Pathology-supported genetic testing as a method for disability prevention in multiple sclerosis (MS). Part II. Insights from two MS cases.

In Part I of this Review we evaluated the scientific evidence for a Metabolic Model of multiple sclerosis (MS). Part II outlines the implementation of an adaptive pathology-supported genetic testing (PSGT) algorithm aimed at preventing/reversing disability in two illustrative MS cases, starting with a questionnaire-based risk assessment, including family history and lifestyle factors. Measurement of iron, vitamin B12, vitamin D, cholesterol and homocysteine levels identified biochemical deficits in both cases. Case 1, after following the PSGT program for 15 years, had an expanded disability status scale (EDSS) of 2.0 (no neurological sequelae) together with preserved brain volume on magnetic resonance imaging (MRI). A novel form of iron deficiency was identified in Case 1, as biochemical testing at each hospital submission due to MS symptoms showed low serum iron, ferritin and transferrin saturation, while hematological status and erythrocyte sedimentation rate measurement of systemic inflammation remained normal. Case 2 was unable to walk unaided until her EDSS improved from 6.5 to 4.0 over 12 months after implementation of the PSGT program, with amelioration of her suboptimal biochemical markers and changes to her diet and lifestyle, allowing her to regain independence. Genotype-phenotype correlation using a pathway panel of functional single nucleotide variants (SNVs) to facilitate clinical interpretation of whole exome sequencing (WES), elucidated the underlying metabolic pathways related to the biochemical deficits. A cure for MS will remain an elusive goal if separated from nutritional support required for production and maintenance of myelin, which can only be achieved by a lifelong investment in wellness.

Targeted next-generation sequencing identifies novel variants in candidate genes for Parkinson's disease in Black South African and Nigerian patients.

BACKGROUND: The prevalence of Parkinson's disease (PD) is increasing in sub-Saharan Africa, but little is known about the genetics of PD in these populations. Due to their unique ancestry and diversity, sub-Saharan African populations have the potential to reveal novel insights into the pathobiology of PD. In this study, we aimed to characterise the genetic variation in known and novel PD genes in a group of Black South African and Nigerian patients. METHODS: We recruited 33 Black South African and 14 Nigerian PD patients, and screened them for sequence variants in 751 genes using an Ion AmpliSeq™ Neurological Research panel. We used bcftools to filter variants and annovar software for the annotation. Rare variants were prioritised using MetaLR and MetaSVM prediction scores. The effect of a variant on ATP13A2's protein structure was investigated by molecular modelling. RESULTS: We identified 14,655 rare variants with a minor allele frequency ≤ 0.01, which included 2448 missense variants. Notably, no common pathogenic mutations were identified in these patients. Also, none of the known PD-associated mutations were found highlighting the need for more studies in African populations. Altogether, 54 rare variants in 42 genes were considered deleterious and were prioritized, based on MetaLR and MetaSVM scores, for follow-up studies. Protein modelling showed that the S1004R variant in ATP13A2 possibly alters the conformation of the protein. CONCLUSIONS: We identified several rare variants predicted to be deleterious in sub-Saharan Africa PD patients; however, further studies are required to determine the biological effects of these variants and their possible role in PD. Studies such as these are important to elucidate the genetic aetiology of this disorder in patients of African ancestry.

Distinct host-immune response toward species related intracellular mycobacterial killing: A transcriptomic study.

The comparison of the host immune response when challenged with pathogenic and nonpathogenic species of mycobacteria can provide answers to the unresolved question of how pathogens subvert or inhibit an effective response. We infected human monocyte derived macrophages (hMDMs) with different species of mycobacteria, in increasing order of pathogenicity, i.e. M. smegmatis, M. bovis BCG, and M. tuberculosis R179 that had been cultured in the absence of detergents. RNA was isolated post-infection and transcriptomic analysis using amplicons (Ampliseq) revealed 274 differentially expressed genes (DEGs) across three species, out of which we selected 19 DEGs for further validation. We used qRT-PCR to confirm the differential expression of 19 DEGs. We studied biological network through Ingenuity Pathway Analysis® (IPA) which revealed up-regulated pathways of the interferon and interleukin family related to the killing of M. smegmatis. Apart from interferon and interleukin family, we found one up-regulated (EIF2AK2) and two down-regulated (MT1A and TRIB3) genes as unique potential targets found by Ampliseq and qRT-PCR which may be involved in the intracellular mycobacterial killing. The roles of these genes have not previously been described in tuberculosis. Multiplex ELISA of culture supernatants showed increased host immune response toward M. smegmatis as compared to M. bovis BCG and M.tb R179. These results enhance our understanding of host immune response against M.tb infection.

