Improving Diagnostic Precision: Phenotype-Driven Analysis Uncovers a Maternal Mosaicism in an Individual with RYR1-Congenital Myopathy.

Congenital myopathies (CMs) are rare genetic disorders for which the diagnostic yield does not typically exceed 60% . We performed deep phenotyping, histopathological studies, clinical exome and trio genome sequencing and a phenotype-driven analysis of the genomic data, that led to the molecular diagnosis in a child with CM. We identified a heterozygous variant in RYR1 in the affected child, inherited from her asymptomatic mother. Given the alignment of the clinical and histopathological phenotype with RYR1-CM, we considered the potential existence of a missing second variant in trans in the proband, but also hypothesized that the variant might be mosaic in the mother, as subsequently demonstrated. Our study is an example of how heterozygous variants inherited from asymptomatic parents are frequently dismissed. When the genotype-phenotype correlation is strong, it is recommended to consider a parental mosaicism.

Advanced Optical Microscopy: Unveiling Functional Insights Regarding a Novel PPP2R1A Variant and Its Unreported Phenotype.

The number of genes implicated in neurodevelopmental conditions is rapidly growing. Recently, variants in PPP2R1A have been associated with syndromic intellectual disability and a consistent, but still expanding, phenotype. The PPP2R1A gene encodes a protein subunit of the serine/threonine protein phosphatase 2A enzyme, which plays a critical role in cellular function. We report an individual showing pontocerebellar hypoplasia (PCH), microcephaly, optic and peripheral nerve abnormalities, and an absence of typical features like epilepsy and an abnormal corpus callosum. He bears an unreported variant in an atypical region of PPP2R1A. In silico studies, functional analysis using immunofluorescence, and super-resolution microscopy techniques were performed to investigate the pathogenicity of the variant. This analysis involved a comparative analysis of the patient's fibroblasts with both healthy control cells and cells from an individual with the previously described phenotype. The results showed reduced expression of PPP2R1A and the presence of aberrant protein aggregates in the patient's fibroblasts, supporting the pathogenicity of the variant. These findings suggest a potential association between PPP2R1A variants and PCH, expanding the clinical spectrum of PPP2R1A-related neurodevelopmental disorder. Further studies and descriptions of additional patients are needed to fully understand the genotype-phenotype correlation and the underlying mechanisms of this novel phenotype.

Coexistence of junctional epidermolysis bullosa, autosomal recessive deafness type 57, and Angelman syndrome: A case report.

Key Clinical Message: The presence of more than one genetic/genomic disorder is not uncommon. It is therefore essential to continuously consider new signs and symptoms over time. Administration of gene therapy could be extremely difficult in particular situations. Abstract: A 9-month-old boy presented to our department for evaluation of developmental delay. We found that he was affected by intermediate junctional epidermolysis bullosa (COL17A1, c.3766 + 1G > A, homozygous), Angelman syndrome (5,5 Mb deletion of 15q11.2-q13.1), and autosomal recessive deafness type 57 (PDZD7, c.883C > T, homozygous).

Variants in DTNA cause a mild, dominantly inherited muscular dystrophy.

DTNA encodes α-dystrobrevin, a component of the macromolecular dystrophin-glycoprotein complex (DGC) that binds to dystrophin/utrophin and α-syntrophin. Mice lacking α-dystrobrevin have a muscular dystrophy phenotype, but variants in DTNA have not previously been associated with human skeletal muscle disease. We present 12 individuals from four unrelated families with two different monoallelic DTNA variants affecting the coiled-coil domain of α-dystrobrevin. The five affected individuals from family A harbor a c.1585G > A; p.Glu529Lys variant, while the recurrent c.1567_1587del; p.Gln523_Glu529del DTNA variant was identified in the other three families (family B: four affected individuals, family C: one affected individual, and family D: two affected individuals). Myalgia and exercise intolerance, with variable ages of onset, were reported in 10 of 12 affected individuals. Proximal lower limb weakness with onset in the first decade of life was noted in three individuals. Persistent elevations of serum creatine kinase (CK) levels were detected in 11 of 12 affected individuals, 1 of whom had an episode of rhabdomyolysis at 20 years of age. Autism spectrum disorder or learning disabilities were reported in four individuals with the c.1567_1587 deletion. Muscle biopsies in eight affected individuals showed mixed myopathic and dystrophic findings, characterized by fiber size variability, internalized nuclei, and slightly increased extracellular connective tissue and inflammation. Immunofluorescence analysis of biopsies from five affected individuals showed reduced α-dystrobrevin immunoreactivity and variably reduced immunoreactivity of other DGC proteins: dystrophin, α, ß, δ and γ-sarcoglycans, and α and ß-dystroglycans. The DTNA deletion disrupted an interaction between α-dystrobrevin and syntrophin. Specific variants in the coiled-coil domain of DTNA cause skeletal muscle disease with variable penetrance. Affected individuals show a spectrum of clinical manifestations, with severity ranging from hyperCKemia, myalgias, and exercise intolerance to childhood-onset proximal muscle weakness. Our findings expand the molecular etiologies of both muscular dystrophy and paucisymptomatic hyperCKemia, to now include monoallelic DTNA variants as a novel cause of skeletal muscle disease in humans.

