Diagnostic utility and reporting recommendations for clinical DNA methylation episignature testing in genetically undiagnosed rare diseases.

PURPOSE: This study aims to assess the diagnostic utility and provide reporting recommendations for clinical DNA methylation episignature testing based on the cohort of patients tested through the EpiSign Clinical Testing Network. METHODS: The EpiSign assay utilized unsupervised clustering techniques and a support vector machine-based classification algorithm to compare each patient's genome-wide DNA methylation profile with the EpiSign Knowledge Database, yielding the result that was reported. An international working group, representing distinct EpiSign Clinical Testing Network health jurisdictions, collaborated to establish recommendations for interpretation and reporting of episignature testing. RESULTS: Among 2399 cases analyzed, 1667 cases underwent a comprehensive screen of validated episignatures, imprinting, and promoter regions, resulting in 18.7% (312/1667) positive reports. The remaining 732 referrals underwent targeted episignature analysis for assessment of sequence or copy-number variants (CNVs) of uncertain significance or for assessment of clinical diagnoses without confirmed molecular findings, and 32.4% (237/732) were positive. Cases with detailed clinical information were highlighted to describe various utility scenarios for episignature testing. CONCLUSION: Clinical DNA methylation testing including episignatures, imprinting, and promoter analysis provided by an integrated network of clinical laboratories enables test standardization and demonstrates significant diagnostic yield and clinical utility beyond DNA sequence analysis in rare diseases.

Improving newborn screening test performance for metachromatic leukodystrophy: Recommendation from a pre-pilot study that identified a late-infantile case for treatment.

Metachromatic leukodystrophy (MLD) is a devastating rare neurodegenerative disease. Typically, loss of motor and cognitive skills precedes early death. The disease is characterised by deficient lysosomal arylsulphatase A (ARSA) activity and an accumulation of undegraded sulphatide due to pathogenic variants in the ARSA gene. Atidarsagene autotemcel (arsa-cel), an ex vivo haematopoietic stem cell gene therapy was approved for use in the UK in 2021 to treat early-onset forms of pre- or early-symptomatic MLD. Optimal outcomes require early diagnosis, but in the absence of family history this is difficult to achieve without newborn screening (NBS). A pre-pilot MLD NBS study was conducted as a feasibility study in Manchester UK using a two-tiered screening test algorithm. Pre-established cutoff values (COV) for the first-tier C16:0 sulphatide (C16:0-S) and the second-tier ARSA tests were evaluated. Before the pre-pilot study, initial test validation using non­neonatal diagnostic bloodspots demonstrated ARSA pseudodeficiency status was associated with normal C16:0-S results for age (n = 43) and hence not expected to cause false positive results in this first-tier test. Instability of ARSA in bloodspot required transfer of NBS bloodspots from ambient temperature to -20°C storage within 7-8 days after heel prick, the earliest possible in this UK pre-pilot study. Eleven of 3687 de-identified NBS samples in the pre-pilot were positive for C16:0-S based on the pre-established COV of ≥170 nmol/l or ≥ 1.8 multiples of median (MoM). All 11 samples were subsequently tested negative determined by the ARSA COV of <20% mean of negative controls. However, two of 20 NBS samples from MLD patients would be missed by this C16:0-S COV. A further suspected false negative case that displayed 4% mean ARSA activity by single ARSA analysis for the initial test validation was confirmed by genotyping of this NBS bloodspot, a severe late infantile MLD phenotype was predicted. This led to urgent assessment of this child by authority approval and timely commencement of arsa-cel gene therapy at 11 months old. Secondary C16:0-S analysis of this NBS bloodspot was 150 nmol/l or 1.67 MoM. This was the lowest result reported thus far, a new COV of 1.65 MoM is recommended for future pilot studies. Furthermore, preliminary data of this study showed C16:1-OH sulphatide is more specific for MLD than C16:0-S. In conclusion, this pre-pilot study adds to the international evidence that recommends newborn screening for MLD, making it possible for patients to benefit fully from treatment through early diagnosis.

Personalised virtual gene panels reduce interpretation workload and maintain diagnostic rates of proband-only clinical exome sequencing for rare disorders.

