Oculocutaneous albinism

Oculocutaneous albinism (OCA) is a group of inherited disorders of melanin biosynthesis characterized by a generalized reduction in pigmentation of hair, skin and eyes. The prevalence of all forms of albinism varies considerably worldwide and has been estimated at approximately 1/17,000, suggesting that about 1 in 70 people carry a gene for OCA. The clinical spectrum of OCA ranges, with OCA1A being the most severe type with a complete lack of melanin production throughout life, while the milder forms OCA1B, OCA2, OCA3 and OCA4 show some pigment accumulation over time. Clinical manifestations include various degrees of congenital nystagmus, iris hypopigmentation and translucency, reduced pigmentation of the retinal pigment epithelium, foveal hypoplasia, reduced visual acuity usually (20/60 to 20/400) and refractive errors, color vision impairment and prominent photophobia. Misrouting of the optic nerves is a characteristic finding, resulting in strabismus and reduced stereoscopic vision. The degree of skin and hair hypopigmentation varies with the type of OCA. The incidence of skin cancer may be increased. All four types of OCA are inherited as autosomal recessive disorders. At least four genes are responsible for the different types of the disease (TYR, OCA2, TYRP1 and MATP). Diagnosis is based on clinical findings of hypopigmentation of the skin and hair, in addition to the characteristic ocular symptoms. Due to the clinical overlap between the OCA forms, molecular diagnosis is necessary to establish the gene defect and OCA subtype. Molecular genetic testing of TYR and OCA2 is available on a clinical basis, while, at present, analysis of TYRP1 and MATP is on research basis only. Differential diagnosis includes ocular albinism, Hermansky-Pudlak syndrome, Chediak-Higashi syndrome, Griscelli syndrome, and Waardenburg syndrome type II. Carrier detection and prenatal diagnosis are possible when the disease causing mutations have been identified in the family. Glasses (possibly bifocals) and dark glasses or photocromic lenses may offer sufficient help for reduced visual activity and photophobia. Correction of strabismus and nystagmus is necessary and sunscreens are recommended. Regular skin checks for early detection of skin cancer should be offered. Persons with OCA have normal lifespan, development, intelligence and fertility

Human CCS gene: genomic organization and exclusion as a candidate for amyotrophic lateral sclerosis (ALS)

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a progressive lethal disorder of large motor neurons of the spinal cord and brain. In approximately 20% of the familial and 2% of sporadic cases the disease is due to a defect in the gene encoding the cytosolic antioxidant enzyme Cu, Zn-superoxide dismutase (SOD1). The underlying molecular defect is known only in a very small portion of the remaining cases and therefore involvement of other genes is likely. As SOD1 receives copper, essential for its normal function, by the copper chaperone, CCS (Copper Chaperone for SOD), we considered CCS as a potential candidate gene for ALS. RESULTS: We have characterized the genomic organization of CCS and determined exon-intron boundaries. The 823 bp coding region of the CCS is organized in 8 exons. We have evaluated involvement of the CCS in ALS by sequencing the entire coding region for mutations in 20 sporadic ALS patients. CONCLUSIONS: No causative mutations for the ALS have been detected in the CCS gene in 20 sporadic ALS patients analyzed, but an intragenic single nucleotide polymorphism has been identified

Elimination of the Warburg effect in Chinese hamster ovary (CHO) cells improves cell phenotype as a protein production platform

Lactate is a common metabolite and is central to many important processes. One of its more prominent roles is in the Warburg effect, in which cancer cells exhibit high rates of glycolytic flux followed by secretion of lactate, even in the presence of oxygen. This fermentation of pyruvate to lactate via lactate dehydrogenase (Ldh) accompanies increased proliferation of cancer cells and several other types of rapidly proliferating cell types in immune cell activation and embryonic development. Aerobic glycolysis is also prominent in biotherapeutic protein production, where mammalian production cells often secrete high levels of lactate. The accumulation of lactate is deleterious for cell growth, viability, product formation, and quality, both directly via acidification of the media and indirectly through base addition to control culture pH. Despite a clear genetic target, efforts to eliminate lactate secretion via knockout of Ldh(s) in mammalian cells have been unsuccessful, pointing to the essentiality of Ldh mediated NAD regeneration. A wide variety of approaches have been utilized to limit lactate accumulation in culture, including knockdown or inhibition of Ldh, replacement of glucose with alternate sugars, controlled feeding strategies, and many others, however none have proven successful in eliminating the Warburg effect. We report the elimination of the Warburg effect in a CHO cell line by using CRISPR/Cas9-based engineering to simultaneously knockout enzymes responsible for lactate production and ancillary regulators. The resulting cell lines remain proliferative while consuming significantly less glucose and can be used to generate protein producing lines using standard industrial processes. In a pH-controlled fedbatch process, the Warburg null cells require minimal base addition to maintain a stable pH, allowing an extended growth phase. The knockout strategy was also successfully applied to a CHO cell line producing Rituximab, again resulting in a prolonged growth phase. Additionally, protein production was maintained, while product quality was improved with increased glycan galactosylation. Thus, CHO cells without the capacity of Warburg metabolism may be useful for engineering production cell lines with enhanced bioproduction traits

