Background 
Exosomes are extracellular vesicle (EV) nanocarriers responsible for delivering biologics including lipids, proteins, and nucleic acids. EVs are an attractive therapeutic weapon for delivering drugs and biologics to cells and tissues. Utilizing EVs for clinical applications is based on their low toxicity, ability to target to specific cells and tissues, and can be bioengineered to enhance their activities. Significant challenges remain before exosomes can be widely used in clinical settings. These include developing improved methods for purifying exosomes, enhancing the targeting of exosomes to diseased tissue, and boosting the efficiency of engineering exosomes for disease-specific applications. 
Exosomes need to be engineered in a more efficient way for broad therapeutic application. The lack of efficient intracellular bioengineering technologies to modify produced exosomes is a major obstacle to the widespread use of EVs for therapeutic applications. 
Advances in mammalian synthetic chromosome design and engineering offer a potential improvement in EV manufacturing. Our aim is to adopt the Artificial Chromosome Expression System (ACE System), an autonomous chromosome-based “circuit-board” designed to carry single or multiple copies of genes or DNA elements of interest for EV engineering.

Methods 
We have demonstrated that human miRNAs expressed from the ACE platform residing in a CHO-based cell line failed to process human miRNAs into CHO exosomes. This suggests that the processing of miRNAs from primary gene transcripts may be regulated in a species-specific manner. Thus, the ACE chromosome has been transferred into human HEK293 cells. Engineered miRNAs have been cloned downstream of a modified green fluorescent protein (AcGFP1) expressed from a doxycycline inducible promoter system retrofitted to contain ACE platform recombination system. 

Results 
Our results indicated that the ACE synthetic chromosome platform can be successfully engineered to express miRNAs as part of an initial doxycycline inducible AcGFP1 transcript. Furthermore, isolation of resulting EVs from the ACE engineered HEK293 cells indicated the presence of the expressed miRNAs. 

Conclusion 
From our work, we propose that the ACE synthetic platform has the potential to serve as a genetic circuit-board for modifying EVs of therapeutic value. In addition, the ACE synthetic chromosome is autonomous to the host cellular genome, i.e., non-integrating and non-mutagenic, thus would provide an increasing safety profile for using our developed synthetic chromosome technology in an ex vivo cellular therapy format. Future efforts will focus on enhancing the efficiency of our bioengineering approach for a specific disease indication.

The Engineering of Human Synthetic Chromosomes for Exosome-based Therapeutics

Distal renal tubular acidosis is an uncommon disorder in adults. The primary defect in distal RTA is
impaired distal acidification. Causes of distal RTA in adults are autoimmune diseases, hypercalciuria, and
medications. This is an interesting case of a young woman found to have distal renal tubular acidosis in
the setting of Sjogren syndrome and nephrolithiasis.

Distal Renal Tubular Acidosis in the Setting of Sjogren Syndrome and
Nephrolithiasis

Background:​
Wilson Disease, also called hepatolenticular degeneration, is a rare autosomal recessive mutation of ATP7B, which links copper to ceruloplasmin for elimination. In symptomatic patients, unbound ceruloplasmin is degraded in the bloodstream and free copper deposits in tissues, notably the basal ganglia, kidneys, and cornea. Despite dietary management and copper chelation, it is progressive and incurable, so early identification is key.​
Case Presentation:​
A 68-year-old Caucasian male presented for increasing peripheral neuropathy, myelopathy, and Parkinsonism. Labs revealed serum ceruloplasmin of 5.7 mg/dL; serum copper of 22 ug/dL; normal urine copper. Neurology suggested liver biopsy but his team was reluctant to pursue Wilson given his age and lack of Keyser-Fleischer rings. A second opinion from external neurology suggested copper supplementation, suspecting Menke. A Rowan medical geneticist suggested utilizing 23&Me data for deeper investigation of the ATP7A and ATP7B genes. Visualization of this data through genomic servers showed poor coverage, with sequence gaps and potential mutations of uncertain significance in ATP7A and ATP7B possibly contributing to Menke or Wilson; two mutations in PANK2; and none in ceruloplasmin or other copper metabolic genes. These unsatisfactory 23&Me results prompted the patient to privately obtain 40x whole genome sequencing, which demonstrated a likely pathogenic ATP7B c.3243+1G>A variant, not previously reported, predicted to disrupt a consensus splice site, and a variant of unknown significance, ATP7B c3243+5G>A, previously reported within an intron in Wilson Disease patients. His liver biopsy was fibrosis-negative; quantitative liver tissue copper testing he requested from gastroenterology showed 402 mcg/g dry weight, roughly 8 times the normal level; 250 mcg is diagnostic. He therefore self-referred to the Seattle Children’s Hospital Wilson Disease Center of Excellence, where DAT scan showed decreased radiotracer uptake in right caudate and bilateral right greater than left putamina consistent with Wilson’s Disease or Parkinson’s. He worsened slightly on trientine, but plateaued on zinc. SynOne testing was positive for Parkinson's and moderately improved on carbidopa-levodopa. ​
Discussion:​
Despite being a retired physician living near one of 18 centers for this condition nationwide, he encountered many barriers to diagnosis, followup, and management. Overreliance on pathognomonic signs may have contributed to missing this case. This was the first patient at Rowan-Virtua medical genetics identified for specific testing and follow up based on consumer genetic testing. On 7/11/23, the ATP7B c3243+5G>A allele was reclassified as likely pathogenic. Increased copper accumulation is associated with Parkinsonism.

