Debunking Occam's razor: Diagnosing multiple genetic diseases in families by whole-exome sequencing.

BACKGROUND: Recent clinical whole exome sequencing (WES) cohorts have identified unanticipated multiple genetic diagnoses in single patients. However, the frequency of multiple genetic diagnoses in families is largely unknown. AIMS: We set out to identify the rate of multiple genetic diagnoses in probands and their families referred for analysis in two national research programs in Canada. MATERIALS & METHODS: We retrospectively analyzed WES results for 802 undiagnosed probands referred over the past 5 years in either the FORGE or Care4Rare Canada WES initiatives. RESULTS: Of the 802 probands, 226 (28.2%) were diagnosed based on mutations in known disease genes. Eight (3.5%) had two or more genetic diagnoses explaining their clinical phenotype, a rate in keeping with the large published studies (average 4.3%; 1.4 - 7.2%). Seven of the 8 probands had family members with one or more of the molecularly diagnosed diseases. Consanguinity and multisystem disease appeared to increase the likelihood of multiple genetic diagnoses in a family. CONCLUSION: Our findings highlight the importance of comprehensive clinical phenotyping of family members to ultimately provide accurate genetic counseling.

Utility of whole-exome sequencing for those near the end of the diagnostic odyssey: time to address gaps in care.

An accurate diagnosis is an integral component of patient care for children with rare genetic disease. Recent advances in sequencing, in particular whole-exome sequencing (WES), are identifying the genetic basis of disease for 25-40% of patients. The diagnostic rate is probably influenced by when in the diagnostic process WES is used. The Finding Of Rare Disease GEnes (FORGE) Canada project was a nation-wide effort to identify mutations for childhood-onset disorders using WES. Most children enrolled in the FORGE project were toward the end of the diagnostic odyssey. The two primary outcomes of FORGE were novel gene discovery and the identification of mutations in genes known to cause disease. In the latter instance, WES identified mutations in known disease genes for 105 of 362 families studied (29%), thereby informing the impact of WES in the setting of the diagnostic odyssey. Our analysis of this dataset showed that these known disease genes were not identified prior to WES enrollment for two key reasons: genetic heterogeneity associated with a clinical diagnosis and atypical presentation of known, clinically recognized diseases. What is becoming increasingly clear is that WES will be paradigm altering for patients and families with rare genetic diseases.

Hemizygous deletions on chromosome 1p21.3 involving the DPYD gene in individuals with autism spectrum disorder.

We describe the identification and clinical presentation of four individuals from three unrelated families with hemizygous deletions involving the DPYD gene at chromosome 1p21.3. DPYD encodes dihydropyrimidine dehydrogenase, which is the initial and rate-limiting enzyme in the catabolism of pyrimidine bases. All four individuals described met diagnostic criteria for autism spectrum disorder with severe speech delay. Patient 1's deletion was originally reported in 2008, and more detailed clinical information is provided. Subsequently, this male individual was found to have a missense mutation in the X-linked PTCHD1 autism susceptibility gene, which may also contribute to the phenotype. Patients 2 and 3 are siblings with a novel deletion encompassing the DPYD gene. In their mother, the genomic region deleted from chromosome 1p21.3 was inserted into chromosome 10. A fourth proband had a novel 10-kb intragenic deletion of exon 6 of the DPYD gene detected on a higher resolution microarray. Our study suggests that hemizygous deletions involving the DPYD locus present with variable phenotypes which can include speech delay and autistic features, and may also be influenced by additional mutations in other genes, issues which need to be considered in genetic counseling.

Holoprosencephaly and cleidocranial dysplasia in a patient due to two position-effect mutations: case report and review of the literature.