PURPOSE: The increased adoption of genomic strategies in the clinic makes it imperative for diagnostic laboratories to improve the efficiency of variant interpretation. Clinical exome sequencing (CES) is becoming a valuable diagnostic tool, capable of meeting the diagnostic demand imposed by the vast array of different rare monogenic disorders. We have assessed a clinician-led and phenotype-based approach for virtual gene panel generation for analysis of targeted CES in patients with rare disease in a single institution. METHODS: Retrospective survey of 400 consecutive cases presumed by clinicians to have rare monogenic disorders, referred on singleton basis for targeted CES. We evaluated diagnostic yield and variant workload to characterise the usefulness of a clinician-led approach for generation of virtual gene panels that can incorporate up to three different phenotype-driven gene selection methods. RESULTS: Abnormalities of the nervous system (54.5%), including intellectual disability, head and neck (19%), skeletal system (16%), ear (15%) and eye (15%) were the most common clinical features reported in referrals. Combined phenotype-driven strategies for virtual gene panel generation were used in 57% of cases. On average, 7.3 variants (median=5) per case were retained for clinical interpretation. The overall diagnostic rate of proband-only CES using personalised phenotype-driven virtual gene panels was 24%. CONCLUSIONS: Our results show that personalised virtual gene panels are a cost-effective approach for variant analysis of CES, maintaining diagnostic yield and optimising the use of resources for clinical genomic sequencing in the clinic.

Truncating and zinc-finger variants in GLI2 are associated with hypopituitarism.

Variants in transcription factor GLI2 have been associated with hypopituitarism and structural brain abnormalities, occasionally including holoprosencephaly (HPE). Substantial phenotypic variability and nonpenetrance have been described, posing difficulties in the counseling of affected families. We present three individuals with novel likely pathogenic GLI2 variants, two with truncating and one with a de novo missense variant p.(Ser548Leu), and review the literature for comprehensive phenotypic descriptions of individuals with confirmed pathogenic (a) intragenic GLI2 variants and (b) chromosome 2q14.2 deletions encompassing only GLI2. We show that most of the 31 missense variants previously reported as pathogenic are likely benign or, at most, low-risk variants. Four Zn-finger variants: p.(Arg479Gly), p.(Arg516Pro), p.(Gly518Lys), and p.(Tyr575His) were classified as likely pathogenic, and three other variants as possibly pathogenic: p.(Pro253Ser), p.(Ala593Val), and p.(Pro1243Leu). We analyze the phenotypic descriptions of 60 individuals with pathogenic GLI2 variants and evidence a morbidity spectrum that includes hypopituitarism (58%), HPE (6%) or other brain structure abnormalities (15%), orofacial clefting (17%) and dysmorphic facial features (35%). We establish that truncating and Zn-finger variants in GLI2 are associated with a high risk of hypopituitarism, and that a solitary median maxillary central incisor is part of the GLI2-related phenotypic variability. The most prevalent phenotypic feature is post-axial polydactyly (65%) which is also the mildest phenotypic expression of the condition, reported in many parents of individuals with systemic findings. Our approach clarifies clinical risks and the important messages to discuss in counseling for a pathogenic GLI2 variant.

A Nonadaptive Combinatorial Group Testing Strategy to Facilitate Health Care Worker Screening during the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) Outbreak.

Routine testing for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in health care workers (HCWs) is critical. Group testing strategies to increase capacity facilitate mass population testing but do not prioritize turnaround time, an important consideration for HCW screening. We propose a nonadaptive combinatorial (NAC) group testing strategy to increase throughput while facilitating rapid turnaround. NAC matrices were constructed for sample sizes of 700, 350, and 250. Matrix performance was tested by simulation under different SARS-CoV-2 prevalence scenarios of 0.1% to 10%. NAC matrices were compared versus Dorfman sequential (DS) group testing approaches. NAC matrices performed well at low prevalence levels, with an average of 97% of samples resolved after a single round of testing via the n = 700 matrix at a prevalence of 1%. In simulations of low to medium (0.1% to 3%) prevalence, all NAC matrices were superior to the DS strategy, measured by fewer repeated tests required. At very high prevalence levels (10%), the DS matrix was marginally superior, although both group testing approaches performed poorly at high prevalence levels. This strategy maximizes the proportion of samples resolved after a single round of testing, allowing prompt return of results to HCWs. This methodology may allow laboratories to adapt their testing scheme based on required throughput and the current population prevalence, facilitating a data-driven testing strategy.