A Case in Verdigris: Late-Life Onset of Wilson Disease Identified by Genomic Data

Background
Chromosome-level mutations play an essential role in the mechanisms behind speciation and disease. Analyzing massive genomic data to elucidate orthologous chromosome pairings between primate species is currently limited by computational constraints. Specifically, computational approaches have been limited by their inability to determine balanced chromosomal mutations rapidly, accurately, and cost-effectively. As the Alu family of transposable elements is widely conserved and prevalent (10%) among primate genomes, it is ideal for interspecies comparison and employed in our novel method. Our investigation employs unique, advanced data science techniques to classify orthologous chromosomes across primates, detect chromosomal inversions, and identify chromosomal translocations. We hypothesize that this unique approach will produce accurate results when compared to established literature, perform in less time relative to current methodologies, and be more cost-effective than its counterparts. 

Methods
We obtained and subsequently aligned Alu elements from 11 distinct primate species’ genomes on the University of California Santa Cruz’s Genome Table Browser. After computationally aggregating and statistically analyzing all primate Alu sequences, orthologous chromosome pairs were identified based on the highest frequency of homologous pairings across any two compared primate species. Using the orthologous chromosomes’ loci-specific pairing data, we detected statistically significant balanced inversions and translocations between species. 

Results
We successfully classified orthologous chromosomes between two primates with high z-scores (minimum z-score of 2.5 and maximum above 4), confirmed with previously published lists. Graphical representations of primate species, utilizing both subfamily-specific and all Alu elements, allowed for clear, statistically significant detection of chromosomal inversions, which were then validated with previously published genomic differences (10/10). Further loci-specific analysis identified every reported chromosomal translocation or change in orientation greater than 5 Mbp translocations (maximum p < 0.007 and regularly p < 1e–4). 

Conclusion
Our novel approach can be used to detect chromosomal abnormalities in primate genomes, making it a powerful tool for evolutionary and oncological research. Furthermore, it does so in a manner that is far less expensive, lengthy, and resource-intensive, while retaining the accuracy of chromosomal mutation detection. As primates serve as an important model organism for basic science research in various subspecialties of medicine, this approach could have a significant impact on numerous biomedical fields. Future investigations will explore specific data-size constraints in interspecies comparisons and the method’s applicability in detecting mutational patterns in diseases affecting humans and other primate species.

Primate Chromosomal Abnormalities Detection Through Selective Sampling Across Large-Scale Genomic Data

Single cell RNA sequencing (scRNA-seq) can detect gene expression and its dynamics in groups of cells. Its application to time-series samples can thus identify temporal gene expression programs active in different cell types. However, current scRNA-seq analysis needs improvement with regards to low gene detection, low replicate count, and inflated statistics when using cells as individual sampling data points. To address these issues, we explore whether “metacells” can be used as “replicates” through time. Toward this, we applied the use of metacells to a time-series scRNA-seq dataset from PBMCs after SARS-Cov-2 infection, which were then used in maSigPro for quadratic regression to find significantly differentially expressed genes (DEGs) over time, followed by clustering analysis of the expression velocity trends. Results demonstrated that this strategy could overcome the limitation of 1-2 replicates, as it correctly identified the known ISG15 interferon response program in almost all PBMC cell types. Its application further led to the uncovering of additional gene expression programs that potentially modulate different levels of host response after infection.

