BACKGROUND: The Limb Girdle Muscular Dystrophies (LGMDs) are characterized by progressive weakness of the shoulder and hip girdle muscles as a result of over 30 different genetic mutations. This study is designed to develop clinical outcome assessments across the group of disorders.
METHODS: The primary goal of this study is to evaluate the utility of a set of outcome measures on a wide range of LGMD phenotypes and ability levels to determine if it would be possible to use similar outcomes between individuals with different phenotypes. We will perform a multi-center, 12-month study of 188 LGMD patients within the established Genetic Resolution and Assessments Solving Phenotypes in LGMD (GRASP-LGMD) Research Consortium, which is comprised of 11 sites in the United States and 2 sites in Europe. Enrolled patients will be clinically affected and have mutations in CAPN3 (LGMDR1), ANO5 (LGMDR12), DYSF (LGMDR2), DNAJB6 (LGMDD1), SGCA (LGMDR3), SGCB (LGMDR4), SGCD (LGMDR6), or SGCG (LGMDR5, or FKRP-related (LGMDR9).
CONCLUSIONS: To the best of our knowledge, this will be the largest consortium organized to prospectively validate clinical outcome assessments (COAs) in LGMD at its completion. These assessments will help clinical trial readiness by identifying reliable, valid, and responsive outcome measures as well as providing data driven clinical trial decision making for future clinical trials on therapeutic agents for LGMD. The results of this study will permit more efficient clinical trial design. All relevant data will be made available for investigators or companies involved in LGMD therapeutic development upon conclusion of this study as applicable.
BACKGROUND: Clinicaltrials.gov NCT03981289; Date of registration: 6/10/2019.

Limb-girdle muscular dystrophy R9 (LGMDR9) is a progressive and disabling genetic muscle disease. Sleep is relevant in the patient care as it impacts on health, functioning, and well-being. LGMDR9 may potentially affect sleep by physical or emotional symptoms, myalgia, or sleep-disordered breathing (SDB) through cardiorespiratory involvement. The objective was to investigate the occurrence of insomnia and unrecognized or untreated SDB in LGMDR9, associated factors, and relationships with fatigue and health-related quality of life (HRQoL). All 90 adults in a Norwegian LGMDR9 cohort received questionnaires on sleep, fatigue, and HRQoL. Forty-nine of them underwent clinical assessments and 26 without mask-based therapy for respiration disorders additionally underwent polysomnography (PSG) and capnometry. Among 77 questionnaire respondents, 31% received mask-based therapy. The prevalence of insomnia was 32% of both those with and without such therapy but was significantly increased in fatigued respondents (54% vs 21%). Insomnia levels correlated inversely with mental HRQoL. Among 26 PSG candidates, an apnea-hypopnea index (AHI) ≥ 5/h was observed in 16/26 subjects (≥ 15/h in 8/26) with median 6.8 obstructive apneas and 0.2 central apneas per hour of sleep. The AHI was related to advancing age and an ejection fraction < 50%. Sleep-related hypoventilation was detected in one subject. Fatigue severity did not correlate with motor function or nocturnal metrics of respiration or sleep but with Maximal Inspiratory Pressure (r = - 0.46). The results indicate that insomnia and SDB are underrecognized comorbidities in LGMDR9 and associated with HRQoL impairment and heart failure, respectively. We propose an increased attention to insomnia and SDB in the interdisciplinary care of LGMDR9. Insomnia and pulmonary function should be examined in fatigued patients.

We aimed to investigate the epidemiology and natural history of FKRP-related limb-girdle muscular dystrophy R9 (LGMDR9) in Norway. We identified 153 genetically confirmed subjects making the overall prevalence 2.84/100,000, the highest reported figure worldwide. Of the 153 subjects, 134 (88 %) were homozygous for FKRP c.826C>A giving a carrier frequency for this variant of 1/101 in Norway. Clinical questionnaires and patient notes from 101 subjects, including 88 c.826C>A homozygotes, were reviewed, and 43/101 subjects examined clinically. Age of onset in c.826C>A homozygotes demonstrated a bimodal distribution. Female subjects showed an increased cumulative probability of wheelchair dependency and need for ventilatory support. Across the cohort, the need for ventilatory support preceded wheelchair dependency in one third of the cases, usually due to sleep apnea. In c.826C>A homozygotes, occurrence of cardiomyopathy correlated positively with male gender but not with age or disease stage. This study highlights novel gender differences in both loss of ambulation, need for ventilatory support and the development of cardiomyopathy. Our results confirm the need for vigilance in order to detect respiratory insufficiency and cardiac involvement, but indicate that these events affect males and females differently.