Subtelomeric study of 132 patients with mental retardation reveals 9 chromosomal anomalies and contributes to the delineation of submicroscopic deletions of 1pter, 2qter, 4pter, 5qter and 9qter

BACKGROUND: Cryptic chromosome imbalances are increasingly acknowledged as a cause for mental retardation and learning disability. New phenotypes associated with specific rearrangements are also being recognized. Techniques for screening for subtelomeric rearrangements are commercially available, allowing the implementation in a diagnostic service laboratory. We report the diagnostic yield in a series of 132 subjects with mental retardation, and the associated clinical phenotypes. METHODS: We applied commercially available subtelomeric fluorescence in situ hybridization (FISH). All patients referred for subtelomeric screening in a 5-year period were reviewed and abnormal cases were further characterized clinically and if possible molecularly. RESULTS: We identified nine chromosomal rearrangements (two of which were in sisters) corresponding to a diagnostic yield of approx. 7%. All had dysmorphic features. Five had imbalances leading to recognizable phenotypes. CONCLUSION: Subtelomeric screening is a useful adjunct to conventional cytogenetic analyses, and should be considered in mentally retarded subjects with dysmorphic features and unknown cause

Deletions and rearrangements of the H19/IGF2 enhancer region in patients with Silver-Russell syndrome and growth retardation

Silver–Russell syndrome (SRS) is characterised by prenatal and postnatal growth retardation, dysmorphic facial features, and body asymmetry. In 35–60% of SRS cases the paternally methylated imprinting control region (ICR) upstream of the H19 gene (H19-ICR) is hypomethylated, leading to downregulation of IGF2 and bi-allelic expression of H19. H19 and IGF2 are reciprocally imprinted genes on chromosome 11p15. The expression is regulated by the imprinted methylation of the ICR, which modulates the transcription of H19 and IGF2 facilitated by enhancers downstream of H19. A promoter element of IGF2, IGF2P0, is differentially methylated equivalently to the H19-ICR, though in a small number of SRS cases this association is disrupted—that is, hypomethylation affects either H19-ICR or IGF2P0. Three pedigrees associated with hypomethylation of IGF2P0 in the probands are presented here, two with paternally derived deletions, and one with a balanced translocation of inferred paternal origin. They all have a breakpoint within the H19/IGF2 enhancer region. One proband has severe growth retardation, the others have SRS. This is the first report of paternally derived structural chromosomal mutations in 11p15 causing SRS. These cases define a novel aetiology of the growth retardation in SRS, namely, dissociation of IGF2 from its enhancers. <br/

The effect of casein glycomacropeptide versus free synthetic amino acids for early treatment of phenylketonuria in a mice model

