENTATION: We present a rare cause of male fetal hydrops in the context of IPEX syndrome and discuss FOXP3 gene variants as a differential for 'unexplained' fetal hydrops that may present after the first trimester. DISCUSSION AND CONCLUSIONS: In all similar cases, the pathological process begins during intrauterine life. Furthermore, there are no survivors described. Consequently, this variant should be considered as a severe one, associated with intrauterine life onset and fatal course, i.e., the most severe IPEX phenotype.

Diagnostic yield of rare skeletal dysplasia conditions in the radiogenomics era.

BACKGROUND: Skeletal dysplasia (SD) conditions are rare genetic diseases of the skeleton, encompassing a heterogeneous group of over 400 disorders, and represent approximately 5% of all congenital anomalies. Developments in genetic and treatment technologies are leading to unparalleled therapeutic advances; thus, it is more important than ever to molecularly confirm SD conditions. Data on 'rates-of-molecular yields' in SD conditions, through exome sequencing approaches, is limited. Figures of 39% and 52.5% have been reported in the USA (n = 54) and South Korea (n = 185) respectively. METHODS: We discuss a single-centre (in the UK) experience of whole-exome sequencing (WES) in a cohort of 15 paediatric patients (aged 5 months to 12 years) with SD disorders previously molecularly unconfirmed. Our cohort included patients with known clinical diagnoses and undiagnosed skeletal syndromes. Extensive phenotyping and expert radiological review by a panel of international SD radiology experts, coupled with a complex bioinformatics pipeline, allowed for both gene-targeted and gene-agnostic approaches. RESULTS: Significant variants leading to a likely or confirmed diagnosis were identified in 53.3% (n = 8/15) of patients; 46.7% (n = 7/15) having a definite molecular diagnosis and 6.7% (n = 1/15) having a likely molecular diagnosis. We discuss this in the context of a rare disease in general and specifically SD presentations. Of patients with known diagnoses pre-WES (n = 10), molecular confirmation occurred in 7/10 cases, as opposed to 1/5 where a diagnosis was unknown pre-test. Thus, diagnostic return is greatest where the diagnosis is known pre-test. For WGS (whole genome sequencing, the next iteration of WES), careful case selection (ideally of known diagnoses pre-test) will yield highest returns. CONCLUSIONS: Our results highlight the cost-effective use of WES-targeted bioinformatic analysis as a diagnostic tool for SD, particularly patients with presumed SD, where detailed phenotyping is essential. Thorough co-ordinated clinical evaluation between clinical, radiological, and molecular teams is essential for improved yield and clinical care. WES (and WGS) yields will increase with time, allowing faster diagnoses, avoiding needless investigations, ensuring individualised patient care and patient reassurance. Further diagnoses will lead to increased information on natural history/mechanistic details, and likely increased therapies and clinical trials.

Impaired P1 Habituation and Mismatch Negativity in Children with Autism Spectrum Disorder.

Passive testing of auditory function is an important objective in individuals with ASD due to known difficulties in understanding and/or following task instructions. In present study the habituation to standard tones following deviants and the auditory discriminative processes were examined in two conditions: electronic and human sounds, in a sample of 16 ASD children. ASD children presented a reduced habituation in the P1 component and a decrease in the amplitude of the mismatch negativity indicating a lower auditory discrimination with respect to controls. MMN amplitude was related to sensory sensitivity. Results suggest an increased activation to repeatedly auditory stimulus and a poor auditory discrimination, for both: electronic and human sounds with consequences on the impaired sensory behavior of ASD subjects.

Toward an Individualized Neural Assessment of Receptive Language in Children.