Hereditary myopathies with limb-girdle muscular weakness (LGW) are a genetically heterogeneous group of disorders, in which molecular diagnosis remains challenging. Our aim was to present a detailed clinical and genetic characterization of a large cohort of patients with LGW.
This nationwide cohort study included patients with LGW suspected to be associated with hereditary myopathies. Parameters associated with specific genetic aetiologies were evaluated, and we further assessed how they predicted the detection of causative variants by conducting genetic analyses.
Molecular diagnoses were identified in 62.0% (75/121) of the cohort, with a higher proportion of patients diagnosed by next-generation sequencing (NGS) than by single-gene testing (77.3% vs. 22.7% of solved cases). The median (interquartile range) time from onset to genetic diagnosis was 8.9 (3.7-19.9) and 17.8 (7.9-27.8) years for single-gene testing and NGS, respectively. The most common diagnoses were myopathies associated with variants in CAPN3 (n = 9), FKRP (n = 9), ANO5 (n = 8), DYSF (n = 8) and SGCA (n = 5), which together accounted for 32.2% of the cohort. Younger age at disease onset (p = 0.043), >10× elevated creatine kinase activity levels (p = 0.024) and myopathic electromyography findings (p = 0.007) were significantly associated with the detection of causative variants.
Our findings suggest that an earlier use of NGS in patients with LGW is needed to avoid long diagnostic delays. We further present parameters predictive of a molecular diagnosis that may help to select patients for genetic analyses, especially in centres with limited access to sequencing.

In this study we report the results of a phase Ib/IIa, open-label, multiple ascending-dose trial of domagrozumab, a myostatin inhibitor, in patients with fukutin-related protein (FKRP)-associated limb-girdle muscular dystrophy.
Nineteen patients were enrolled and assigned to one of three dosing arms (5, 20, or 40 mg/kg every 4 weeks). After 32 weeks of treatment, participants receiving the lowest dose were switched to the highest dose (40 mg/kg) for an additional 32 weeks. An extension study was also conducted. The primary endpoints were safety and tolerability. Secondary endpoints included muscle strength, timed function testing, pulmonary function, lean body mass, pharmacokinetics, and pharmacodynamics. As an exploratory outcome, muscle fat fractions were derived from whole-body magnetic resonance images.
Serum concentrations of domagrozumab increased in a dose-dependent manner and modest levels of myostatin inhibition were observed in both serum and muscle tissue. The most frequently occurring adverse events were injuries secondary to falls. There were no significant between-group differences in the strength, functional, or imaging outcomes studied.
We conclude that, although domagrozumab was safe in patients in limb-girdle muscular dystrophy type 2I/R9, there was no clear evidence supporting its efficacy in improving muscle strength or function.

Glycosyltransferases (GTs) are a ubiquitous group of enzymes that catalyze the synthesis of glycosidic bonds. In this work, we focused on the retained reaction catalyzed by xyloside α-1,3-xylosyltransferase (XXYLT1) from Mus musculus. Our calculations revealed that the xylose transfer reaction follows the SNi-like mechanism, which involves a short-lived oxocarbenium-phosphate ion-pair intermediate (IP). The previously obtained crystal structure of the UDP-Xyl ternary Michaelis reaction complex was found to be an inactive form. Accordingly, the β-phosphate oxygen O3B of the donor should first undergo a conformational change to reach an active state where the donor forms a strong hydrogen bond with the acceptor, facilitating the departure of the phosphate group. Our calculations also revealed that two predicated transition states for the sugar-phosphate bond cleavage and glycosidic bond formation are structurally similar to the short-lived intermediate, which contains a three-member ring formed by the β-phosphate oxygen, the hydroxyl oxygen in the acceptor, and the anomeric carbon. It can be considered as a typical characteristic of the SNi-like mechanism. In addition, a nearby polar residue, Q330, is responsible for stabilizing the short-lived intermediate by electrostatic interactions. Thus, the Q330A mutant can abolish the activity of XXYLT1. In addition, using UDP-glucose as the donor, our calculations revealed that glucose transfer would correspond to a higher energy barrier owing to the steric repulsion between the glucosyl moiety and the nearby residue L327, indicating the requirement of active site architecture for glucose transfer. These findings not only explain the experimental observations but also are meaningful for clarifying the mechanism of GTs.

The complete mutational spectrum of dystrophinopathies and limb-girdle muscular dystrophy (LGMD) remains unknown in Mexican population. Seventy-two unrelated Mexican male patients (73% of pediatric age) with clinical suspicion of muscular dystrophy and no evidence of DMD gene deletion on multiplex polymerase chain reaction (mPCR) analysis were analyzed by multiplex ligation-dependent probe amplification (MLPA). Those with a normal result were subjected to Sanger sequencing or to next-generation sequencing for DMD plus 10 selected LGMD-related genes. We achieved a diagnostic genotype in 80.5% (n = 58/72) of patients with predominance of dystrophinopathy-linked genotypes (68%, n = 49/72), followed by autosomal recessive LGMD-related genotypes (types 2A-R1, 2C-R5, 2E-R4, 2D-R3 and 2I-R9; 12.5%, n = 9/72). MLPA showed 4.2% of false-negatives for DMD deletions assessed by mPCR. Among the small DMD variants, 96.5% (n = 28/29) corresponded to null-alleles, most of which (72%) were inherited through a carrier mother. The FKRP p.[Leu276Ile]; [Asn463Asp] genotype is reported for the first time in Mexican patients as being associated with dilated cardiomyopathy. Absence of dysferlinopathies could be related to the small sample size and/or the predominantly pediatric age of patients. The employed strategy seems to be an affordable diagnosis approach for Mexican muscular dystrophy male patients and their families.

