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Faisal Gulzar,1,2 Muhammad Shoaib Akhtar,1 Rafshan Sadiq,3 Sajid Bashir,1 Sajida Jamil,2 Shahid Mahmood Baig4,5 1Department of Pharmacology, College of Pharmacy, University of Sargodha, Sargodha, Punjab, Pakistan; 2Department of Pharmacology, Faculty of Pharmacy, The University of Lahore, Lahore, Punjab, Pakistan; 3Punjab Institute for Nuclear Medicines (PINUM) Cancer Hospital, Faisalabad, Punjab, Pakistan; 4Human Molecular Genetics Laboratory, Health Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Punjab, Pakistan; 5Pakistan Institute of Engineering and Applied Sciences (PIEAS), Islamabad, Punjab, Pakistan Background: Delay in seeking health care by breast cancer patients is associated with advanced stage of disease at presentation and poor survival rates. This study aimed to identify the reasons for delayed presentation and their association with various sociodemographic variables. Methods: A total of 200 female patients with abnormal clinical findings, ie, lump or palpable mass, were consecutively invited for this study. Diagnostic delay was defined as a consultation with a health care provider more than 3 months from the appearance of the first symptoms. Sociodemographic variables, presenting symptoms, knowledge about diseases and its symptoms, time between seeking medical attention after appearance of symptoms and causes of delayed presentation were investigated. Chi-squared and logistic regression tests for significance and associations were used. Results: Among 125 women with breast cancer fulfilling the inclusion criteria, aged 24–75 years, 88.8% (n=111) presented late (≥3 months) and 59% presented with advanced stage of disease (stage III/IV). The majority (65.6%) were older than 40 years of age, 99.2% were married, 60.8% had

Autosomal recessive primary microcephaly ( MCPH) is caused by mutations in at least eight different genes involved either in cell division or DNA repair. Most mutations are identified in consanguine families from Pakistan, Iran and India. To further assess their genetic heterogeneity and mutational spectra, we have analyzed 57 consanguine Pakistani MCPH families. In 34 MCPH families, we detected linkage to five out of the eight well-characterized disease loci and identified mutations in 27 families, leaving seven families without mutations in the coding exons of the presumably underlying MCPH genes. In the MCPH cohort 23 families could not be linked to any of the known loci, pointing to remarkable locus heterogeneity. The majority of mutations were found in ASPM followed by WDR62, CENPJ, CEP152 and MCPH1. One ASPM mutation (p.Trp1326*) was found in as many as eight families suggesting a Pakistani founder mutation. One third of the families were linked to ASPM followed by WDR62 confirming previous data. We identified three novel ASPM mutations, four novel WDR62 mutations, one novel MCPH1 mutation and two novel CEP152 mutations. CEP152 mutations have not been described before in the Pakistani population. [ABSTRACT FROM AUTHOR]

Congenital heart defects (CHD) appear in almost one percent of live births. Asian countries have the highest birth prevalence of CHD in the world. Recessive genotypes may represent a CHD risk factor in Asian populations with a high degree of consanguineous marriages. Genetic analysis of consanguineous families may represent a relatively unexplored source for investigating CHD etiology. To obtain insight into the contribution of recessive genotypes in CHD we analysed a cohort of forty-nine Pakistani CHD probands, originating from consanguineous unions. The majority (82%) of patient's malformations were septal defects. We identified protein altering, rare homozygous variants (RHVs) in the patient's coding genome by whole exome sequencing. The patients had a median of seven damaging RHVs each, and our analysis revealed a total of 758 RHVs in 693 different genes. By prioritizing these genes based on variant severity, loss-of-function intolerance and specific expression in the developing heart, we identified a set of 23 candidate disease genes. These candidate genes were significantly enriched for genes known to cause heart defects in recessive mouse models (P < 2.4e-06). In addition, we found a significant enrichment of cilia genes in both the initial set of 693 genes (P < 5.4e-04) and the 23 candidate disease genes (P < 5.2e-04). Functional investigation of ADCY6 in cell- and zebrafish-models verified its role in heart development. Our results confirm a significant role for cilia genes in recessive forms of CHD and suggest important functions of cilia genes in cardiac septation., (© 2024. The Author(s).)

Erythrocyte Membrane Protein Band 4.1 Like 3 (EPB41L3: NM&#95;012307.5), also known as DAL-1, encodes the ubiquitously expressed, neuronally enriched 4.1B protein, part of the 4.1 superfamily of membrane-cytoskeleton adaptors. 4.1B plays key roles in cell spreading, migration, and cytoskeletal scaffolding that support oligodendrocyte axon adhesions essential for proper myelination. We herein describe six individuals from five unrelated families with global developmental delay, intellectual disability, seizures, hypotonia, neuroregression, and delayed myelination. Exome sequencing identified biallelic variants in EPB41L3 in all affected individuals: two nonsense (c.466C>T, p.(R156*); c.2776C>T, p.(R926*)) and three frameshift (c.666delT, p.(F222Lfs*46); c.2289dupC, p.(V764Rfs*19); c.948&#95;949delTG, p.(A317Kfs*33)). Quantitative-real time PCR and Western blot analysis in human fibroblasts harbouring EPB41L3:c.666delT, p.(F222Lfs*46) indicate ablation of EPB41L3 mRNA and 4.1B protein expression. Inhibition of the nonsense mediated decay (NMD) pathway led to an upregulation of EPB41L3:c.666delT transcripts, supporting NMD as a pathogenic mechanism. Epb41l3-deficient mouse oligodendroglia cells showed significant reduction in mRNA expression of key myelin genes, reduced branching, and increased apoptosis. Our report provides the first clinical description of an autosomal recessive disorder associated with variants in EPB41L3, which we refer to as EPB41L3-associated developmental disorder (EADD). Moreover, our functional studies substantiate the pathogenicity of EPB41L3 hypothesized loss-of-function variants., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background & Objectives: Ataxia is usually caused by cerebellar pathology or a decrease in vestibular or proprioceptive afferent input to the cerebellum. It is characterized by uncoordinated walking, truncal instability, body or head tremors, uncontrolled coordination of the hands, dysarthria, and aberrant eye movements. The objective of the current investigation was to identify the underlying genetic cause of the hereditary ataxia that affects the Pakistani population., Methods: We studied numerous consanguineous Pakistani families whose members had ataxia-related clinical symptoms to varying degrees. The families were chosen from the Punjab province, and the neurophysician conducted a clinical examination. Peripheral blood samples from both sick and healthy members of the family were taken after obtaining informed consent. Genomic DNA was used to find potential variations in probands using whole exome sequencing. The study was carried out at the University Hospital of Tübingen, Germany, and Government College University in Faisalabad, Pakistan, during 2018-2023., Results: The molecular analysis of these families identified different variants including SGCB : c.902C>T, c.668G>A, ATM : c.6196&#95;6197insGAA, SPG11 : c.5769del, SETX c.5525&#95;5533del, and ATM : c.7969A>T. A noteworthy mutation in ATM and SETX was observed among them, and its symptoms were shown to cause ataxia in these families., Conclusion: The current study broadens the mutation spectrum of several hereditary ataxia types and suggests the next generation sequencing in conjunction with clinical research for a more accurate diagnosis of overlapping phenotypes of this disorder in the Pakistani population., Competing Interests: Conflicts of Interest: None., (Copyright: © Pakistan Journal of Medical Sciences.)