Holoprosencephaly (HPE) is a genetically heterogeneous developmental field defect in which midline cleavage of the forebrain and craniofacial structures is impaired. Based on the analysis of HPE patients with chromosome rearrangements, at least six loci for the disorder have been assigned. The sonic hedgehog gene (SHH) at 7q36 has been identified as the HPE3 locus. Cleidocranial dysplasia (CCD) is an autosomal dominant skeletal disorder characterized by clavicular, pelvic and dental anomalies. It is caused by mutations in the osteoblast-specific transcription factor CBFA1/RUNX2, which maps to 6p21. We report a 20-year-old female with premaxillary agenesis (part of the HPE spectrum), as well as skeletal abnormalities and impacted teeth reminiscent of CCD. She carries a de novo 6;7 reciprocal translocation, with breakpoints at 6p21.1 and 7q36. We have shown previously that the 7q36 breakpoint maps 15 kb telomeric to the 5' end of SHH, which explains the patient's HPE phenotype. Now, using fluorescence in situ hybridization, we have identified a P1 artificial chromosome clone 800 kb upstream of CBFA1/RUNX2 that spans the 6p breakpoint. We propose that the proband's complex phenotype is due to two position-effect (PE) mutations, one at each translocation breakpoint, which have altered the expression of the SHH and CBFA1/RUNX2 genes. The role of PE mutations in human disease is also reviewed.

[Primary liver cancer. Its epidemiological, clinical and biochemical characteristics]. / Cáncer hepático primario. Características epidemiológicas, clínicas y bioquímicas.

Epidemiological, clinical, biochemical and topographic features of primary hepatic cancer (PHC) were reviewed retrospective and prospectively in this study. This review consisted of 76 patients from 1971 to 1990. Forty nine males and 27 females. The mean age was 66.1 +/- 11.7 years. Hepatocellular carcinoma (HC) was the most frequent histological type (84.1%), followed by cholangiocarcinoma (87.7%). Mixed carcinoma and hepatoblastoma were 4.3 and 2.9% respectively. The prevalence af PHC among 1485 autopsies was 0.74%. The most frequent sites af metastasis were the lungs (66%) and portal vein (50%). Hepatocellular carcinoma was associated to cirrhosis in 80% of the cases. A syndrome including asthenia, weight loss, hepatomegaly and cholestasis was identified in most of the patients, and alkaline phosphatase was the most frequently disturbed laboratory test. 60% of tumors were bilateral and none of the solitary tumors had less than 5 cms in diameter. 20% of HC showed normal serum levels of AFP (< 20 ng/ml). 40% had at least one of the markers of B virus hepatitis in serum.

Surfactant-based ordered media in analytical atomic spectrometry.

During the last 10 years or so we have witnessed an enormous growth of interest and applications of surfactant-based ordered media in analytical chemistry. However, their use in analytical atomic spectroscopy (AAS) has been rather scarce and often controversial. The utilization of surfactants in this latter field is discussed here along two main lines: one refers to the favourable manipulation of physical properties of the sample solutions (Part A) while the other, demonstrated very recently, refers to the adequate manipulation of chemical reactions and/or interactions of analytes in solution by resorting to surfactants use (Part B). The control of physical properties of sample solutions, e.g. manipulation of the surface tension, allows three main applications of surfactants in atomic methods: possible increases of nebulization/atomization efficiencies in flame-AAS, improvement of aqueous/organic solvent compatibility (emulsification applications) and enhancement of the wettability of graphitic solid surfaces. The facts and controversies existing today on this method of utilization of surfactants to enhance atomic methodologies is critically discussed. The ability of surfactant-based "ordered media" to organize reactants at the molecular level has also been applied to enhance chemical generation of volatile species (e.g. hydride generation or cold Hg vapour generation) used in atomic methods. The analytical potential and usefulness of micelles and vesicles to improve the detection power of hydride generation ICP-AES methodologies are summarized for the determination of arsenic, lead and cadmium by plasma emission. Increases up to two-fold in the sensitivity of As and Pb have been observed by addition of organized media. A volatile Cd species is formed very easily in cationic vesicles with NaBH(4). This Cd species can be used to increase by five times the detectability of Cd by ICP-AES. Moreover, synergic combinations of liquid chromatography separations/atomic detection are possible by resorting to the use of micellar or vesicular mobile phases. The successful application of this principle to the modern problem of toxic arsenic HPLC speciation by using a vesicular solution [as mobile phase for the HPLC separation of As(III), As(V), monomethylarsonic and dimethylarsinic acids] and "on-line" surfactant-enhanced arsine generation is also described in detail and completes the whole picture of the present interface between analytical atomic spectroscopy and surfactant assemblies.