Identification of an iron-responsive subtype in two children diagnosed with relapsing-remitting multiple sclerosis using whole exome sequencing.

BACKGROUND: Multiple sclerosis is a disorder related to demyelination of axons. Iron is an essential cofactor in myelin synthesis. Previously, we described two children (males of mixed ancestry) with relapsing-remitting multiple sclerosis (RRMS) where long-term remission was achieved by regular iron supplementation. A genetic defect in iron metabolism was postulated, suggesting that more advanced genetic studies could shed new light on disease pathophysiology related to iron. METHODS: Whole exome sequencing (WES) was performed to identify causal pathways. Blood tests were performed over a 10 year period to monitor the long-term effect of a supplementation regimen. Clinical wellbeing was assessed quarterly by a pediatric neurologist and regular feedback was obtained from the schoolteachers. RESULTS: WES revealed gene variants involved in iron absorption and transport, in the transmembrane protease, serine 6 (TMPRSS6) and transferrin (TF) genes; multiple genetic variants in CUBN, which encodes cubilin (a receptor involved in the absorption of vitamin B12 as well as the reabsorption of transferrin-bound iron and vitamin D in the kidneys); SLC25A37 (involved in iron transport into mitochondria) and CD163 (a scavenger receptor involved in hemorrhage resolution). Variants were also found in COQ3, involved with synthesis of Coenzyme Q10 in mitochondria. Neither of the children had the HLA-DRB1*1501 allele associated with increased genetic risk for MS, suggesting that the genetic contribution of iron-related genetic variants may be instrumental in childhood MS. In both children the RRMS has remained stable without activity over the last 10 years since initiation of nutritional supplementation and maintenance of normal iron levels, confirming the role of iron deficiency in disease pathogenesis in these patients. CONCLUSION: Our findings highlight the potential value of WES to identify heritable risk factors that could affect the reabsorption of transferrin-bound iron in the kidneys causing sustained iron loss, together with inhibition of vitamin B12 absorption and vitamin D reabsorption (CUBN) and iron transport into mitochondria (SLC25A37) as the sole site of heme synthesis. This supports a model for RRMS in children with an apparent iron-deficient biochemical subtype of MS, with oligodendrocyte cell death and impaired myelination possibly caused by deficits of energy- and antioxidant capacity in mitochondria.

IMA Genome-F 11: Draft genome sequences of Fusarium xylarioides, Teratosphaeria gauchensis and T. zuluensis and genome annotation for Ceratocystis fimbriata.

Draft genomes of the fungal species Fusarium xylarioides, Teratosphaeria gauchensis and T. zuluensis are presented. In addition an annotation of the genome of Ceratocystis fimbriata is presented. Overall these genomes provide a valuable resource for understanding the molecular processes underlying pathogenicity and potential management strategies of these economically important fungi.

Complete mitochondrial genome of the freshwater prawn Palaemon capensis (Crustacea: Palaemonidae).

The complete mitogenome of Palaemon capensis is presented here. The mitogenome is 15,925 bp in length and comprises 13 protein coding genes, 2 ribosomal subunit genes, 22 transfer RNAs, and a non-coding AT-rich region. The PCGs were used to perform a phylogenetic analysis together with other Caridea representatives with mitogenome data from GenBank, placing P. capensis sister to a clade comprising P. serenus, P. gravieri, and P. carinicauda in the family Palaemonidae.

Overrepresentation of the founder PPOX gene mutation R59W in a South African patient with severe clinical manifestation of porphyria.

A patient, who presented with abdominal pain and severe photosensitivity that resulted in scarring and mutilation of the fingers, nose and ears, was referred for biochemical assessment of porphyria and DNA screening. Although these clinical manifestations were suggestive of both acute porphyria and congenital erythropoietic porphyria, the biochemical profile was consistent with variegate porphyria (VP). Analysis of the protoporphyrinogen oxidase (PPOX) gene underlying VP resulted in the identification of the founder mutation R59W in a heterozygous state in this patient. Despite extensive mutation analysis, no other potential disease-causing genetic alterations could be detected in the PPOX gene or the uroporphyrinogen III synthase gene. Slight overrepresentation of the mutant PPOX allele was however, observed repeatedly in DNA of the proband compared to other R59W heterozygotes, including his mother who also tested positive for mutation R59W using restriction enzyme analysis and direct DNA sequencing. Confirmation of this phenomenon by real-time polymerase chain reaction analysis and microsatellite analysis, using highly informative markers flanking the PPOX gene, raised the possibility of partial homozygosity for VP in this patient. This study represents the first report of overrepresentation of mutation R59W in a patient with a severe form of VP. A homozygote for the R59W mutation has never been detected, and the severe clinical manifestation observed in our patient is consistent with the hypothesis that such a genotype will not be compatible with life.