Purpose: Neurodevelopmental disorders exhibit clinical and genetic heterogeneity, ergo manifest dysfunction in components of diverse cellular pathways; the precise pathomechanism for the majority remains elusive., Methods: We studied 5 affected individuals from 3 unrelated families manifesting global developmental delay, postnatal microcephaly, and hypotonia. We used exome sequencing and prioritized variants that were subsequently characterized using immunofluorescence, immunoblotting, pulldown assays, and RNA sequencing., Results: We identified biallelic variants in ZFTRAF1, encoding a protein of yet unknown function. Four affected individuals from 2 unrelated families segregated 2 homozygous frameshift variants in ZFTRAF1, whereas, in the third family, an intronic splice site variant was detected. We investigated ZFTRAF1 at the cellular level and signified it as a nucleocytoplasmic protein in different human cell lines. ZFTRAF1 was completely absent in the fibroblasts of 2 affected individuals. We also identified 110 interacting proteins enriched in mRNA processing and autophagy-related pathways. Based on profiling of autophagy markers, patient-derived fibroblasts show irregularities in the protein degradation process., Conclusion: Thus, our findings suggest that biallelic variants of ZFTRAF1 cause a severe neurodevelopmental disorder., Competing Interests: Conflict of Interest The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier Inc.)

Background: Autosomal recessive primary microcephaly (MCPH) is a rare neurodevelopmental and genetically heterogeneous disorder, characterized by small cranium size (> - 3 SD below mean) and often results in varying degree of intellectual disability. Thirty genes have been identified for the etiology of this disorder due to its clinical and genetic heterogeneity., Methods and Results: Here, we report two consanguineous Pakistani families affected with MCPH exhibiting mutation in WDR62 gene. The investigation approach involved Next Generation Sequencing (NGS) gene panel sequencing coupled with linkage analysis followed by validation of identified variants through automated Sanger sequencing and Barcode-Tagged (BT) sequencing. The molecular genetic analysis revealed one novel splice site variant (NM&#95;001083961.2(WDR62):c.1372-1del) in Family A and one known exonic variant NM&#95;001083961.2(WDR62):c.3936dup (p.Val1313Argfs*18) in Family B. Magnetic Resonance Imaging (MRI) scans were also employed to gain insights into the structural architecture of affected individuals. Neurological assessments showed the reduced gyral and sulcal patterns along with normal corpus callosum in affected individuals harboring novel variant. In silico assessments of the identified variants were conducted using different tools to confirm the pathogenicity of these variants. Through In silico analyses, both variants were identified as disease causing and protein modeling of exonic variant indicates subtle conformational alterations in prophesied protein structure., Conclusion: This study identifies a novel variant (c.1372-1del) and a recurrent pathogenic variant c.3936dup (p.Val1313Argfs*18) in the WDR62 gene among the Pakistani population, expanding the mutation spectrum for MCPH. These findings emphasize the importance of genetic counseling and awareness to reduce consanguinity and address the burden of this disorder., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Based on a limited number of reported families, biallelic CA8 variants have currently been associated with a recessive neurological disorder named, cerebellar ataxia, mental retardation, and dysequilibrium syndrome 3 (CAMRQ-3)., Objectives: We aim to comprehensively investigate CA8-related disorders (CA8-RD) by reviewing existing literature and exploring neurological, neuroradiological, and molecular observations in a cohort of newly identified patients., Methods: We analyzed the phenotype of 27 affected individuals from 14 families with biallelic CA8 variants (including data from 15 newly identified patients from eight families), ages 4 to 35 years. Clinical, genetic, and radiological assessments were performed, and zebrafish models with ca8 knockout were used for functional analysis., Results: Patients exhibited varying degrees of neurodevelopmental disorders (NDD), along with predominantly progressive cerebellar ataxia and pyramidal signs and variable bradykinesia, dystonia, and sensory impairment. Quadrupedal gait was present in only 10 of 27 patients. Progressive selective cerebellar atrophy, predominantly affecting the superior vermis, was a key diagnostic finding in all patients. Seven novel homozygous CA8 variants were identified. Zebrafish models demonstrated impaired early neurodevelopment and motor behavior on ca8 knockout., Conclusion: Our comprehensive analysis of phenotypic features indicates that CA8-RD exhibits a wide range of clinical manifestations, setting it apart from other subtypes within the category of CAMRQ. CA8-RD is characterized by cerebellar atrophy and should be recognized as part of the autosomal-recessive cerebellar ataxias associated with NDD. Notably, the presence of progressive superior vermis atrophy serves as a valuable diagnostic indicator. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society., (© 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.)