Purpose We aimed to develop a noninvasive neural test of language comprehension to use with nonspeaking children for whom standard behavioral testing is unreliable (e.g., minimally verbal autism). Our aims were threefold. First, we sought to establish the sensitivity of two auditory paradigms to elicit neural responses in individual neurotypical children. Second, we aimed to validate the use of a portable and accessible electroencephalography (EEG) system, by comparing its recordings to those of a research-grade system. Third, in light of substantial interindividual variability in individuals' neural responses, we assessed whether multivariate decoding methods could improve sensitivity. Method We tested the sensitivity of two child-friendly covert N400 paradigms. Thirty-one typically developing children listened to identical spoken words that were either strongly predicted by the preceding context or violated lexical-semantic expectations. Context was given by a cue word (Experiment 1) or sentence frame (Experiment 2), and participants either made an overall judgment on word relatedness or counted lexical-semantic violations. We measured EEG concurrently from a research-grade system, Neuroscan's SynAmps2, and an adapted gaming system, Emotiv's EPOC+. Results We found substantial interindividual variability in the timing and topology of N400-like effects. For both paradigms and EEG systems, traditional N400 effects at the expected sensors and time points were statistically significant in around 50% of individuals. Using multivariate analyses, detection rate increased to 88% of individuals for the research-grade system in the sentences paradigm, illustrating the robustness of this method in the face of interindividual variations in topography. Conclusions There was large interindividual variability in neural responses, suggesting interindividual variation in either the cognitive response to lexical-semantic violations and/or the neural substrate of that response. Around half of our neurotypical participants showed the expected N400 effect at the expected location and time points. A low-cost, accessible EEG system provided comparable data for univariate analysis but was not well suited to multivariate decoding. However, multivariate analyses with a research-grade EEG system increased our detection rate to 88% of individuals. This approach provides a strong foundation to establish a neural index of language comprehension in children with limited communication. Supplemental Material https://doi.org/10.23641/asha.12606311.

Post-mortem Characterisation of a Case With an ACTG1 Variant, Agenesis of the Corpus Callosum and Neuronal Heterotopia.

Cytoplasmic Actin Gamma 1 (ACTG1) gene variant are autosomal dominant and can cause CNS anomalies (Baraitser Winter Malformation Syndrome; BWMS). ACTG1 anomalies in offspring include agenesis of the corpus callosum (ACC) and neuronal heterotopia which are ectopic nodules of nerve cells that failed to migrate appropriately. Subcortical and periventricular neuronal heterotopia have been described previously in association with ACC. In this case report, we investigated a neonatal brain with an ACTG1 gene variant and a phenotype of ACC, and neuronal heterotopia (ACC-H) which was diagnosed on antenatal MR imaging and was consistent with band heterotopia seen on post-mortem brain images. Histologically clusters of neurons were seen in both the subcortical and periventricular white matter (PVWM) brain region that coincided with impaired abnormalities in glial formation. Immunohistochemistry was performed on paraffin-embedded brain tissue blocks from this case with ACTG1 variant and an age-matched control. Using tissue sections from the frontal lobe, we examined the distribution of neuronal cells (HuC/HuD, calretinin, and parvalbumin), growth cone (drebrin), and synaptic proteins (synaptophysin and SNAP-25). Additionally, we investigated how the ACTG1 variant altered astroglia (nestin, GFAP, vimentin); oligodendroglia (OLIG2) and microglia (Iba-1) in the corpus callosum, cortex, caudal ganglionic eminence, and PVWM. As predicted in the ACTG1 variant case, we found a lack of midline radial glia and glutamatergic fibers. We also found disturbances in the cortical region, in glial cells and a lack of extracellular matrix components in the ACTG1 variant. The caudal ganglionic eminence and the PVWM regions in the ACTG1 variant lacked several cellular components that were identified in a control case. Within the neuronal heterotopia, we found evidence of glutamatergic and GABAergic neurons with apparent synaptic connections. The data presented from this case study with BWMS with variants in the ACTG1 gene provides insight as to the composition of neuronal heterotopia, and how disturbances of important migratory signals may dramatically affect ongoing brain development.

A novel custom high density-comparative genomic hybridization array detects common rearrangements as well as deep intronic mutations in dystrophinopathies.