Determination of arsenic by inductively-coupled plasma atomic emission spectrometry enhanced by hydride generation from organized media.

A method is described for the determination of arsenic, which combines a continuous flow hydride generation technique with an inductively coupled plasma atomic emission detection system. Some atomic absorption preliminary studies are described as well. Arsine is generated with NaBH(4) from a didodecyldimethylammonium bromide (DDBA) vesicular medium. The analytical performance of this vesicles-enhanced method is superior to the generation of the hydride from aqueous media: the detection limit (0.6 ppb) is improved by a factor of 2 and greater tolerance to interferences is observed for arsine generation from DDBA vesicles. Precision of As determinations is also improved. The proposed method has been validated for low As levels determinations in two Certified Reference Materials (CRM) sediments with satisfactory results. The potential of organized media to improve hydride generation is addressed.

Thermal denaturation profiles of deoxypolynucleotide-ligand complexes: semiempirical studies.

We have semiempirically studied the thermal denaturation profiles of complexes formed between double strand polynucleotides and pure stabilizer nonspecific binding ligands. By using the McGhee model (J. D. McGhee, (1976) Biopolymers 15, 1345-1375) we have found a simple, analytical relationship between the melting temperature (Tm) and the Kh (intrinsic association constant), nh (apparent site size), and wh (cooperativity constant) values of the interaction. The validity of this approach strongly depends on the sigma value (sigma being the nucleation parameter of the DNA). Through the equation so obtained it is possible to calculate the Kh, nh, and wh values from the melting temperature of three experimental thermal denaturation profiles at different r (ligand/polynucleotide ratio) values. The method has been checked by studying the thermal denaturation profiles of daunomycin-poly(d(A-T)).poly(d(A-T)) complexes in two different salt concentrations. The results so obtained are compared with those previously described using other techniques. The applicability of the method here developed is discussed in relation with both the nature of the ligands and the value of the nucleation parameter (sigma).

Interactions of the high-mobility-group-like Ceratitis capitata C1 proteins with DNA.

We have studied the interactions of the high-mobility-group-like proteins (C1a1, C1a2 and C1b) from the fruit fly Ceratitis capitata with DNA. Nitrocellulose filter binding assays, thermal denaturation studies and spectrofluorimetry of the complexes revealed the existence of specific and nonspecific interactions. Thermal denaturation curves showed that the three proteins stabilized the DNA, thus suggesting a preferential binding to double-stranded DNA. The calculation of the thermodynamic parameters of the interactions showed that the nonspecific bindings were characterized by low association constants (Ka) with values ranging from 2.7 X 10(4) M-1 to 2.0 X 10(6) M-1. Also, the cooperativity of these interactions was relatively high (cooperativity factor, w, values ranging over 20-35), and the number of nucleotides involved was low (1-3 base pairs). On the other hand, the existence of specific interactions between C1 proteins and DNA was suggested by two facts: the retention of C. capitata [3H]DNA in nitrocellulose filters was only a low percentage of total input DNA and there was a marked size dependence of the binding (25% retention of a 40-kb DNA and only 3% retention with a DNA of 1 kb). The specific bindings had higher Ka values than the nonspecific ones, and they also were cooperative. Some differences were observed between C1b and the C1a proteins about the way they interact with C. capitata DNA.

Kinetics of conformational changes in the globular head of calf thymus H1 upon tryptic digestion.

The influence of the N- and C-terminal tails on the conformation of the globular domain of histone H1 from calf thymus has been investigated by monitoring the changes in absorption far UV circular dichroism and fluorescence spectra due to the tryptic digestion of the molecule. The values of kinetic constants obtained for the time course of the reaction followed by different above mentioned techniques seem to indicate that whereas the relaxation in the tyrosine environment occurs due to the digestion of the polar tails, the alpha-helix content increases in the earlier phase of the digestion.