Diversifying Data, Diagnostics, and Treatment Options for Genetic Disease

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Platform sessions are abstract driven sessions with 6 talks per session. These talks are 10 minutes in length and are cross-topical in nature to represent the broad discipline our field of genetics and genomics represent. After each talk, there will be a 5-minute Q&A with each speaker. For information on each individual session, please view the "Details" tab. 

Recorded session from the 2021 virtual meeting.

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Long-Term systemic expression and cross-correction ability of HMI-203: Investigational gene therapy candidate for mucopolysaccharidosis type II or Hunter Syndrome

Mucopolysaccharidosis type II (MPS II), or Hunter syndrome, is a rare X-linked lysosomal storage disorder caused by mutations in the iduronate-2-sulfatase (IDS) gene, resulting in loss of I2S activity leading to systemic (peripheral organs and central nervous system (CNS)) toxic lysosomal accumulation of glycosaminoglycans (GAGs). GAGs are large polysaccharides made of repeating disaccharide units responsible for providing structure and hydration to the cell. The disease results in skeletal dysplasia, joint stiffness, organomegaly, airway obstruction and, in severe cases, neurocognitive deficits. Hunter syndrome occurs in approximately 1 in 100,000 to 1 in 170,000 males, and causes significantly reduced lifespan, with the severe form leading to life expectancy of 10 to 20 years The proposed therapeutic mechanism of gene therapy candidate HMI‑203 is based on both intracellular expression and synthesis of active I2S, as well as high levels of expression and secretion of active I2S enzyme to support cross correction. Herein, we report preclinical data where a single intravenous dose of HMI-203 delivering human IDS via a rAAVHSC vector in the MPS II murine model resulted in dose-dependent and long-term transduction, IDS expression and I2S enzymatic activity in the evaluated tissues, e.g., liver, brain and serum through 52 weeks post-dose. A significant correlation was observed between liver and serum I2S activity, suggesting that the liver was likely the major contributor to the elevated levels of active I2S in the serum. The circulating I2S protein in the serum was functionally active (i.e., 90 kDa form) and cross-correction activity via a mannose-6-phosphate receptor dependent pathway was demonstrated using an in vitro competition assay. The robust and broad IDS tissue expression, along with demonstrated cross-correction significantly reduced GAG heparan sulfate (GAG-HS) to wild type (WT) levels in all evaluated organs associated with the disease, cerebrospinal fluid (CSF) and urine. In addition, lysosomal-associated membrane protein-1 (LAMP1) levels were significantly reduced to WT-like levels in the peripheral organs and CNS tissues. Of note, positive and significant correlations were observed between reduction in GAG-HS and LAMP1 levels in the CNS and brain and CSF GAG-HS levels, suggesting that CSF GAG-HS levels could be indicative of overall brain GAG and lysosomal burden levels in the clinic. Taken together, we have demonstrated that HMI-203 combines transduction and expression with the potential for cross-correction. These HMI-203 IND-enabling studies support HMI-203 as a gene therapy candidate for the treatment of MPS II. 

Kruti Patel
Homology Medicines, Inc.

Tasimelteon Safely and Effectively Improves Sleep in Smith Magenis Syndrome: results from a Double-Blind Randomized Trial Followed by an Open-Label Extension

Smith-Magenis Syndrome (SMS; OMIM #182290) is a rare genetic disorder that results from an interstitial deletion of 17p11.2 and, in rare cases, from a retinoic acid induced 1 (RAI1) gene variant (Slager et al 2003). Currently, the prevailing theory is that there is an underlying circadian pathophysiology causing sleep disturbances in these patients, as they exhibit low overall melatonin concentrations and abnormal timing of peak plasma melatonin concentrations. This abnormal inverted circadian rhythm is estimated to occur in 95% of individuals with SMS (Boone et al., 2011; Spruyt et al., 2016). To assess the efficacy of tasimelteon, a melatonin receptor agonist, to improve sleep in SMS, a 9-week, double-blind, randomized, two-period crossover study was conducted at four U.S. clinical centers. Genetically-confirmed SMS patients, aged 3 to 39, with sleep complaints participated in the study. Patients were assigned to treatment with tasimelteon or placebo in a 4-week crossover study with a one week washout between treatments. Eligible patients participated in an open label study and were followed for > 3 months. Improvement of sleep quality (DDSQ50) and total sleep time (DDTST50) on the worst 50% of nights were primary endpoints. Secondary measures included actigraphy and behavioral parameters. Over three years, fifty-two patients were screened and twenty-five patients completed the randomized portion of the study. DDSQ50 significantly improved over placebo (0.4, p=0.0139) and DDTST50 also improved (18.5 min, p=0.0556). Average sleep quality (0.3, p=0.0155) and actigraphy-based total sleep time (21.1 min, p=0.0134) improved significantly, consistent with the primary outcomes. Patients treated for ≥ 90 days in the open label study showed persistent efficacy. Adverse events were similar between placebo and tasimelteon. Tasimelteon safely and effectively improved sleep in SMS. The 17p11.2 deletion encompasses RAI1, leading to haploinsufficiency, which is considered the primary cause for most features of SMS, including dysregulation of the molecular clock via its effect on CLOCK expression. ChIP-Chip and reporter studies suggest that RAI1 binds, directly or in a complex, to the 1st intron of CLOCK, enhancing transcriptional activity, resulting in reduced CLOCK expression in SMS patient-derived cells (Williams et al 2012). The results of this study suggest that treatment with a the circadian regulator can, in part, ameliorate the circadian deficiencies caused by RAI1 haploinsufficiency, providing further evidence of a critical role for RAI1 in the regulation of circadian rhythms. 

