Expanded carrier screening for inherited genetic disease using exome and genome sequencing.

The goal of this study was to assess the feasibility of using exome (ES) and genome sequencing (GS) in guiding preconception genetic screening (PCGS) for couples who are planning to conceive by creating a workflow for identifying risk alleles for autosomal recessive (AR) and X-linked (XL) disorders without the constraints of a predetermined, targeted gene panel. There were several limitations and challenges related to reporting and the technical aspects of ES and GS, which are listed in the discussion. We selected 150 couples from a cohort of families (trios) enrolled in a research protocol where the goal was to define the genetic etiology of disease in an affected child. Pre-existing, de-identified parental sequencing data were analyzed to define variants that would place the couple at risk of having a child affected by an AR or XL disorder. We identified 17 families who would be selected for counseling about risk alleles. We noted that only 3 of these at-risk couples would be identified if we limited ourselves to the current ACMG-recommended expanded carrier screening gene panel. ES and GS successfully identified couples who are at risk of having a child with a rare AR or XL disorder that would have been missed by the current recommended guidelines. Current limitations of this approach include ethical concerns, difficulties in reporting results including variant calling due to the rare nature of some of the variants, determining which disorders to report, as well as technical difficulties in detecting certain variants such as repeat expansions.

Okur-Chung neurodevelopmental syndrome-linked CK2α variants have reduced kinase activity.

The Okur-Chung neurodevelopmental syndrome, or OCNDS, is a newly discovered rare neurodevelopmental disorder. It is characterized by developmental delay, intellectual disability, behavioral problems (hyperactivity, repetitive movements and social interaction deficits), hypotonia, epilepsy and language/verbalization deficits. OCNDS is linked to de novo mutations in CSNK2A1, that lead to missense or deletion/truncating variants in the encoded protein, the protein kinase CK2α. Eighteen different missense CK2α mutations have been identified to date; however, no biochemical or cell biological studies have yet been performed to clarify the functional impact of such mutations. Here, we show that 15 different missense CK2α mutations lead to varying degrees of loss of kinase activity as recombinant purified proteins and when mutants are ectopically expressed in mammalian cells. We further detect changes in the phosphoproteome of three patient-derived fibroblast lines and show that the subcellular localization of CK2α is altered for some of the OCNDS-linked variants and in patient-derived fibroblasts. Our data argue that reduced kinase activity and abnormal localization of CK2α may underlie the OCNDS phenotype.

Ascertaining the paternal lineage in crossbred calves.

Karyotyping is one among the culling parameter used for taking up culling decisions. Cytogenetic screening of breeding bulls has been recommended to screen for chromosomal abnormalities before semen production in artificial insemination. The chromosomal analysis of a Holstein Friesian crossbred bull revealed the presence of acrocentric Y-chromosome, which was further confirmed by CBG banding. The shape of the Y-chromosome determining that male line used for crossbreeding was from indigenous origin. Karyotyping is a best and reliable technique for the identification of crossbred calves born to the indigenous bulls.

A de novo splice site mutation in CASK causes FG syndrome-4 and congenital nystagmus.

Mutations in CASK cause X-linked intellectual disability, microcephaly with pontine and cerebellar hypoplasia, optic atrophy, nystagmus, feeding difficulties, GI hypomotility, and seizures. Here we present a patient with a de novo carboxyl-terminus splice site mutation in CASK (c.2521-2A>G) and clinical features of the rare FG syndrome-4 (FGS4). We provide further characterization of genotype-phenotype correlations in CASK mutations and the presentation of nystagmus and the FGS4 phenotype. There is considerable variability in clinical phenotype among patients with a CASK mutation, even among variants predicted to have similar functionality. Our patient presented with developmental delay, nystagmus, and severe gastrointestinal and gastroesophageal complications. From a cognitive and neuropsychological perspective, language skills and IQ are within normal range, although visual-motor, motor development, behavior, and working memory were impaired. The c.2521-2A>G splice mutation leads to skipping of exon 26 and a 9 base-pair deletion associated with a cryptic splice site, leading to a 28-AA and a 3-AA in-frame deletion, respectively (p.Ala841_Lys843del and p.Ala841_Glu868del). The predominant mutant transcripts contain an aberrant guanylate kinase domain and thus are predicted to degrade CASK's ability to interact with important neuronal and ocular development proteins, including FRMD7. Upregulation of CASK as well as dysregulation among a number of interactors is also evident by RNA-seq. This is the second CASK mutation known to us as cause of FGS4. © 2017 Wiley Periodicals, Inc.

New treatments for diabetic retinopathy.