Adequacy of the 10 mg/kg Daily Dose of Antituberculosis Drug Isoniazid in Infants under 6 Months of Age.

In 2010, the WHO recommended an increase in the daily doses of first-line anti-tuberculosis medicines in children. We aim to characterize the pharmacokinetics of the once-daily isoniazid (INH) dose at 10 mg/kg of body weight in infants <6 months of age. We performed a multicenter pharmacokinetic study in Spain. The N-acetyltransferase 2 gene was analyzed to determine the acetylation status. Samples were analyzed using a validated UPLC-UV assay. A non-compartmental pharmacokinetic analysis was performed. Twenty-three pharmacokinetic profiles were performed in 20 infants (8 females) at a median (IQR) age of 19.0 (12.6-23.3) weeks. The acetylator statuses were homozygous fast (n = 1), heterozygous intermediate (n = 12), and homozygous slow (n = 7). INH median (IQR) Cmax and AUC0-24h values were 4.8 (3.7-6.7) mg/L and 23.5 (13.4-36.7) h*mg/L and the adult targets (>3 mg/L and 11.6-26.3 h*mg/L) were not reached in three and five cases, respectively. The age at assessment or acetylator status had no impact on Cmax values, but a larger INH AUC0-24h (p = 0.025) and trends towards a longer half-life (p = 0.055) and slower clearance (p = 0.070) were observed in homozygous slow acetylators. Treatment was well tolerated; mildly elevated alanine aminotransferase levels were observed in three cases. In our series of young infants receiving isoniazid, no major safety concerns were raised, and the target adult levels were reached in most patients.

The landscape of submicroscopic structural variants at the OPN1LW/OPN1MW gene cluster on Xq28 underlying blue cone monochromacy.

Blue cone monochromacy (BCM) is an X-linked retinal disorder characterized by low vision, photoaversion, and poor color discrimination. BCM is due to the lack of long-wavelength-sensitive and middle-wavelength-sensitive cone photoreceptor function and caused by mutations in the OPN1LW/OPN1MW gene cluster on Xq28. Here, we investigated the prevalence and the landscape of submicroscopic structural variants (SVs) at single-base resolution in BCM patients. We found that about one-third (n = 73) of the 213 molecularly confirmed BCM families carry an SV, most commonly deletions restricted to the OPN1LW/OPN1MW gene cluster. The structure and precise breakpoints of the SVs were resolved in all but one of the 73 families. Twenty-two families-all from the United States-showed the same SV, and we confirmed a common ancestry of this mutation. In total, 42 distinct SVs were identified, including 40 previously unreported SVs, thereby quadrupling the number of precisely mapped SVs underlying BCM. Notably, there was no "region of overlap" among these SVs. However, 90% of SVs encompass the upstream locus control region, an essential enhancer element. Its minimal functional extent based on deletion mapping in patients was refined to 358 bp. Breakpoint analyses suggest diverse mechanisms underlying SV formation as well as in one case the gene conversion-based exchange of a 142-bp deletion between opsin genes. Using parsimonious assumptions, we reconstructed the composition and copy number of the OPN1LW/OPN1MW gene cluster prior to the mutation event and found evidence that large gene arrays may be predisposed to the occurrence of SVs at this locus.

Intellectual-disability-associated mutations in the ceramide transport protein gene CERT1 lead to aberrant function and subcellular distribution.

The lipid molecule ceramide is transported from the endoplasmic reticulum to the Golgi apparatus for sphingomyelin production via the ceramide transport protein (CERT), encoded by CERT1. Hyperphosphorylation of CERT's serine-repeat motif (SRM) decreases its functionality. Some forms of inherited intellectual disability (ID) have been associated with a serine-to-leucine substitution in the SRM (S132L mutation) and a glycine-to-arginine substitution outside the SRM (G243R mutation) in CERT; however, it is unclear if mutations outside the SRM disrupt the control of CERT functionality. In the current investigation, we identified a new CERT1 variant (dupAA) in a patient with mild ID that resulted from a frameshift at the C-terminus of CERT1. However, familial analysis revealed that the dupAA variant was not associated with ID, allowing us to utilize it as a disease-matched negative control for CERT1 variants that are associated with ID. Biochemical analysis showed that G243R and S132L, but not dupAA, impair SRM hyperphosphorylation and render the CERT variants excessively active. Additionally, both S132L and G243R mutations but not dupAA caused the proteins to be distributed in a punctate subcellular manner. On the basis of these findings, we infer that the majority of ID-associated CERT variants may impair SRM phosphorylation-dependent repression, resulting in an increase in sphingomyelin production concurrent with CERT subcellular redistribution.