Genomic and healthcare dynamics of nosocomial SARS-CoV-2 transmission.

Understanding the effectiveness of infection control methods in reducing and preventing SARS-CoV-2 transmission in healthcare settings is of high importance. We sequenced SARS-CoV-2 genomes for patients and healthcare workers (HCWs) across multiple geographically distinct UK hospitals, obtaining 173 high-quality SARS-CoV-2 genomes. We integrated patient movement and staff location data into the analysis of viral genome data to understand spatial and temporal dynamics of SARS-CoV-2 transmission. We identified eight patient contact clusters (PCC) with significantly increased similarity in genomic variants compared to non-clustered samples. Incorporation of HCW location further increased the number of individuals within PCCs and identified additional links in SARS-CoV-2 transmission pathways. Patients within PCCs carried viruses more genetically identical to HCWs in the same ward location. SARS-CoV-2 genome sequencing integrated with patient and HCW movement data increases identification of outbreak clusters. This dynamic approach can support infection control management strategies within the healthcare setting.

First report of somatic mosaicism for mutations in STK11 in four patients with Peutz-Jeghers syndrome.

Peutz-Jeghers syndrome (PJS) is an autosomal dominant cancer predisposition syndrome characterised by gastrointestinal polyposis and mucocutaneous pigmentation. Mutations in STK11, a serine-threonine protein kinase, have been associated with PJS in up to 100 % of published series. The hypothesis that a further genetic locus for PJS exists is controversial. No mutations in any other genes have been described in association with PJS. To date, no instances of somatic mosaicism for STK11 have been described. DNA extracted from peripheral lymphocytes and buccal cells was screened by sequence analysis for mutations in STK11. Dosage analysis was undertaken by multiplex ligation-dependent probe amplification (MLPA). Four patients have been shown to have mosaicism in STK11: two had mosaic deletions of specific exons (2-3 and 3-10) of the STK11 gene; one had a mosaic nonsense mutation in exon 5; and one had a mosaic frameshift mutation in exon 8. This report details the first four reported cases of somatic mosaicism for STK11 associated with PJS. This shows that techniques in addition to direct sequencing such as MLPA must be used to assess for large scale genomic deletions in patients meeting clinical diagnostic criteria for PJS. This also adds further weight to the hypothesis of a single genetic locus for PJS.

Prenatal diagnosis in a family with X-linked hydrocephalus.

The neural cell adhesion molecule L1 is a transmembrane glycoprotein belonging to the immunoglobulin superfamily of cell adhesion molecules (CAMs). Its expression is essential during embryonic development of the nervous system and it is involved in cognitive function and memory. Mutations in the L1CAM gene are responsible for four related L1 disorders; X-linked hydrocephalus/HSAS (Hydrocephalus as a result of Stenosis of the Aqueduct of Sylvius), MASA (Mental retardation, Aphasia, Shuffling gait, and Adducted thumbs) syndrome, X-linked complicated spastic paraplegia type I (SPG1) and X-linked Agenesis of the Corpus Callosum (ACC). These four disorders represent a clinical spectrum that varies both between and within families. The main clinical features of this spectrum are Corpus callosum hypoplasia, mental Retardation, Adducted thumbs, Spastic paraplegia and Hydrocephalus (CRASH syndrome). Since there is no biochemically assayed disease marker, molecular analysis of the L1CAM gene is the only means of confirming a clinical diagnosis. Most L1CAM mutations reported to date are point mutations (missense, nonsense, splice site) and only a few patients with larger rearrangements have been documented. We have characterised a rare intragenic deletion of the L1CAM gene in a sample of DNA extracted from a chorionic villus biopsy (CVB) performed at 12 weeks' gestation. =