INTRODUCTION: Management of phenylketonuria (PKU) is mainly achieved through dietary control with limited intake of phenylalanine (Phe) from food, supplemented with low protein (LP) food and a mixture of free synthetic (FS) amino acids (AA) (FSAA). Casein glycomacropeptide (CGMP) is a natural peptide released in whey during cheese making by the action of the enzyme chymosin. Because CGMP in its pure form does not contain Phe, it is nutritionally suitable as a supplement in the diet for PKU when enriched with specific AAs. Lacprodan(®) CGMP-20 (= CGMP) used in this study contained only trace amounts of Phe due to minor presence of other proteins/peptides. OBJECTIVE: The aims were to address the following questions in a classical PKU mouse model: Study 1, off diet: Can pure CGMP or CGMP supplemented with Large Neutral Amino Acids (LNAA) as a supplement to normal diet significantly lower the content of Phe in the brain compared to a control group on normal diet, and does supplementation of selected LNAA results in significant lower brain Phe level?. Study 2, on diet: Does a combination of CGMP, essential (non-Phe) EAAs and LP diet, provide similar plasma and brain Phe levels, growth and behavioral skills as a formula which alone consist of FSAA, with a similar composition?. MATERIAL AND METHODS: 45 female mice homozygous for the Pah(enu2) mutation were treated for 12 weeks in five different groups; G1(N-CGMP), fed on Normal (N) casein diet (75%) in combination with CGMP (25%); G2 (N-CGMP-LNAA), fed on Normal (N) casein diet (75%) in combination with CGMP (19,7%) and selected LNAA (5,3% Leu, Tyr and Trp); G3 (N), fed on normal casein diet (100%); G4 (CGMP-EAA-LP), fed on CGMP (70,4%) in combination with essential AA (19,6%) and LP diet; G5 (FSAA-LP), fed on FSAA (100%) and LP diet. The following parameters were measured during the treatment period: Plasma AA profiles including Phe and Tyr, growth, food and water intake and number of teeth cut. At the end of the treatment period, a body scan (fat and lean body mass) and a behavioral test (Barnes Maze) were performed. Finally, the brains were examined for content of Phe, Tyr, Trp, dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT) and 5-hydroxyindole-acetic acid (5-HIAA), and the bone density and bone mineral content were determined by dual-energy x-ray absorptiometry. RESULTS: Study 1: Mice off diet supplemented with CGMP (G1 (N-CGMP)) or supplemented with CGMP in combination with LNAA (G2 (N-CGMP-LNAA)) had significantly lower Phe in plasma and in the brain compared to mice fed only casein (G3 (N)). Extra LNAA (Tyr, Trp and Leu) to CGMP did not have any significant impact on Phe levels in the plasma and brain, but an increase in serotonin was measured in the brain of G2 mice compared to G1. Study 2: PKU mice fed with mixture of CGMP and EAA as supplement to LP diet (G4 (CGMP-EAA-LP)) demonstrated lower plasma-Phe levels but similar brain- Phe levels and growth as mice fed on an almost identical combination of FSAA (G5 (FSAA-LP)). CONCLUSION: CGMP can be a relevant supplement for the treatment of PKU

Elimination of the “Essential” Warburg effect in CHO cells through a multiplex genome engineering strategy

The Warburg effect has posed a constant challenge in mammalian bioprocessing since the field began. Indeed, the predisposition of mammalian cells to secrete large quantities of lactic acid through the Warburg effect leads to premature cell death, reduced product yields, and often lower quality products. Thus, over the past decades, numerous innovations in the mammalian cell culture field have focused on mitigating lactate secretion, including through media optimization, feeding control, chemical inhibition, etc. Despite extensive efforts from many researchers, complete elimination of lactic acid production has not yet been obtained. Specifically, several independent efforts to knock out lactate dehydrogenase (the enzyme responsible for producing lactic acid from pyruvate) have been unsuccessful, as it has seemed essential for immortalized cell growth. Here I present our work in which we discovered a panel of genes involved in a genetic feedback circuit that controls lactic acid secretion in mammalian cells. Knocking out individual genes in serial was unsuccessful since LdhA and other targets are essential for CHO cell growth. However, we knocked out these genes simultaneously and overcame the “essentiality” of these genes, leading to the successful elimination of lactic acid secretion in Chinese hamster ovary cells. Since many hypotheses have been proposed regarding the essentiality of lactic acid secretion for rapid cell proliferation in cancer, immune cell activation, and embryonic development, we were interested to study how the complete elimination of the Warburg effect impacts CHO cells. Surprisingly, the cells show improved metabolic and growth phenotypes, despite the elimination of this fundamental metabolic activity. To understand how immortalized mammalian cells can cope without this seemingly essential metabolic process, we conducted a comprehensive analysis of these cell lines using time-course RNA-Seq, metabolomics, and analysis with a genome-scale metabolic network model developed for Chinese hamster ovary cells1. We further characterized its impact on recombinant drug production yields and quality. Thus, through a multiplex metabolic engineering effort and comprehensive systems biology analysis, we have been able to engineer out a leading challenge in protein biotherapeutic development and begin to understand now a cell can survive without a seemingly essential process. 1. Hefzi, H. et al. A Consensus Genome-scale Reconstruction of Chinese Hamster Ovary Cell Metabolism. Cell Syst. 3, 434–443.e8 (2016)