Background: Cockayne syndrome (CS) is a rare neurodegenerative disorder characterized by impaired neurological functions, cachectic dwarfism, microcephaly and photosensitivity. Complementation assays identify two groups of this disorder, CS type I (CSA) and CS type II (CSB), caused by mutations in ERCC8 and ERCC6, respectively., Objectives: This study aimed to investigate the genetic basis of a consanguineous Pakistani family with three affected individuals presenting with typical clinical symptoms of CS., Methods: We employed whole exome sequencing of the proband and then Sanger sequenced all the family members to confirm its segregation in the family. Different bioinformatics tools were used to predict pathogenicity of this variant., Results: Variants were filtered according to the pedigree structure. We identified a novel homozygous variant (c.202A>T; p.Ile68Phe) in ERCC8 gene in the proband. The variant was found to segregate in the family., Conclusions: These findings add to the genetic heterogeneity of ERCC8 and expands the mutation spectrum. Also, identification of this variant can facilitate prenatal diagnosis/genetic counselling set ups in Pakistan where this disease largely remains undiagnosed.

Background: Hereditary neurodevelopmental disorders (NDDs) are prevalent in poorly prognostic pediatric diseases, but the pathogenesis of NDDs is still unclear. Irregular myelination could be one of the possible causes of NDDs., Case Presentation: Here, whole exome sequencing was carried out for a consanguineous Pakistani family with NDDs to identify disease-associated variants. The co-segregation of candidate variants in the family was validated using Sanger sequencing. The potential impact of the gene on NDDs has been supported by conservation analysis, protein prediction, and expression analysis. A novel homozygous variant DOP1A(NM&#95;001385863.1):c.2561A>G was identified. It was concluded that the missense variant might affect the protein-protein binding sites of the critical MEC interaction region of DOP1A, and DOP1A-MON2 may cause stability deficits in Golgi-endosome protein traffic. Proteolipid protein (PLP) and myelin-associate glycoprotein (MAG) could be targets of the DOP1A-MON2 Golgi-endosome traffic complex, especially during the fetal stage and the early developmental stages. This further supports the perspective that disorganized myelinogenesis due to congenital DOP1A deficiency might cause neurodevelopmental disorders (NDDs)., Conclusion: Our case study revealed the potential pathway of myelinogenesis-relevant NDDs and identified DOP1A as a potential NDDs-relevant gene in humans., Competing Interests: Author WZ was employed by BGI Genomics, BGI-Shenzhen. Authors JS and XF were employed by BGI-Shenzhen. Author JS and XF were employed by China National GeneBank, BGI-Shenzhen. Author SH was employed by BGI-Shijiazhuang Medical Laboratory. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zhang, Tariq, Roy, Shen, Khan, Altaf Malik, He, Baig, Fang and Zhang.)

A novel patterning method achieves two-dimensional nano-patterning of metal nanofibers by depositing a platinum-cerium alloy film on a silicon wafer and inducing phase separation in an oxygen-carbon monoxide atmosphere. The resulting nano-patterned thin film, Pt#CeO 2 /Si, consists of platinum and cerium oxide with an average pattern width of 50 nm and exhibits potential as a hydrogen sensor with sensitive electrical responses to hydrogen ad/desorption. The patterning method introduced herein addresses the challenge of wavelength limitations in traditional optical lithography, offering a scalable approach for sub-50 nm patterns, which are crucial for advanced sensor and electronic applications., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Background: Adequate glucose supply is essential for brain function, therefore hypoglycemic states may lead to seizures. Since blood glucose supply for brain is buffered by liver glycogen, an impairment of liver glycogen synthesis by mutations in the liver glycogen synthase gene (GYS2) might result in a substantial neurological involvement. Here, we describe the phenotypes of affected siblings of two families harboring biallelic mutations in GYS2., Methods: Two suspected families - a multiplex Pakistani family (family A) with three affected siblings and a family of Moroccan origin (family B) with a single affected child who presented with seizures and reduced fasting blood glucose levels were genetically characterized. Whole exome sequencing (WES) was performed on the index patients, followed by Sanger sequencing-based segregation analyses on all available members of both families., Results: The variant prioritization of WES and later Sanger sequencing confirmed three mutations in the GYS2 gene (12p12.1) consistent with an autosomal recessive pattern of inheritance. A homozygous splice acceptor site variant (NM&#95;021957.3, c. 1646 -2A>G) segregated in family A. Two novel compound heterozygous variants (NM&#95;021957.3: c.343G>A; p.Val115Met and NM&#95;021957.3: c.875A>T; p.Glu292Val) were detected in family B, suggesting glycogen storage disorder. A special diet designed to avoid hypoglycemia, in addition to change of the anti-seizure medication led to reduction in seizure frequency., Conclusions: This study suggests that the seizures in patients initially diagnosed with epilepsy might be directly caused, or influenced by hypoglycemia due to pathogenic variants in the GYS2 gene., Competing Interests: Conflict of interest There is no conflict of interest by any author., (Copyright © 2023 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.)

Background: Autosomal Recessive Primary Microcephaly (MCPH) is a rare, neurodevelopmental disorder associated with mild to severe mental retardation. It is characterized by reduced cerebral cortex that ultimately leads to reduction in skull size less than - 3 S.D below the mean for normal individuals having same age and sex. Till date, 30 known loci have been reported for MCPH., Methods: In the present study, Sanger sequencing was performed followed by linkage analysis to validate the mutation in ASPM gene of the consanguineous Pakistani clans. Bioinformatics tools were also used to confirm the pathogenicity of the diseased variant in the gene. MRI scan was used to compare the brain structure of both the affected individuals (Aslam et al. in Kinnaird's 2nd International Conference on Science, Technology and Innovation, Lahore, 2023)., Results: Our study described a consanguineous family with two patients with a known ASPM (MCPH5) variant c.8508&#95;8509delGA causing a frameshift mutation in exon 18 which located in calmodulin-binding IQ domain of the ASPM protein. The salient feature of this study is that a single variant led to significantly distinct changes in the architecture of brain of both siblings which is further confirmed by MRI results. The computation analysis showed that the change in the conservation of this residue cause this variant highly pathogenic. Carrier screening and genetic counselling were also remarkable features of this study (Aslam et al. in Kinnaird's 2nd International Conference on Science, Technology and Innovation, Lahore, 2023)., Conclusion: This study explores the extraordinary influence of a single ASPM variant on divergent brain structure in consanguineous siblings and enable us to reduce the incidence of further microcephalic cases in this Pakistani family (Aslam et al. in Kinnaird's 2nd International Conference on Science, Technology and Innovation, Lahore, 2023)., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