Christos Polymeropoulos
Vanda Pharmaceuticals Inc.

Unravelling African genomes: Whole-genome sequencing of 1000 Nigerian samples spanning 50 tribal groups provides new insights into diversity and admixture

The lack of adequate representation of diverse genomes in human genomics research may limit insights that can be made about variants influencing disease susceptibility and trait variability across populations. We are helping to address this gap by performing germline whole genome sequencing of a Nigerian cohort. Nigeria represents one of the most diverse and populous regions on earth, with a population of over 200 million and over 250 unique tribal groups. We coordinate data generation in Lagos with analysis by staff around the world by leveraging cloud resources and deploying a scalable, robust, portable pipeline for alignment and variant calling. We present results from an initial round of whole-genome sequencing of ~1000 subjects from 50 tribal groups in Nigeria. We describe patterns of variation across tribes including variants of different functional classes and frequencies. We survey patterns of autozygosity across groups and compare these to 1000 Genomes samples. We highlight genetic distances between tribes and reveal evidence of admixture with European and northern African populations. We compare frequencies within our dataset to those reported in publicly available data (e.g. 1000 Genomes) for specific loci of clinical utility, e.g. those associated with drug response, highlighting noteworthy differences. Lastly, we find widespread, tribe-specific differences in allele frequency for medically-relevant variation, underscoring the importance of variant discovery and replication in non-European ancestry cohorts. Our results add to the growing body of genomic data from diverse populations, investigating understudied groups and the unique opportunities for discovery that they represent. We highlight opportunities for precision medicine, and reveal insights about variants of most clinical importance within and between human populations. 

Colm O'Dushlaine
54gene

NOTCH3 p.Arg1231Cys is Present in 1 in 92 Pakistani and Associated with Stroke

Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is an autosomal dominant Mendelian disorder characterized by early onset of migraine with aura, recurrent stroke, and dementia. Pathogenic CADASIL variants either add or remove a cysteine (Cys+/-) residue in one of 34 epidermal growth factor like repeats (EGFR) in the extra-cellular domain (ECD) or NOTCH3. Exome-wide association analysis of 4,882 stroke cases and 6,094 controls recruited in the Pakistan Genomic Resource (PGR) from Pakistan identified one such variant, p.Arg1231Cys, associated with subcortical stroke; p value 2.18e-8, odds ratio (OR) 2.97, 95% confidence interval (CI) 2.03 to 4.35, minor allele frequency (MAF) 7.1e-3. Analyses of the larger PGR cohort comprising of 80,000 participants identified additional heterozygous and homozygous carriers of this variant; call back studies of the carriers and their family members identified a high mortality in family members and a high prevalence of stroke. The Cys allele was found to disrupt a highly conserved domain (91% overall sequence identity between human and mouse), was predicted deleterious by PolyPhen2 (score 0.843 of 1), and was risk-increasing (cases MAF 0.016, controls MAF 0.0053). The p.Arg1231Cys variant was observed at a similar MAF in other South Asian populations sequenced by Regeneron Genetics Center, and present but orders of magnitude rarer in European populations. Despite rare prevalence in Europe, p.Arg1231Cys was associated with ischemic stroke in 450 thousand UK Biobank (UKB) participants; p value 8.8e-4, OR 3.38, CI 1.65 to 6.94, MAF 2.0e-4. In addition, p.Arg1231Cys was associated with multiple brain MRI phenotypes relevant to CADASIL in a 40K subset of UKB, such as mean diffusivity in the external capsule; p value 5.41e-10, OR 1.4, CI 0.96 to 1.8, MAF 2.8e-4. Consistent with CADASIL pathogenicity, a burden test limited to Cys+/- variants in the NOTCH3 ECD (including p.Arg1231Cys) strengthened associations in both Pakistan (subcortical stroke p value 1.5e-10, OR 3.39, CI 2.32 to 4.91) and UKB (ischemic stroke p value 9.3e-8, OR 3.38, CI 1.74 to 2.98). In both cohorts, p.Arg1231Cys was the most common Cys+/- variant in the NOTCH3 ECD. Taken together, these findings have major implications for precision medicine in South Asia, given that an estimated 1 in 92 (over 20 million of 1.9 billion) individuals are carriers for this variant and are at approximately 3-fold elevated risk for stroke. Our estimates suggest that around 2% of strokes in Pakistan may be attributable to NOTCH3 p.Arg1231Cys. 