Identifying cell type transcriptome changes in COVID-19 patient PBMCs using metacells

Background
X-linked Creatine Transporter Deficiency (CTD) is caused by pathogenic variants in SLC6A8 and is characterized by intellectual disability, behavioral / developmental disorders, and seizures. The SLC6A8 protein has critical roles in synthesis and distribution of creatine throughout the body. The central nervous system is especially reliant on SLC6A8 to allow creatine to cross the blood brain barrier. Cerebral creatine deficiency disorders are found in up to 2.6% of males with intellectual disability, but few female cases have been reported. Small cohorts have suggested that supplementation of creatine, arginine, and glycine can stop disease progression in males and improve symptoms in females with CTD, though only four females on this therapy have been reported in the medical literature. We offer a case of a female with a de novo pathogenic SLC6A8 variant who significantly improved with supplementation. 

Case Presentation
At time of diagnosis, the reported patient had weight loss, mild intellectual disability, attention deficit hyperactivity disorder, generalized anxiety disorder, and intermittent explosive disorder. Magnetic resonance spectroscopy (MRS) of the brain showed reduced creatine on all acquired spectra. Based on literature review of previously treated female patients, the reported patient was started on creatine-monohydrate at 200 mg/kg/day, L-arginine at 400 mg/kg/day, and L-glycine at 150 mg/kg/day. Within three weeks of starting supplementation, her caregiver reported that she was “doing better in almost every way.” She gained 5 kg and symptom improvement was also seen through her mood: she was sleeping through the night, was more alert during the day, and was showing more appropriate behaviors. After 8 months of supplementation, MRS showed improved creatine concentrations with normalizing semi-quantitative ratios with other brain metabolites. 

Discussion
Previous small cohort studies of male patients with SLC6A8 deficiency demonstrated neither improvement nor decline on treatment. One female supplemented only with creatine showed increased aggression and worsening of self-injurious behaviors. However, two female patients in the literature supplemented with dosages of creatine, arginine, and glycine demonstrated improvement in neuropsychiatric symptoms. The reported patient provides an additional example of a female who responded to supplementation. Females are thought to better respond to supplementation because they are presumed to have at least one partially functioning copy of the SLC6A8 transporter on their second X chromosome, while more severely impacted males are more likely to have no residual transporter function and do not respond to supplementation. Clinicians treating females with CTD should consider treatment with creatine, arginine, and glycine supplementation.

Arginine, Glycine, and Creatine Supplementation Improves Symptoms in a Female with SLC6A8 Creatine Transporter Deficiency

Introduction
Renal medullary carcinoma (RMC) is an exceedingly rare and devastating malignancy that primarily affects adolescents and young adults between the ages of 11 and 39 years with sickle cell trait (SCT) or sickle cell disease. RMC is characterized by early and widespread metastases, leading to late-stage diagnosis and poor prognosis. The tumor suppressor gene SMARCB1/INI1 is inactivated in all cases, resulting in the loss of expression of INI1 protein. RMC also shares similarities with collecting duct carcinoma (CDC), including a predilection for the right kidney and the renal medulla as the epicenter, as well as aggressive clinical behavior.
Case Report
We present the case of a 27-year-old male with a medical history of asthma, HIV, and
nephrolithiasis. The patient presented with worsening right flank pain, testicular swelling, rapid weight loss, and a history of hematuria. Physical examination revealed mild tenderness in the right flank and scrotal edema. Imaging studies demonstrated infiltrative liver lesions, moderate pleural effusion, and renal abnormalities. Biopsy confirmed INI-deficient renal cell carcinoma associated with sickle cell trait. Palliative chemotherapy was initiated. RMC is an exceptionally rare malignancy that is often diagnosed at advanced stages. Loss of INI1 and its correlation with sickle cell trait are diagnostic hallmarks. Our case demonstrates an aggressive and rapidly progressive clinical presentation, including right-sided involvement and metastasis to the liver, lung pleura, cardiac pericardia, and omentum. The treatment options for RMC are limited, and the prognosis remains unfavorable.
Discussion
The discussion highlights the rarity and diagnostic challenges of RMC, which has fewer than 220 reported cases in the literature. RMC is distinguished by the presence of SCT and loss of SMARCB1/INI1 expression. It primarily affects African Americans and shows a right-sided predominance, often presenting with advanced disease and widespread metastases. Clinical manifestations include flank pain, hematuria, abdominal mass, and weight loss. Histologically, RMC displays anastomosing tubules and cords forming microcytic structures with a reticular appearance.
This case report emphasizes the importance of early recognition and prompt management of RMC. Despite its rarity, clinicians should consider RMC in young patients with sickle cell trait presenting with renal abnormalities. Timely collaboration between various medical specialties is essential for tailored therapeutic approaches, considering the aggressive nature of this malignancy. Further research is warranted to explore more effective treatment strategies and improve outcomes for this devastating disease.

