Expanding the genetic spectrum of hereditary motor sensory neuropathies in Pakistan.

BACKGROUND: Hereditary motor and sensory neuropathy (HMSN) refers to a group of inherited progressive peripheral neuropathies characterized by reduced nerve conduction velocity with chronic segmental demyelination and/or axonal degeneration. HMSN is highly clinically and genetically heterogeneous with multiple inheritance patterns and phenotypic overlap with other inherited neuropathies and neurodegenerative diseases. Due to this high complexity and genetic heterogeneity, this study aimed to elucidate the genetic causes of HMSN in Pakistani families using Whole Exome Sequencing (WES) for variant identification and Sanger sequencing for validation and segregation analysis, facilitating accurate clinical diagnosis. METHODS: Families from Khyber Pakhtunkhwa with at least two members showing HMSN symptoms, who had not previously undergone genetic analysis, were included. Referrals for genetic investigations were based on clinical features suggestive of HMSN by local neurologists. WES was performed on affected individuals from each family, with Sanger sequencing used to validate and analyze the segregation of identified variants among family members. Clinical data including age of onset were assessed for variability among affected individuals, and the success rate of genetic diagnosis was compared with existing literature using proportional differences and Cohen's h. RESULTS: WES identified homozygous pathogenic variants in GDAP1 (c.310 + 4 A > G, p.?), SETX (c.5948_5949del, p.(Asn1984Profs*30), IGHMBP2 (c.1591 C > A, p.(Pro531Thr) and NARS1 (c.1633 C > T, p.(Arg545Cys) as causative for HMSN in five out of nine families, consistent with an autosomal recessive inheritance pattern. Additionally, in families with HMSN, a SETX variant was found to cause cerebellar ataxia, while a NARS1 variant was linked to intellectual disability. Based on American College of Medical Genetics and Genomics criteria, the GDAP1 variant is classified as a variant of uncertain significance, while variants in SETX and IGHMBP2 are classified as pathogenic, and the NARS1 variant is classified as likely pathogenic. The age of onset ranged from 1 to 15 years (Mean = 5.13, SD = 3.61), and a genetic diagnosis was achieved in 55.56% of families with HMSN, with small effect sizes compared to previous studies. CONCLUSIONS: This study expands the molecular genetic spectrum of HMSN and HMSN plus type neuropathies in Pakistan and facilitates accurate diagnosis, genetic counseling, and clinical management for affected families.

Investigating the genetic basis of hereditary spastic paraplegia and cerebellar Ataxia in Pakistani families.

BACKGROUND: Hereditary Spastic Paraplegias (HSPs) and Hereditary Cerebellar Ataxias (HCAs) are progressive neurodegenerative disorders encompassing a spectrum of neurogenetic conditions with significant overlaps of clinical features. Spastic ataxias are a group of conditions that have features of both cerebellar ataxia and spasticity, and these conditions are frequently clinically challenging to distinguish. Accurate genetic diagnosis is crucial but challenging, particularly in resource-limited settings. This study aims to investigate the genetic basis of HSPs and HCAs in Pakistani families. METHODS: Families from Khyber Pakhtunkhwa with at least two members showing HSP or HCA phenotypes, and who had not previously been analyzed genetically, were included. Families were referred for genetic analysis by local neurologists based on the proband's clinical features and signs of a potential genetic neurodegenerative disorder. Whole Exome Sequencing (WES) and Sanger sequencing were then used to identify and validate genetic variants, and to analyze variant segregation within families to determine inheritance patterns. The mean age of onset and standard deviation were calculated to assess variability among affected individuals, and the success rate was compared with literature reports using differences in proportions and Cohen's h. RESULTS: Pathogenic variants associated with these conditions were identified in five of eight families, segregating according to autosomal recessive inheritance. These variants included previously reported SACS c.2182 C > T, p.(Arg728*), FA2H c.159_176del, p.(Arg53_Ile58del) and SPG11 c.2146 C > T, p.(Gln716*) variants, and two previously unreported variants in SACS c.2229del, p.(Phe743Leufs*8) and ZFYVE26 c.1926_1941del, p.(Tyr643Metfs*2). Additionally, FA2H and SPG11 variants were found to have recurrent occurrences, suggesting a potential founder effect within the Pakistani population. Onset age among affected individuals ranged from 1 to 14 years (M = 6.23, SD = 3.96). The diagnostic success rate was 62.5%, with moderate effect sizes compared to previous studies. CONCLUSIONS: The findings of this study expand the genotypic and phenotypic spectrum of HSPs and HCAs in Pakistan and emphasize the importance of utilizing exome/genome sequencing for accurate diagnosis or support accurate differential diagnosis. This approach can improve genetic counseling and clinical management, addressing the challenges of diagnosing neurodegenerative disorders in resource-limited settings.