MED27 is a subunit of the Mediator multiprotein complex, which is involved in transcriptional regulation. Biallelic MED27 variants have recently been suggested to be responsible for an autosomal recessive neurodevelopmental disorder with spasticity, cataracts and cerebellar hypoplasia. We further delineate the clinical phenotype of MED27-related disease by characterizing the clinical and radiological features of 57 affected individuals from 30 unrelated families with biallelic MED27 variants. Using exome sequencing and extensive international genetic data sharing, 39 unpublished affected individuals from 18 independent families with biallelic missense variants in MED27 have been identified (29 females, mean age at last follow-up 17 ± 12.4 years, range 0.1-45). Follow-up and hitherto unreported clinical features were obtained from the published 12 families. Brain MRI scans from 34 cases were reviewed. MED27-related disease manifests as a broad phenotypic continuum ranging from developmental and epileptic-dyskinetic encephalopathy to variable neurodevelopmental disorder with movement abnormalities. It is characterized by mild to profound global developmental delay/intellectual disability (100%), bilateral cataracts (89%), infantile hypotonia (74%), microcephaly (62%), gait ataxia (63%), dystonia (61%), variably combined with epilepsy (50%), limb spasticity (51%), facial dysmorphism (38%) and death before reaching adulthood (16%). Brain MRI revealed cerebellar atrophy (100%), white matter volume loss (76.4%), pontine hypoplasia (47.2%) and basal ganglia atrophy with signal alterations (44.4%). Previously unreported 39 affected individuals had seven homozygous pathogenic missense MED27 variants, five of which were recurrent. An emerging genotype-phenotype correlation was observed. This study provides a comprehensive clinical-radiological description of MED27-related disease, establishes genotype-phenotype and clinical-radiological correlations and suggests a differential diagnosis with syndromes of cerebello-lental neurodegeneration and other subtypes of 'neuro-MEDopathies'., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Biallelic variants in genes for seven out of eight subunits of the conserved oligomeric Golgi complex (COG) are known to cause recessive congenital disorders of glycosylation (CDG) with variable clinical manifestations. COG3 encodes a constituent subunit of the COG complex that has not been associated with disease traits in humans. Herein, we report two COG3 homozygous missense variants in four individuals from two unrelated consanguineous families that co-segregated with COG3-CDG presentations. Clinical phenotypes of affected individuals include global developmental delay, severe intellectual disability, microcephaly, epilepsy, facial dysmorphism, and variable neurological findings. Biochemical analysis of serum transferrin from one family showed the loss of a single sialic acid. Western blotting on patient-derived fibroblasts revealed reduced COG3 and COG4. Further experiments showed delayed retrograde vesicular recycling in patient cells. This report adds to the knowledge of the COG-CDG network by providing collective evidence for a COG3-CDG rare disease trait and implicating a likely pathology of the disorder as the perturbation of Golgi trafficking., (© 2023 SSIEM.)

Bardet-Biedl syndrome (BBS), is an emblematic ciliopathy hallmarked by pleiotropy, phenotype variability, and extensive genetic heterogeneity. BBS is a rare (~1/140,000 to ~1/160,000 in Europe) autosomal recessive pediatric disorder characterized by retinal degeneration, truncal obesity, polydactyly, cognitive impairment, renal dysfunction, and hypogonadism. Twenty-eight genes involved in ciliary structure or function have been implicated in BBS, and explain the molecular basis for ~75%-80% of individuals. To investigate the mutational spectrum of BBS in Romania, we ascertained a cohort of 24 individuals in 23 families. Following informed consent, we performed proband exome sequencing (ES). We detected 17 different putative disease-causing single nucleotide variants or small insertion-deletions and two pathogenic exon disruptive copy number variants in known BBS genes in 17 pedigrees. The most frequently impacted genes were BBS12 (35%), followed by BBS4, BBS7, and BBS10 (9% each) and BBS1, BBS2, and BBS5 (4% each). Homozygous BBS12 p.Arg355* variants were present in seven pedigrees of both Eastern European and Romani origin. Our data show that although the diagnostic rate of BBS in Romania is likely consistent with other worldwide cohorts (74%), we observed a unique distribution of causal BBS genes, including overrepresentation of BBS12 due to a recurrent nonsense variant, that has implications for regional diagnostics., (© 2023 The Authors. American Journal of Medical Genetics Part A published by Wiley Periodicals LLC.)

Intellectual developmental disorder with paroxysmal dyskinesia or seizures (IDDPADS, OMIM#619150) is an ultra-rare childhood-onset autosomal recessive movement disorder manifesting paroxysmal dyskinesia, global developmental delay, impaired cognition, progressive psychomotor deterioration and/or drug-refractory seizures. We investigated three consanguineous Pakistani families with six affected individuals presenting overlapping phenotypes partially consistent with the reported characteristics of IDDPADS. Whole exome sequencing identified a novel missense variant in Phosphodiesterase 2A (PDE2A): NM&#95;002599.4: c.1514T > C p.(Phe505Ser) that segregated with the disease status of individuals in these families. Retrospectively, we performed haplotype analysis that revealed a 3.16 Mb shared haplotype at 11q13.4 among three families suggesting a founder effect in this region. Moreover, we also observed abnormal mitochondrial morphology in patient fibroblasts compared to controls. Belonging to diverse age groups (13 years-60 years), patients presented paroxysmal dyskinesia, developmental delay, cognitive abnormalities, speech impairment, and drug-refractory seizures with variable onset of disease (as early as 3 months of age to 7 years). Together with the previous reports, we observed that intellectual disability, progressive psychomotor deterioration, and drug-refractory seizures are consistent outcomes of the disease. However, permanent choreodystonia showed variability. We also noticed that the later onset of paroxysmal dyskinesia manifests severe attacks in terms of duration. Being the first report from Pakistan, we add to the clinical and mutation spectrum of PDE2A-related recessive disease raising the total number of patients from six to 12 and variants from five to six. Together, with our findings, the role of PDE2A is strengthened in critical physio-neurological processes., (© 2023 The Authors. Clinical Genetics published by John Wiley & Sons Ltd.)