Juan L Rodriguez-Flores
Regeneron Genetics Center

A high-resolution panel for uncovering repeat expansions that cause ataxias

The hereditary ataxias are a group of rare neurological diseases with similar symptoms. Many of these ataxic syndromes are caused by expansions of short tandem repeat (STR) in a number of different genes. Molecular genetic testing to accurately determine the genetic cause of known ataxias is often employed to support clinical diagnoses. Advances in therapeutic strategies (e.g., antisense oligonucleotides) to target repeat expansions underscore the importance of understanding the genetic context and sequence complexity of ataxic repeat expansions. Further highlighting the importance of molecular genetic testing, several studies have shown that repeat sequence interruptions in certain ataxia expansions play important roles in modifying the penetrance of the disease and age of onset. PCR and Southern blotting assays are currently the most employed methods in commercially available ataxia repeat expansion panels for clinical testing. Although these electrophoresis-based methods could detect repeat expansions above pathogenic threshold, accurate sizing of the repeat expansion is difficult to achieve when the length of repeat sequence is longer than a few hundred bases. Sequence interruption information is also not available with these approaches. We have recently developed an ataxia expansion panel using the PacBio No-Amp targeted sequencing approach to capture and sequence repeat expansion loci associated with fifteen ataxia diseases. The method utilizes CRISPR-Cas9 nuclease and pairs of guide RNAs to excise DNA fragments containing the repeat sequences within ataxia genes. This approach eliminates PCR amplification artifacts, amplification bias, and preserves native DNA for base modification detection. In this study, we sequenced samples with known or unknown diagnosis for ataxia with the No-Amp targeted sequencing panel utilizing PacBio highly accurate long reads - HiFi reads. The high accuracy of HiFi reads provides both certainty in sizing of the repeat expansion and repeat sequence interruption within the expansion sequences. Sequencing results demonstrate the potential of using this repeat expansion panel for eventual genetic testing. As additional ataxia, and related neurological diseases, caused by STR expansions are discovered and studied, the No-Amp targeted sequencing panel could be expanded to include additional targets. The ability to multiplex samples from different patients also makes the method a potentially cost-effective option for molecular genetic screening in the future. 

Yu-Chih Tsai
Pacific Biosciences

Deployment of clinical whole genome sequencing in support of more than 1,000 resource-limited patients: four years of the iHope Program

Patients with a suspected genetic disease are often unable to obtain a timely molecular diagnosis, and those in resource-limited locations face even greater challenges. Clinical whole genome sequencing (cWGS) shows promise as a comprehensive test which may shorten the diagnostic odyssey regardless of setting. The iHope Program is a philanthropic effort to provide cWGS to patients who are unable to obtain precision testing due to resource-limitations.
From June 2016 through June 9, 2021, 1004 individuals pursued cWGS test through the iHope Program. Cases were received from 23 partner iHope clinical sites spanning seven countries. Forty percent of cases (n=403) were received from global partners in Mexico (n=205), Peru (n=93), Italy (n=50), Democratic Republic of Congo (n=40), New Zealand (n=10), and the United Arab Emirates (n=5). Most testing was performed on duos and trios. Proband phenotypes were complex, with nervous system, head and neck, skeletal, eye, and digestive the five most frequently identified Human Phenotype Ontology root ancestor terms.
Variants were reported in 67% (n=677) of cases, of which 40.5% (n=407) conferred a definitive molecular diagnosis. Reported variants per case ranged from 0 to 5, and in 33 cases (3.3%), multiple molecular diagnoses were observed. Variants spanned 468 unique single genes. Of 1020 reported variants, a majority were nuclear SNVs or MNVs (n=693, 67.9%), followed by CNVs (n=175,17.2%), small indels (n=127, 12.5%), short tandem repeats (n=12, 1.2%), mitochondrial SNVs (n=10, 1%) and uniparental disomy (n=2, 0.1%). Copy number variants ranged in size from 3 kb to full aneuploidies. In fifteen individuals from eleven families, findings were suggestive of a structural chromosomal rearrangement.
At least ninety days after cWGS report delivery, a clinical utility survey was requested of the ordering clinician to assess effects on care and management. To date, surveys have been obtained for 581 patients (58%), representing one of the largest cWGS clinical utility datasets in a pediatric outpatient population. Data collection is ongoing, but initial analysis indicates that cWGS results prompted follow-up such as imaging, laboratory or physiological testing, referral for specialty consultation or other evaluations in 40% (233/581) of patients. In 56.6% (329/581), cWGS results contributed to counseling about prognosis, recurrence risks, reproductive screening/testing options and screening/testing recommendations or options for family members. These findings suggest that deployment of cWGS in support of resource-limited patients is tractable globally and can have a substantial impact on patient management. 

Erin Thorpe
Illumina, Inc.