TECPR2-related hereditary sensory and autonomic neuropathy in two siblings from Palestine.

Due to the majority of currently available genome data deriving from individuals of European ancestry, the clinical interpretation of genomic variants in individuals from diverse ethnic backgrounds remains a major diagnostic challenge. Here, we investigated the genetic cause of a complex neurodevelopmental phenotype in two Palestinian siblings. Whole exome sequencing identified a homozygous missense TECPR2 variant (Chr14(GRCh38):g.102425085G>A; NM_014844.5:c.745G>A, p.(Gly249Arg)) absent in gnomAD, segregating appropriately with the inheritance pattern in the family. Variant assessment with in silico pathogenicity prediction and protein modeling tools alongside population database frequencies led to classification as a variant of uncertain significance. As pathogenic TECPR2 variants are associated with hereditary sensory and autonomic neuropathy with intellectual disability, we reviewed previously published candidate TECPR2 missense variants to clarify clinical outcomes and variant classification using current approved guidelines, classifying a number of published variants as of uncertain significance. This work highlights genomic healthcare inequalities and the challenges in interpreting rare genetic variants in populations underrepresented in genomic databases. It also improves understanding of the clinical and genetic spectrum of TECPR2-related neuropathy and contributes to addressing genomic data disparity and inequalities of the genomic architecture in Palestinian populations.

TMEM63C mutations cause mitochondrial morphology defects and underlie hereditary spastic paraplegia.

The hereditary spastic paraplegias (HSP) are among the most genetically diverse of all Mendelian disorders. They comprise a large group of neurodegenerative diseases that may be divided into 'pure HSP' in forms of the disease primarily entailing progressive lower-limb weakness and spasticity, and 'complex HSP' when these features are accompanied by other neurological (or non-neurological) clinical signs. Here, we identified biallelic variants in the transmembrane protein 63C (TMEM63C) gene, encoding a predicted osmosensitive calcium-permeable cation channel, in individuals with hereditary spastic paraplegias associated with mild intellectual disability in some, but not all cases. Biochemical and microscopy analyses revealed that TMEM63C is an endoplasmic reticulum-localized protein, which is particularly enriched at mitochondria-endoplasmic reticulum contact sites. Functional in cellula studies indicate a role for TMEM63C in regulating both endoplasmic reticulum and mitochondrial morphologies. Together, these findings identify autosomal recessive TMEM63C variants as a cause of pure and complex HSP and add to the growing evidence of a fundamental pathomolecular role of perturbed mitochondrial-endoplasmic reticulum dynamics in motor neurone degenerative diseases.

Dominant mitochondrial membrane protein-associated neurodegeneration (MPAN) variants cluster within a specific C19orf12 isoform.

Mitochondria membrane protein-associated neurodegeneration (MPAN) neurodegenerative disorder is typically associated with biallelic C19orf12 variants. Here we describe a new and review candidate previous monoallelic de novo C19orf12 variants to define loss of function mutations located in the putative non-membrane spanning C19orf12 isoform as the potential basis of monoallelic MPAN.

Datasets of whole cell and mitochondrial oxysterols derived from THP-1, SH-SY5Y and human peripheral blood mononuclear cells using targeted metabolomics.