Craniosynostosis is characterized by the premature fusion and ossification of one or more of the sutures of the calvaria, often resulting in abnormal features of the face and the skull. In cases in which growth of the brain supersedes available space within the skull, developmental delay or cognitive impairment can occur. A complex interplay of different cell types and multiple signaling pathways are required for correct craniofacial development. In this study, we report on two siblings with craniosynostosis and a homozygous missense pathogenic variant within the IL11RA gene (c.919 T > C; p.W307R). The patients present with craniosynostosis, exophthalmos, delayed tooth eruption, mild platybasia, and a basilar invagination. The p.W307R variant is located within the arginine-tryptophan-zipper within the D3 domain of the IL-11R, a structural element known to be important for the stability of the cytokine receptor. Expression of IL-11R-W307R in cells shows impaired maturation of the IL-11R, no transport to the cell surface and intracellular retention. Accordingly, cells stably expressing IL-11R-W307R do not respond when stimulated with IL-11, arguing for a loss-of-function mutation. In summary, the IL-11R-W307R variant, reported here for the first time to our knowledge, is most likely the causative variant underlying craniosynostosis in these patients., (© 2023. Springer Nature Limited.)

Spinocerebellar disorders are a vast group of rare neurogenetic conditions, generally characterized by overlapping clinical symptoms including progressive cerebellar ataxia, spastic paraparesis, cognitive deficiencies, skeletal/muscular and ocular abnormalities. The objective of the present study is to identify the underlying genetic causes of the rare spinocerebellar disorders in the Pakistani population. Herein, nine consanguineous families presenting different spinocerebellar phenotypes have been investigated using whole exome sequencing. Sanger sequencing was performed for segregation analysis in all the available individuals of each family. The molecular analysis of these families identified six novel pathogenic/likely pathogenic variants; ZFYVE26 : c.1093del, SACS : c.1201C>T, BICD2 : c.2156A>T, ALS2 : c.2171-3T>G, ALS2 : c.3145T>A, and B4GALNT1 : c.334&#95;335dup, and three already reported pathogenic variants; FA2H : c.159&#95;176del, APTX : c.689T>G, and SETX : c.5308&#95;5311del. The clinical features of all patients in each family are concurrent with the already reported cases. Hence, the current study expands the mutation spectrum of rare spinocerebellar disorders and implies the usefulness of next-generation sequencing in combination with clinical investigation for better diagnosis of these overlapping phenotypes.

3D printing has been one of the recent buzzwords, along with Machine learning and AI. The combination of these three provides a great deal of improvisation in health education and healthcare management techniques. This paper studies various implementations of 3D printing solutions. Shortly, AI combined with 3D printing would revolutionize the healthcare industry in most areas, not just limited to human implants, pharmaceuticals, tissue engineering/regenerative medicine, education, and other evidence-based decision support systems. 3D printing is a manufacturing method in which objects are made by fusion or depositing materials such as plastic, metals, ceramics, powder, liquids, or even living cells in layers to produce a desired 3D-Object.

Intellectual disability (ID) is a condition of significant limitation of cognitive functioning and adaptive behavior, with 50% of etiology attributed to genetic predisposition. We recruited two consanguineous Pakistani families manifesting severe ID and developmental delay. The probands were subjected to whole exome sequencing (WES) and variants were further prioritized based on population frequency, predicted pathogenicity and functional relevance. The WES data analysis identified homozygous pathogenic variants in genes MBOAT7 and TRAPPC9 . The pathogenicity of the variants was supported by co-segregation analysis and in silico tool. The findings of this study expand mutation spectrum and provide additional evidence to the role of MBOAT7 and TRAPPC9 in causation of ID.

Hereditary neurological disorders (HNDs) are a clinically and genetically heterogeneous group of disorders. These disorders arise from the impaired function of the central or peripheral nervous system due to aberrant electrical impulses. More than 600 various neurological disorders, exhibiting a wide spectrum of overlapping clinical presentations depending on the organ(s) involved, have been documented. Owing to this clinical heterogeneity, diagnosing these disorders has been a challenge for both clinicians and geneticists and a large number of patients are either misdiagnosed or remain entirely undiagnosed. Contribution of genetics to neurological disorders has been recognized since long; however, the complete picture of the underlying molecular bases are under-explored. The aim of this study was to accurately diagnose 11 unrelated Pakistani families with various HNDs deploying NGS as a first step approach. Using exome sequencing and gene panel sequencing, we successfully identified disease-causing genomic variants these families. We report four novel variants, one each in, ECEL1, NALCN, TBR1 and PIGP in four of the pedigrees. In the rest of the seven families, we found five previously reported pathogenic variants in POGZ, FA2H, PLA2G6 and CYP27A1. Of these, three families segregate a homozygous 18 bp in-frame deletion of FA2H, indicating a likely founder mutation segregating in Pakistani population. Genotyping for this mutation can help low-cost population wide screening in the corresponding regions of the country. Our findings not only expand the existing repertoire of mutational spectrum underlying neurological disorders but will also help in genetic testing of individuals with HNDs in other populations., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Microcephaly (MCPH) is a developmental anomaly of the brain known by reduced cerebral cortex and underdeveloped intellectual disability without additional clinical symptoms. It is a genetically and clinically heterogenous disorder. Twenty-five genes (involved in spindle positioning, Wnt signaling, centriole biogenesis, DNA repair, microtubule dynamics, cell cycle checkpoints, and transcriptional regulation) causing MCPH have been identified so far. Pakistani population has contributed in the identification of many MCPH genes. WES of three large consanguineous families revealed three pathogenic variants of MCPH1 , CENPJ , and CASK . One novel (c.1254delT) deletion variant of MCPH1 and one known (c.18delC) deletion variant of CENPJ were identified in family 1 and 2, respectively. In addition to this, we also identified a missense variant (c.1289G>A) of CASK in males individuals in family 3. Missense mutation in the CASK gene is frequent in the boys with intellectual disability and autistic traits which are the common features that are associated with FG Syndrome 4. The study reports novel and reported mutant alleles disrupting the working of genes vital for normal brain functioning. The findings of this study enhance our understanding about the genetic architecture of primary microcephaly in our local pedigrees and add to the allelic heterogeneity of 3 known MCPH genes. The data generated will help to develop specific strategies to reduce the high incidence rate of MCPH in Pakistani population., Competing Interests: The authors declare that they have no conflict of interests., (Copyright © 2022 Niaz Muhammad Khan et al.)