INI Deficiency Unveiled: An Aggressive Case of Renal Cell Medullary Carcinoma.

Abstract Title:
SLCO1B1*5 allele is associated with atorvastatin discontinuation and adverse muscle symptoms in the context of routine care

Background
SLCO1B1 genotype is known to influence patient adherence to statin therapy, in part by increasing the risk for statin-associated musculoskeletal symptoms (SAMS). For simvastatin specifically, the SLCO1B1*5 allele has previously been associated with SAMS. For atorvastatin, prior studies assessing the impact of SLCO1B1*5 on SAMS have yielded inconclusive findings, due to insufficient power.

Methods
This study quantifies the impact of SLCO1B1*5 on atorvastatin discontinuation and SAMS in a large observational cohort using electronic medical record (EMR) data from a single health care system.

Results
In the study cohort (n = 1,627 patients exposed to atorvastatin during the course of routine clinical care), 56% (n = 912 of 1,627 patients) discontinued atorvastatin, and 18% (n = 303 of 1,627 patients) developed SAMS. A univariate analysis revealed that SLCO1B1*5 increased the likelihood that patients would stop atorvastatin during routine care (Odds Ratio 1.2, 95% confidence interval [C.I.]: 1.1 - 1.5, p = 0.04). Further analyses using a multivariate Cox proportional hazards model demonstrated that this same variant was associated with time to atorvastatin discontinuation (Hazard Ratio 1.2, C.I. 1.1 - 1.4, p = 0.004). Additional time-to-event analyses also revealed that SCLO1B1*5 was associated with SAMS (Hazard Ratio 1.4, C.I. 1.1 - 1.7, p = 0.02).

Conclusion
Atorvastatin discontinuation was associated with SAMS (Odds Ratio 1.67, p = 0.0001) in this cohort. It is plausible that SLCO1B1-informed statin therapy with the aim of reducing muscle side effects may lead to improved adherence for this important class of drugs.

Genetic Variation in SLCO1B1 Increases Risk for Atorvastatin Muscle Side Effects

Background: Familial Mediterranean fever (FMF) is a rare autosomal recessive, autoinflammatory disease that causes recurrent attacks of fever and acute inflammation in the body. Here we have a case of a clinically diagnosed FMF pt who has a monoallelic mutation of the MEFV gene. This case is unique as the specific monoallelic mutation found has not been reported to cause FMF on its own.

Case Presentation: 13 yo male presents with 2-year history of frequent fever occurring 2-4 times per week accompanied with feeling unwell. Fevers range from 100.5-101 F, resolve with Tylenol, and can occur with sore throat and abdominal pain. No chest pain, joint pain, or rashes present. Genetic testing and evaluation revealed pt is a heterozygous carrier of a pathogenic variant in the MEFV gene, c.2177T>C (p.Val726Ala). FHx of Ashkenazi Jewish ancestry and SLE on maternal side. Workup of UA, CRP/ESR, liver + BMP, and CBC were unremarkable. Diagnosed patient with FMF type I and started on colchicine trial of 0.9 mg daily which improved symptoms. Raised to 1.5 mg daily after pt tolerated trial and symptoms significantly improved. Clinical diagnosis for FMF based on Tel Hashomer Criteria and improvement of symptoms with colchicine trial.

Discussion: FMF is the most common of the known autoinflammatory diseases. The recessive gene for FMF, MEFV, is carried by Ashkenazi Jewish, Turkish, Arabian, Mediterranean, and Middle Eastern populations. This gene encodes pyrin, a protein thought to regulate inflammation through the IL-1 signaling pathway. Severe heterozygous mutations can sometimes cause FMF in a monoallelic form, but this patient’s specific mutation has previously not been found to cause FMF. This is a unique case which demonstrates a Val726Ala heterozygous mutation to cause clinically significant FMF. Also, this case provides an opportunity to demonstrate proper treatment for this patient presentation. 


A Unique Monoallelic Presentation of Familial Mediterranean Fever

Background
FMR1-related disorders is a classification of X-linked trinucleotide repeat conditions which includes Fragile X syndrome. Preimplantation genetic testing for monogenic diseases (PGT-M) is classically considered a linkage-based test for determination of the allele present in an at-risk embryo. FMR1 expansion size modifies interpretation of embryo disease risk, where >200 CGG repeats is consistent with a diagnosis of Fragile X syndrome. This study is the first collection of embryo outcomes that includes triplet repeat size determination.