The raw datasets of oxysterol quantifications from whole cell and mitochondrial fractions of THP-1 monocytes and macrophages, neuronal-like SH-SH5Y cells and human peripheral blood mononuclear cells are presented. Oxysterols were quantified using a new liquid chromatography-mass spectrometry (LC-MS) and multiple reaction monitoring analysis published in the article "A quantitative LC-MS/MS method for analysis of mitochondrial-specific oxysterol metabolism" in Redox Biology [1]. This method showed improved extraction efficiency and recovery of mono and dihydroxycholesterols from cellular matrix. The datasets derived from the three cell lines are included in the appendix. These datasets provide new information about the oxysterol distribution in THP-1 monocytes and macrophages, SH-SY5Y cells and peripheral blood mononuclear cells. These datasets can be used as a guide for oxysterol distribution in the three cell lines for future studies, and can used for future method optimization, and for comparison of oxysterol recovery with other analytical techniques.

A quantitative LC-MS/MS method for analysis of mitochondrial -specific oxysterol metabolism.

Oxysterols are critical regulators of inflammation and cholesterol metabolism in cells. They are oxidation products of cholesterol and may be differentially metabolised in subcellular compartments and in biological fluids. New analytical methods are needed to improve our understanding of oxysterol trafficking and the molecular interplay between the cellular compartments required to maintain cholesterol/oxysterol homeostasis. Here we describe a method for isolation of oxysterols using solid phase extraction and quantification by liquid chromatography-mass spectrometry, applied to tissue, cells and mitochondria. We analysed five monohydroxysterols; 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, 7α-hydroxycholesterol, 7 ketocholesterol and three dihydroxysterols 7α-24(S)dihydroxycholesterol, 7α-25dihydroxycholesterol, 7α-27dihydroxycholesterol by LC-MS/MS following reverse phase chromatography. Our new method, using Triton and DMSO extraction, shows improved extraction efficiency and recovery of oxysterols from cellular matrix. We validated our method by reproducibly measuring oxysterols in mouse brain tissue and showed that mice fed a high fat diet had significantly lower levels of 24S/25diOHC, 27diOHC and 7ketoOHC. We measured oxysterols in mitochondria from peripheral blood mononuclear cells and highlight the importance of rapid cell isolation to minimise effects of handling and storage conditions on oxysterol composition in clinical samples. In addition, in vitro cell culture systems, of THP-1 monocytes and neuronal-like SH-SH5Y cells, showed mitochondrial-specific oxysterol metabolism and profiles were lineage specific. In summary, we describe a robust and reproducible method validated for improved recovery, quantitative linearity and detection, reproducibility and selectivity for cellular oxysterol analysis. This method enables subcellular oxysterol metabolism to be monitored and is versatile in its application to various biological and clinical samples.

Characterization of the watercress (Nasturtium officinale R. Br.; Brassicaceae) transcriptome using RNASeq and identification of candidate genes for important phytonutrient traits linked to human health.

BACKGROUND: Consuming watercress is thought to provide health benefits as a consequence of its phytonutrient composition. However, for watercress there are currently limited genetic resources underpinning breeding efforts for either yield or phytonutritional traits. In this paper, we use RNASeq data from twelve watercress accessions to characterize the transcriptome, perform candidate gene mining and conduct differential expression analysis for two key phytonutritional traits: antioxidant (AO) capacity and glucosinolate (GLS) content. RESULTS: The watercress transcriptome was assembled to 80,800 transcripts (48,732 unigenes); 71 % of which were annotated based on orthology to Arabidopsis. Differential expression analysis comparing watercress accessions with 'high' and 'low' AO and GLS resulted in 145 and 94 differentially expressed loci for AO capacity and GLS respectively. Differentially expressed loci between high and low AO watercress were significantly enriched for genes involved in plant defence and response to stimuli, in line with the observation that AO are involved in plant stress-response. Differential expression between the high and low GLS watercress identified links to GLS regulation and also novel transcripts warranting further investigation. Additionally, we successfully identified watercress orthologs for Arabidopsis phenylpropanoid, GLS and shikimate biosynthesis pathway genes, and compiled a catalogue of polymorphic markers for future applications. CONCLUSIONS: Our work describes the first transcriptome of watercress and establishes the foundation for further molecular study by providing valuable resources, including sequence data, annotated transcripts, candidate genes and markers.