Autosomal recessive limb-girdle muscular dystrophy-1 (LGMDR1) is an autosomal recessive disorder characterized by progressive weakness of the proximal limb and girdle muscles. Biallelic mutations in CAPN3 are reported frequently to cause LGMDR1. Here, we describe 11 individuals from three unrelated consanguineous families that present with typical features of LGMDR1 that include proximal muscle wasting, weakness of the upper and lower limbs, and elevated serum creatine kinase. Whole-exome sequencing identified a rare homozygous CAPN3 variant near the exon 2 splice donor site that segregates with disease in all three families. mRNA splicing studies showed partial retention of intronic sequence and subsequent introduction of a premature stop codon (NM&#95;000070.3: c.379 + 3A>G; p.Asp128Glyfs*15). Furthermore, we observe reduced CAPN3 expression in primary dermal fibroblasts derived from an affected individual, suggesting instability and/or nonsense-mediated decay of mutation-bearing mRNA. Genome-wide homozygosity mapping and single-nucleotide polymorphism analysis identified a shared haplotype and supports a possible founder effect for the CAPN3 variant. Together, our data extend the mutational spectrum of LGMDR1 and have implications for improved diagnostics for individuals of Pakistani origin., (© 2021 Wiley Periodicals LLC.)

The concept of registry-based medical research goes back to the mid of 18th century where data was collected in actual physical registers and analyzed using manual counts in a very primitive way until computing technologies took over to digitize information, to change the process all the way from data collection to data analysis. This digital age of technology can be hypothetically classified in 3 eras; the Digitization Era, the Integration Era, and finally the Futuristic, Smart Intelligence Era. This study would highlight the changes in the fundamental aspects of a medical registry under each of these digital eras.

Background & Objectives: Primary Microcephaly (MCPH) is a rare neurogenetic disease, manifesting congenitally reduced head circumference and non-progressive intellectual disability (ID). To date, twenty-eight genes with biallelic mutations have been reported for this disorder. The study aimed for molecular genetic characterization of Pakistani families segregating MCPH., Methods: We studied two unrelated consanguineous families (family A and B) presenting >2 patients with diagnostic symptoms of MCPH, born to asymptomatic parents. We employed whole-exome sequencing (WES) of probands to find putative causal mutations. The candidate variants were further confirmed and analyzed for co-segregation by Sanger sequencing of all available members of each family. This study was conducted at Government College University, Faisalabad, Pakistan, and Cologne Center for Genomics (CCG), University of Cologne, Germany; during 2017-2020., Results: We identified a novel homozygous variant c.10097&#95;10098delGA, p.(Gly3366Glufs*19) in exon 26 of ASPM gene in family A which presents with moderate intellectual disability, speech impairment, visual abnormalities, seizures, and ptyalism. Family B was found to segregate nonsense, homozygous variant c.448C>T p.(Arg150*) in CDK5RAP2 . The patients also exhibited mild to severe seizures without ptyalism that has not been previously reported in patients with mutations in the CDK5RAP2 gene., Conclusion: We report a novel mutation in ASPM and ultra-rare mutation in the CDK5RAP2 gene, both causing primary microcephaly. The study expands the mutational spectrum of the ASPM gene to 212, and also adds to the clinical spectrum of CDK5RAP2 mutations. It also demonstrated the utility of WES in the investigation and genetic diagnosis of genetically heterogeneous disorders like MCPH. These findings would aid in diagnostic and preventive strategies including carrier screening, cascade testing, and genetic counselling., Competing Interests: Conflict of interest: None., (Copyright: © Pakistan Journal of Medical Sciences.)

Mitochondrial disorders are collectively common, genetically heterogeneous disorders in both pediatric and adult populations. They are caused by molecular defects in oxidative phosphorylation, failure of essential bioenergetic supply to mitochondria, and apoptosis. Here, we present three affected individuals from a consanguineous family of Pakistani origin with variable seizures and intellectual disability. Both females display primary ovarian insufficiency (POI), while the male shows abnormal sex hormone levels. We performed whole exome sequencing and identified a recessive missense variant c.694C > T, p.Arg232Cys in TFAM that segregates with disease. TFAM (mitochondrial transcription factor A) is a component of the mitochondrial replisome machinery that maintains mtDNA transcription and replication. In primary dermal fibroblasts, we show depletion of mtDNA and significantly altered mitochondrial function and morphology. Moreover, we observed reduced nucleoid numbers with significant changes in nucleoid size or shape in fibroblasts from an affected individual compared to controls. We also investigated the effect of tfam impairment in zebrafish; homozygous tfam mutants carrying an in-frame c.141&#95;149 deletion recapitulate the mtDNA depletion and ovarian dysgenesis phenotypes observed in affected humans. Together, our genetic and functional data confirm that TFAM plays a pivotal role in gonad development and expands the repertoire of mitochondrial disease phenotypes., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Schizophrenia is a disabling neuropsychiatric disorder of adulthood onset with high heritability. Worldwide collaborations have identified an association of ~270 common loci, with small individual effects and hence weak clinical implications. The recent technological feasibility of exome sequencing enables the identification of rare variants of high penetrance that refine previous findings and improve risk assessment and prognosis. We recruited two multiplex Pakistani families, having 11 patients and 19 unaffected individuals in three generations. We performed genome-wide SNP genotyping, next-generation mate pairing and whole-exome sequencing of selected members to unveil genetic components. Candidate variants were screened in unrelated cohorts of 508 cases, 300 controls and fifteen families (with 51 affected and 47 unaffected individuals) of Pakistani origin. The structural impact of substituted residues was assessed through in silico modeling using iTASSER. In one family, we identified a rare novel microduplication (5q14.1&#95;q14.2) encompassing critical genes involved in glutamate signaling, such as CMYA5 , HOMER and RasGRF2 . The second family segregates two ultra-rare, predicted pathogenic variants in the GRIN2A (NM&#95;001134407.3: c.3505C>T, (p.R1169W) and in the NRG3 NM&#95;001010848.4: c.1951G>A, (p.E651K). These genes encode for parts of AMPA and NMDA receptors of glutamatergic neurotransmission, respectively, and the variants are predicted to compromise protein function by destabilizing their structures. The variants were absent in the aforementioned cohorts. Our findings suggest that rare, highly penetrant variants of genes involved in glutamatergic neurotransmission are contributing to the etiology of schizophrenia in these families. It also highlights that genetic investigations of multiplex, multigenerational families could be a powerful approach to identify rare genetic variants involved in complex disorders.