Methods
Trophectoderm biopsies were sent to CooperSurgical for PGT-M for FMR1 between January 2018 and February 2021. Samples underwent linkage analysis via Karyomapping (lllumina, USA) and triplet repeat size determination. Patients with FMR1 expansions on both alleles and those with a full expansion (>200 repeats) on one allele were removed. A total of 1130 blastocysts from 318 IVF cycles underwent triplet repeat size determination and linkage analysis. 

Results
48.6% (549/1130) of embryos had the abnormal FMR1 allele. 69.4% (381/549) blastocyst samples inherited the abnormal allele but did not experience a significant expansion, with repeats remaining < 200. 202/381 of samples were euploid. 17.9% of the total study population (202/1130) was euploid, abnormal allele present, and expansion <200.

Conclusion
Embryo triplet repeat size determination increased the number of embryos that could be considered for transfer compared to linkage-only methods by 17.9%. Complete information about CGG sizes in embryos is an asset for genetic counseling about selection for embryo transfer, especially in a patient population where FMR1 carrier status confers a risk for premature ovarian insufficiency. Expanded information of embryo status may reduce the number of total IVF cycles a patient pursues if they choose to transfer a carrier embryo with a repeat size < 200.

Medical Genetics and Genomics - Observed outcomes of FMR1 PGT-M analysis with incorporation of CGG repeat expansion - Medical Genetics and Genomics

Background: Usher syndrome is the most common cause of deafness-blindness in the world. Usher syndrome type 1B (USH1B) is caused by mutations in MYO7A. Patients with USH1B experience deafness, blindness, and vestibular dysfunction. In this study, we applied dual AAV-mediated gene therapy to a mouse model of USH1B, the shaker-1 (Myo7a4626SB/4626SB) mouse. The shaker-1 mouse has a nonsense mutation in Myo7a. It is profoundly deaf throughout life, and it has significant vestibular dysfunction, similar to patients with USH1B. 

Methods: Neonatal (P0 to P5) shaker-1 (Myo7a4626SB/4626SB) mutant mice were used in this study. Due to the ~6.7 kb of the Myo7a cDNA, the dual AAV approach was used for gene delivery via the posterior semicircular canal. Auditory brainstem response (ABR) was used to assess auditory function. Vestibular evoked potential (VsEP) and circling behavior recording were used to assess vestibular function. Immunohistochemistry was used to evaluate viral transduction efficiency, sensory hair cell viability, and stereocilia morphology. 

Results: The shaker-1 mice are profoundly deaf due to significant stereocilia disorganization and rapidly progressive cochlear hair cell loss. Even though vestibular hair cells do not undergo degeneration in shaker-1 mice, they also exhibit significant vestibular dysfunction, due to stereocilia disorganization of the vestibular hair cells as well. When Myo7a cDNA was delivered to shaker-1 inner ears using the dual AAV approach, cochlear hair cell survival was improved. However, stereocilia organization and auditory function were not improved. In the vestibular system, dual AAV-mediated Myo7a delivery caused an improvement in stereocilia organization. In addition, the treated shaker-1 mice also had improved vestibular function, reflected by reduced circling behavior and improved VsEP thresholds. 

Conclusions: Our results showed that dual AAV gene therapy was able to improve the vestibular function in the shaker-1 mutant mice.

Medical Genetics and Genomics - Dual AAV-Mediated Gene Replacement Therapy Improves the Vestibular Function in a Mouse Model of USH1B - Medical Genetics and Genomics

In less than 30 years, NHS genetic diagnostic capability has progressed from low resolution chromosome analysis and sequencing of a very small number of rare diseases associated genes, to the introduction of the routine use of whole genome sequencing at scale, for the first time in any national healthcare system. The primary focus to date, has been the diagnosis of rare heritable diseases including rare cancer predisposition syndromes, with limited therapeutic implications in all but a few examples. The advent of high throughput sequencing technologies and improved bioinformatics along with the reorganisation of existing regional genetic laboratories into a smaller number of Genomic Laboratory Hubs delivering the National Genomic Test Directory supported by Genomic Medicine Service Alliances, will form the National Genomic Medicine Service in the UK. These developments along with progress in other spheres of laboratory diagnostics create exciting possibilities in the advance of truly integrated and personalised diagnostics to support the development of personalised medicine. Collaboration between academic, clinical and industrial partners is essential if these potential developments are to be maximised. This session will explore ways in which these potential opportunities can be realised in the NHS with particular emphasis on the North East and Yorkshire.

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