BACKGROUND: The commonest pathogenic DMD changes are intragenic deletions/duplications which make up to 78% of all cases and point mutations (roughly 20%) detectable through direct sequencing. The remaining mutations (about 2%) are thought to be pure intronic rearrangements/mutations or 5'-3' UTR changes. In order to screen the huge DMD gene for all types of copy number variation mutations we designed a novel custom high density comparative genomic hybridisation array which contains the full genomic region of the DMD gene and spans from 100 kb upstream to 100 kb downstream of the 2.2 Mb DMD gene. RESULTS: We studied 12 DMD/BMD patients who either had no detectable mutations or carried previously identified quantitative pathogenic changes in the DMD gene. We validated the array on patients with previously known mutations as well as unaffected controls, we identified three novel pure intronic rearrangements and we defined all the mutation breakpoints both in the introns and in the 3' UTR region. We also detected a novel polymorphic intron 2 deletion/duplication variation. Despite the high resolution of this approach, RNA studies were required to confirm the functional significance of the intronic mutations identified by CGH. In addition, RNA analysis identified three intronic pathogenic variations affecting splicing which had not been detected by the CGH analysis. CONCLUSION: This novel technology represents an effective high throughput tool to identify both common and rarer DMD rearrangements. RNA studies are required in order to validate the significance of the CGH array findings. The combination of these tools will fully cover the identification of causative DMD rearrangements in both coding and non-coding regions, particularly in patients in whom standard although extensive techniques are unable to detect a mutation.

Simultaneous mutation scanning for gross deletions, duplications and point mutations in the DMD gene.

We have developed a technique to screen for gross deletions/duplications and point mutations using one streamlined approach. Fluorescent multiplex quantitative PCR is used to determine the copy number of each exon, followed by conformation sensitive capillary electrophoresis (CSCE) of the same PCR products on a multi-capillary genetic analyser. We have developed this technique to screen all 79 exons of one of the largest human genes currently known (dystrophin) using 12 multiplex PCR assays. A blind trial of 50 male and 50 female samples, in which 84 mutations had previously been found and characterized by other techniques, showed 100% sensitivity and specificity. We then applied this method to screen over 100 patient samples previously screened for deletions and duplications of 28 exons from the two hotspot regions. Our data show that combining a full deletion/duplication screen with CSCE will detect a mutation in 98% of Duchenne muscular dystrophy patients and 93% of Becker muscular dystrophy patients where the clinical diagnosis is certain. This technique is applicable to any gene and is particularly suited to mutation screening of large genes, decreasing the time taken for a complete gene screen for nearly all mutation types.

Modeling and simulation for toxicity assessment.

The effect of various toxicants on growth/death and morphology of human cells is investigated using the xCELLigence Real-Time Cell Analysis High Troughput in vitro assay. The cell index is measured as a proxy for the number of cells, and for each test substance in each cell line, time-dependent concentration response curves (TCRCs) are generated. In this paper we propose a mathematical model to study the effect of toxicants with various initial concentrations on the cell index. This model is based on the logistic equation and linear kinetics. We consider a three dimensional system of differential equations with variables corresponding to the cell index, the intracellular concentration of toxicant, and the extracellular concentration of toxicant. To efficiently estimate the model's parameters, we design an Expectation Maximization algorithm. The model is validated by showing that it accurately represents the information provided by the TCRCs recorded after the experiments. Using stability analysis and numerical simulations, we determine the lowest concentration of toxin that can kill the cells. This information can be used to better design experimental studies for cytotoxicity profiling assessment.

Low prevalence of BRCA1 exon rearrangements in familial and young sporadic breast cancer patients.

BRCA1 exon deletions and duplications have been reported in a number of studies, and in order to design an effective mutation screening strategy in a diagnostic setting it is import to determine the frequency of this type of mutation in breast and ovarian cancer patients. We have designed and applied quantitative fluorescent PCR (QF-PCR) assays to screen for BRCA1 exon rearrangements in breast cancer patients both with and without a family history. A panel of 182 familial patients was screened, and an exon 3-7 deletion mutation was detected in a patient with a family history of breast and ovarian cancer. Additionally, we detected a duplication of exons 18-19 in an early onset sporadic breast cancer patient from a panel of 100 patients tested. These data indicate that in the absence of any founder mutations, screening for BRCA1 exon rearrangements does not significantly increase the overall BRCA1 mutation detection rate in patients referred to a genetics clinic because of either a family history and/or an early onset of disease.