Purpose: We aimed to define a novel autosomal recessive neurodevelopmental disorder, characterize its clinical features, and identify the underlying genetic cause for this condition., Methods: We performed a detailed clinical characterization of 19 individuals from nine unrelated, consanguineous families with a neurodevelopmental disorder. We used genome/exome sequencing approaches, linkage and cosegregation analyses to identify disease-causing variants, and we performed three-dimensional molecular in silico analysis to predict causality of variants where applicable., Results: In all affected individuals who presented with a neurodevelopmental syndrome with progressive microcephaly, seizures, and intellectual disability we identified biallelic disease-causing variants in Protocadherin-gamma-C4 (PCDHGC4). Five variants were predicted to induce premature protein truncation leading to a loss of PCDHGC4 function. The three detected missense variants were located in extracellular cadherin (EC) domains EC5 and EC6 of PCDHGC4, and in silico analysis of the affected residues showed that two of these substitutions were predicted to influence the Ca 2+ -binding affinity, which is essential for multimerization of the protein, whereas the third missense variant directly influenced the cis-dimerization interface of PCDHGC4., Conclusion: We show that biallelic variants in PCDHGC4 are causing a novel autosomal recessive neurodevelopmental disorder and link PCDHGC4 as a member of the clustered PCDH family to a Mendelian disorder in humans., (© 2021. The Author(s).)

Primary microcephaly (MCPH) is a prenatal condition of small brain size with a varying degree of intellectual disability. It is a heterogeneous genetic disorder with 28 associated genes reported so far. Most of these genes encode centrosomal proteins. Recently, AKNA was recognized as a novel centrosomal protein that regulates neurogenesis via microtubule organization, making AKNA a likely candidate gene for MCPH. Using linkage analysis and whole-exome sequencing, we found a frameshift variant in exon 12 of AKNA (NM&#95;030767.4: c.2737delG) that cosegregates with microcephaly, mild intellectual disability and speech impairment in a consanguineous family from Pakistan. This variant is predicted to result in a protein with a truncated C-terminus (p.(Glu913Argfs*42)), which has been shown to be indispensable to AKNA's localization to the centrosome and a normal brain development. Moreover, the amino acid sequence is altered from the beginning of the second of the two PEST domains, which are rich in proline (P), glutamic acid (E), serine (S), and threonine (T) and common to rapidly degraded proteins. An impaired function of the PEST domains may affect the intracellular half-life of the protein. Our genetic findings compellingly substantiate the predicted candidacy, based on its newly ascribed functional features, of the multifaceted protein AKNA for association with MCPH.

Cardiac conduction disease (CCD), which causes altered electrical impulse propagation in the heart, is a life-threatening condition with high morbidity and mortality. It exhibits genetic and clinical heterogeneity with diverse pathomechanisms, but in most cases, it disrupts the synchronous activity of impulse-generating nodes and impulse-conduction underlying the normal heartbeat. In this study, we investigated a consanguineous Pakistani family comprised of four patients with CCD. We applied whole exome sequencing (WES) and co-segregation analysis, which identified a novel homozygous missense mutation (c.1531T>C;(p.Ser511Pro)) in the highly conserved kinase domain of the cardiac troponin I-interacting kinase (TNNI3K) encoding gene. The behaviors of mutant and native TNNI3K were compared by performing all-atom long-term molecular dynamics simulations, which revealed changes at the protein surface and in the hydrogen bond network. Furthermore, intra and intermolecular interaction analyses revealed that p.Ser511Pro causes structural variation in the ATP-binding pocket and the homodimer interface. These findings suggest p.Ser511Pro to be a pathogenic variant. Our study provides insights into how the variant perturbs the TNNI3K structure-function relationship, leading to a disease state. This is the first report of a recessive mutation in TNNI3K and the first mutation in this gene identified in the Pakistani population.

Different mutations in the Growth/Differentiation Factor 5 gene (GDF5) have been associated with varying types of skeletal dysplasia, including Grebe type chondrodysplasia (GTC), Hunter-Thompson syndrome, Du Pan Syndrome and Brachydactyly type C (BDC). Heterozygous pathogenic mutations exert milder effects, whereas homozygous mutations are known to manifest more severe phenotypes. In this study, we report a GDF5 frameshift mutation (c.404delC) segregating over six generations in an extended consanguineous Pakistani family. The family confirmed that both GTC and BDC are part of the GDF5 mutational spectrum, with severe GTC associated with homozygosity, and with a wide phenotypic variability among heterozygous carriers, ranging from unaffected non-penetrant carriers, to classical BDC and to novel unclassified types of brachydactylies., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Congenital microcephaly is the clinical presentation of significantly reduced head circumference at birth. It manifests as both non-syndromic-microcephaly primary hereditary (MCPH)-and syndromic forms and shows considerable inter- and intrafamilial variability. It has been hypothesized that additional genetic variants may be responsible for this variability, but data are sparse. We have conducted deep phenotyping and genotyping of five Pakistani multiplex families with either MCPH ( n = 3) or Seckel syndrome ( n = 2). In addition to homozygous causal variants in ASPM or CENPJ , we discovered additional heterozygous modifier variants in WDR62, CEP63, RAD50 and PCNT -genes already known to be associated with neurological disorders. MCPH patients carrying an additional heterozygous modifier variant showed more severe phenotypic features. Likewise, the phenotype of Seckel syndrome caused by a novel CENPJ variant was aggravated to microcephalic osteodysplastic primordial dwarfism type II (MOPDII) in conjunction with an additional PCNT variant. We show that the CENPJ missense variant impairs splicing and decreases protein expression. We also observed centrosome amplification errors in patient cells, which were twofold higher in MOPDII as compared to Seckel cells. Taken together, these observations advocate for consideration of additional variants in related genes for their role in modifying the expressivity of the phenotype and need to be considered in genetic counseling and risk assessment.