Recent advances in the study of plant developmental and physiological responses have benefited from tissue-specific approaches, revealing the role of some cell types in these processes. Such approaches have relied on the inactivation of target cells using either toxic compounds or deleterious genes; however, both tissue-specific and truly inducible tools are lacking in order to precisely target a developmental window or specific growth response. We engineered the yeast fluorocytosine deaminase (FCY1) gene by creating a fusion with the bacterial uracil phosphoribosyl transferase (UPP) gene. The recombinant protein converts the precursor 5-fluorocytosine (5-FC) into 5-fluorouracyl, a drug used in the treatment of a range of cancers, which triggers DNA and RNA damage. We expressed the FCY-UPP gene construct in specific cell types using enhancer trap lines and promoters, demonstrating that this marker acts in a cell-autonomous manner. We also showed that it can inactivate slow developmental processes like lateral root formation by targeting pericycle cells. It also revealed a role for the lateral root cap and the epidermis in controlling root growth, a faster response. The 5-FC precursor acts systemically, as demonstrated by its ability to inhibit stomatal movements when supplied to the roots in combination with a guard cell-specific promoter. Finally, we demonstrate that the tissular inactivation is reversible, and can therefore be used to synchronize plant responses or to determine cell type-specific functions during different developmental stages. This tool will greatly enhance our capacity to understand the respective role of each cell type in plant physiology and development.

Genome-wide association studies (GWASs) in European populations have identified more than 100 schizophrenia-associated loci. A schizophrenia GWAS in a unique Indian population offers novel findings.
To discover and functionally evaluate genetic loci for schizophrenia in a GWAS of a unique Indian population.
This GWAS included a sample of affected individuals, family members, and unrelated cases and controls. Three thousand ninety-two individuals were recruited and diagnostically ascertained via medical records, hospitals, clinics, and clinical networks in Chennai and surrounding regions. Affected participants fulfilled DSM-IV diagnostic criteria for schizophrenia. Unrelated control participants had no personal or family history of psychotic disorder. Recruitment, genotyping, and analysis occurred in consecutive phases beginning January 1, 2001. Recruitment was completed on February 28, 2018, and genotyping and analysis are ongoing.
Associations of single-nucleotide polymorphisms and gene expression with schizophrenia.
The study population included 1321 participants with schizophrenia, 885 family controls, and 886 unrelated controls. Among participants with schizophrenia, mean (SD) age was 39.1 (11.4) years, and 52.7% were male. This sample demonstrated uniform ethnicity, a degree of inbreeding, and negligible rates of substance abuse. A novel genome-wide significant association was observed between schizophrenia and a chromosome 8q24.3 locus (rs10866912, allele A; odds ratio [OR], 1.27 [95% CI, 1.17-1.38]; P = 4.35 × 10-8) that attracted support in the schizophrenia Psychiatric Genomics Consortium 2 data (rs10866912, allele A; OR, 1.04 [95% CI, 1.02-1.06]; P = 7.56 × 10-4). This locus has undergone natural selection, with the risk allele A declining in frequency from India (approximately 72%) to Europe (approximately 43%). rs10866912 directly modifies the abundance of the nicotinate phosphoribosyltransferase gene (NAPRT1) transcript in brain cortex (normalized effect size, 0.79; 95% CI, 0.6-1.0; P = 5.8 × 10-13). NAPRT1 encodes a key enzyme for niacin metabolism. In Indian lymphoblastoid cell lines, (risk) allele A of rs10866912 was associated with NAPRT1 downregulation (AA: 0.74, n = 21; CC: 1.56, n = 17; P = .004). Preliminary zebrafish data further suggest that partial loss of function of NAPRT1 leads to abnormal brain development.
Bioinformatic analyses and cellular and zebrafish gene expression studies implicate NAPRT1 as a novel susceptibility gene. Given this gene\'s role in niacin metabolism and the evidence for niacin deficiency provoking schizophrenialike symptoms in neuropsychiatric diseases such as pellagra and Hartnup disease, these results suggest that the rs10866912 genotype and niacin status may have implications for schizophrenia susceptibility and treatment.

Syntheses of α-branched alkyl and aryl substituted 9-[2-(phosphonomethoxy)ethyl]purines from substituted 1,3-dioxolanes have been developed. Key synthetic precursors, α-substituted dialkyl [(2-hydroxyethoxy)methyl]phosphonates were prepared via Lewis acid mediated cleavage of 1,3-dioxolanes followed by reaction with dialkyl or trialkyl phosphites. The best preparative yields were achieved under conditions utilizing tin tetrachloride as Lewis acid and triisopropyl phosphite. Attachment of purine bases to dialkyl [(2-hydroxyethoxy)methyl]phosphonates was performed by Mitsunobu reaction. Final α-branched 9-[2-(phosphonomethoxy)ethyl]purines were tested for antiviral, cytostatic and antiparasitic activity, the latter one determined as inhibitory activity towards Plasmodium falciparum enzyme hypoxanthine-guanine-xanthine phosphoribosyltransfesase. In most cases biological activity was only marginal.