Diabetic retinopathy is the major cause of vision loss in middle-aged adults. Alteration of the blood-retinal barrier (BRB) is the hallmark of diabetic retinopathy and, subsequently, hypoxia may result in retinal neovascularization. Tight control of systemic factors such as blood glucose, blood pressure and blood lipids is essential in the management of this disease. Vascular endothelial growth factor (VEGF) is one of the most important factors responsible for alteration of the BRB. The introduction of anti-VEGF agents has revolutionized the therapeutic strategies used in people with diabetic retinopathy, and the use of laser therapy has been modified. In the present article, we examine the clinical features and pathophysiology of diabetic retinopathy and review the current status of new treatment recommendations for this disease, and also explore some possible future therapies.

Plant regeneration from sorghum anther cultures and field evaluation of progeny.

Embryogenic calli were obtained from a hybrid line of Sorghum bicolor (L.) Moench anthers with mid to late uninucleate pollen cultured in N6 medium supplemented with 3.0% sucrose, 2.0 mg/1 2,4-D, 0.8% agar and incubated at 30°C, which was the optimum temperature. The regeneration of embryos was obtained from the embryogenic calli cultured in modified MS medium supplemented with 3.0% sucrose, 2.0 mg/1 BAP combined with 0.3 mg/1 IAA and 0.8% agar. A total of 248 doubled haploids and 12 haploid plants were regenerated. In a subsequent field study, the selfed progeny from anther culture (designated as the anther culture-2, [A2] generation) derived families was compared with both the F2 and the F1 for agronomic and morphological traits. Significant differences were noticed between the family means of both A2 and F2 for all the quantitative traits studied. The distinctive difference in the behavior of the A2 families in comparison with the F2 was established by within family variance, which was significant in F2 for days to 50 per cent flowering, plant height, panicle length, leaf area index, dry matter production, harvest index and grain yield and was non-significant in A2. Male sterility, one of the potentially important traits, currently exploited in the hybrid seed production of cereals, including CSH5 hybrid sorghum and the morphological traits (panicle shape, compactness, grain color, glume color and nature of the leaf sheath) segregated in the F2. Such segregation was not observed within A2 families and they bred true to their respective A1 plants, indicating the rapid attainment of homozygosity/uniformity. The present study establishes the gametophytic origin of anther culture derived families and indicates the possibility of rapid production of homozygous lines which can be used as recombinant inbreds.

Effect of X-irradiation on the size of the mutated sector in diploid and tetraploid rice.

An investigation was carried out of the effect of X-irradiation on the size of the mutated sector in three diploids (A.S.D.8, T.N. 1 and G.E.B. 24) and the autotetraploid of G.E.B 24 of Oryza sativa L., by analyzing the M2 segregation ratios of chlorophyll mutants. A total of 612 segregating M1 panicle progenies in the diploids and 284 panicle progenies in the tetraploid was studied in the M2 generation for the ratio of normal to chlorophyll mutant seedlings. Of the 573 segregating M1 panicles analysed by chi square test in the diploids, 206 showed good fit with a 3∶1 ratio, while the rest of the progenies (367) deviated significantly in both directions from the expected 3∶1 segregation. This was interpreted as the result of M1 panicles developing from both single cell and multicellular initials following X-irradiation, the former segregating for 3∶1, the latter significantly deviating from 3∶1. An assessment of fertility (as judged from the number of M2 seedlings per mutated M1 panicle), related to the distribution of chi square deviations from the 3∶1 ratio in the diploids, indicated that the M1 panicle progenies deriving from single cell initials had low fertility but those originating from multicellular initials ranged from low to high fertility. A comparison of the distribution pattern of the M2 segregation ratios of chlorophyll mutant phenotypes in the diploid and autotetraploid G.E.B. 24 indicated that, in the tetraploid, a larger sector and only one, or fewer cell initials than in the diploid species, are involved.

A comparison of mutation induction in diploid and tetraploid rice.

A comparative assessment of the frequency and spectrum of chlorophyll mutations in the M 2 and M 3 of three diploids and one tetraploid of rice after X-irradiation was made. As well as a linear relationship of the frequency of mutations with the dose of mutagen, a saturation effect was also evident. Among the three diploids, the maximum frequency of mutations was observed in T. N. 1, followed by G. E. B. 24 and A. S. D. 8. The diploid of G. E. B. 24 showed a higher frequency of mutations than the tetraploid when measured on the M1 plant basis only. The diploids showed a higher rate of mutations in the M 2 than in the M3.There was no relationship between the frequency of different types of chlorophyll mutations and the dose of X-rays. Albina, occurred in greater proportion than the other types of mutations in the diploids as well as in the tetraploid. The diploid showed a wider spectrum in the M 2, whereas the tetraploid recorded the maximum types of mutations in the M 3. Striking differences in the spectrum and the relative frequency of each type were observed among the three diploids and also between the diploid G. E. B. 24 and its tetraploid.The frequency and spectrum of induced mutations in the diploid and autotetraploid provided an insight into the genetic behaviour of the diploid and autotetraploid of G. E. B. 24, indicating that the genetics of diploidisation of the existing diploid rice may give evidence on the nature of the evolutionary pathway.