Neurochondrin (NCDN) is a cytoplasmatic neural protein of importance for neural growth, glutamate receptor (mGluR) signaling, and synaptic plasticity. Conditional loss of Ncdn in mice neural tissue causes depressive-like behaviors, impaired spatial learning, and epileptic seizures. We report on NCDN missense variants in six affected individuals with variable degrees of developmental delay, intellectual disability (ID), and seizures. Three siblings were found homozygous for a NCDN missense variant, whereas another three unrelated individuals carried different de novo missense variants in NCDN. We assayed the missense variants for their capability to rescue impaired neurite formation in human neuroblastoma (SH-SY5Y) cells depleted of NCDN. Overexpression of wild-type NCDN rescued the neurite-phenotype in contrast to expression of NCDN containing the variants of affected individuals. Two missense variants, associated with severe neurodevelopmental features and epilepsy, were unable to restore mGluR5-induced ERK phosphorylation. Electrophysiological analysis of SH-SY5Y cells depleted of NCDN exhibited altered membrane potential and impaired action potentials at repolarization, suggesting NCDN to be required for normal biophysical properties. Using available transcriptome data from human fetal cortex, we show that NCDN is highly expressed in maturing excitatory neurons. In combination, our data provide evidence that bi-allelic and de novo variants in NCDN cause a clinically variable form of neurodevelopmental delay and epilepsy, highlighting a critical role for NCDN in human brain development., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Incontinentia pigmenti (IP) is a rare X-linked dominant disorder. We present a case of an infant with IP who was brought to the operating room for panretinal diode photocoagulation under general anesthesia. The anesthesia team was unable to obtain intravenous access even with instruments such as a vein finder and ultrasound. Anesthesia for IP patients also poses challenges such as prevention of the oculocardiac reflex, obesity and airway management, and preemptive measures for intravenous access due to skin manifestations. Patients with IP may present with many challenges for the anesthesiologist during all phases of anesthetic management., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 International Anesthesia Research Society.)

Diamond-Blackfan Anemia (DBA) is a congenital pure red cell aplasia caused by heterozygous variants in ribosomal protein genes. The hematological features associated with DBA are highly variable and non-hematological abnormalities are common. We report herein on an affected mother and her daughter presenting with transfusion-dependent anemia. The mother showed mild physical abnormalities and entered spontaneous remission at age 13 years. Her daughter was born with occipital meningocele. Exome sequencing of DNA from the mother revealed a heterozygous novel splice site variant (NM&#95;001011.4:c.508-3T > G) in the Ribosomal Protein S7 gene (RPS7) inherited by the daughter. Functional analysis of the RPS7 variant expressed from a mini-gene construct revealed that the exon 7 acceptor splice site was replaced by a cryptic splice resulting in a transcript missing 64 bp of exon 7 (p.Val170Serfs*8). Our study confirms a pathogenic effect of a novel RPS7 variant in DBA associated with spontaneous remission in the mother and meningocele in her daughter, thus adding to the genotype-phenotype correlations in DBA.

Primary microcephaly (MCPH) is characterized by reduced brain size and intellectual disability. The exact pathophysiological mechanism underlying MCPH remains to be elucidated, but dysfunction of neuronal progenitors in the developing neocortex plays a major role. We identified a homozygous missense mutation (p.W155C) in Ribosomal RNA Processing 7 Homolog A, RRP7A, segregating with MCPH in a consanguineous family with 10 affected individuals. RRP7A is highly expressed in neural stem cells in developing human forebrain, and targeted mutation of Rrp7a leads to defects in neurogenesis and proliferation in a mouse stem cell model. RRP7A localizes to centrosomes, cilia and nucleoli, and patient-derived fibroblasts display defects in ribosomal RNA processing, primary cilia resorption, and cell cycle progression. Analysis of zebrafish embryos supported that the patient mutation in RRP7A causes reduced brain size, impaired neurogenesis and cell proliferation, and defective ribosomal RNA processing. These findings provide novel insight into human brain development and MCPH.

Background: Grisel's syndrome is a non-traumatic atlantoaxial subluxation associated with inflammatory conditions of the head and neck, which occurs primarily in children. Increased flexibility of the ligaments during inflammation is implicated in the pathogenesis of the subluxation between the axis and atlas. The potential sequelae may be severe, and early diagnosis and treatment of Grisel's syndrome can prevent tragic outcomes., Case Presentation: We present a case of torticollis in an 8-year-old child. She had a two-week history of a streptococcal throat infection. The patient was treated with several different methods of conservative care, including muscle relaxation, cervical halter traction, and Halo application. However, the torticollis persisted. The patient then required surgical correction involving cervical spine fusion. She had no complications and experienced no reoccurrence of the torticollis to date., Conclusion: Grisel's syndrome is a pathology for which conservative management is successful in most cases. Cases requiring surgical intervention are rarely documented in the literature. Our case is significant, as in spite of aggressive conservative management, the patient required surgical correction. Patients requiring surgical management of Grisel's syndrome may require additional anesthetic exposure for diagnostic interventions like magnetic resonance imaging or neck manipulations for closed reduction. We discuss the features of Grisel's syndrome and specific anesthetic management considerations for procedures such as magnetic resonance imaging, application of cervical traction, and surgical correction of torticollis.