Plasma idebenone monitoring in Friedreich's ataxia patients during a long-term follow-up.

INTRODUCTION AND OBJECTIVES: Despite the growing interest and the potential benefits of idebenone as a repurposed drug for different orphan conditions, data regarding its monitoring are scarce. Our main goal was to report plasma idebenone values in a cohort of Friedreich's ataxia (FRDA) patients during a long-term follow-up. Taking advantage of this, we also assessed cardiological and neurological status together with idebenone values and genetic background. METHODS: Long-term follow-up retrospective study in 27 FRDA patients with a disease onset at the paediatric age treated with idebenone by compassionate use. Plasma idebenone was measured by HPLC with electrochemical detection. RESULTS: Median plasma idebenone values increased when doses were increased, but apparently linearity was lost in the highest dose group. Marked intraindividual and interindividual differences were observed among patients. We did not find a consistent positive effect after analysis of paired data at the beginning and the end of the study. We only found a correlation between some cardiological measures and the duration of idebenone therapy at high doses, but with uncertain significance. CONCLUSIONS: The large variations observed among the different individuals involved in this study should be considered for optimization of individual dosage regimens.

CACNA1A Mutations Causing Early Onset Ataxia: Profiling Clinical, Dysmorphic and Structural-Functional Findings.

The CACNA1A gene encodes the pore-forming α1A subunit of the voltage-gated CaV2.1 Ca2+ channel, essential in neurotransmission, especially in Purkinje cells. Mutations in CACNA1A result in great clinical heterogeneity with progressive symptoms, paroxysmal events or both. During infancy, clinical and neuroimaging findings may be unspecific, and no dysmorphic features have been reported. We present the clinical, radiological and evolutionary features of three patients with congenital ataxia, one of them carrying a new variant. We report the structural localization of variants and their expected functional consequences. There was an improvement in cerebellar syndrome over time despite a cerebellar atrophy progression, inconsistent response to acetazolamide and positive response to methylphenidate. The patients shared distinctive facial gestalt: oval face, prominent forehead, hypertelorism, downslanting palpebral fissures and narrow nasal bridge. The two α1A affected residues are fully conserved throughout evolution and among the whole human CaV channel family. They contribute to the channel pore and the voltage sensor segment. According to structural data analysis and available functional characterization, they are expected to exert gain- (F1394L) and loss-of-function (R1664Q/R1669Q) effect, respectively. Among the CACNA1A-related phenotypes, our results suggest that non-progressive congenital ataxia is associated with developmental delay and dysmorphic features, constituting a recognizable syndromic neurodevelopmental disorder.

The Increasing Impact of Translational Research in the Molecular Diagnostics of Neuromuscular Diseases.

The diagnosis of neuromuscular diseases (NMDs) has been progressively evolving from the grouping of clinical symptoms and signs towards the molecular definition. Optimal clinical, biochemical, electrophysiological, electrophysiological, and histopathological characterization is very helpful to achieve molecular diagnosis, which is essential for establishing prognosis, treatment and genetic counselling. Currently, the genetic approach includes both the gene-targeted analysis in specific clinically recognizable diseases, as well as genomic analysis based on next-generation sequencing, analyzing either the clinical exome/genome or the whole exome or genome. However, as of today, there are still many patients in whom the causative genetic variant cannot be definitely established and variants of uncertain significance are often found. In this review, we address these drawbacks by incorporating two additional biological omics approaches into the molecular diagnostic process of NMDs. First, functional genomics by introducing experimental cell and molecular biology to analyze and validate the variant for its biological effect in an in-house translational diagnostic program, and second, incorporating a multi-omics approach including RNA-seq, metabolomics, and proteomics in the molecular diagnosis of neuromuscular disease. Both translational diagnostics programs and omics are being implemented as part of the diagnostic process in academic centers and referral hospitals and, therefore, an increase in the proportion of neuromuscular patients with a molecular diagnosis is expected. This improvement in the process and diagnostic performance of patients will allow solving aspects of their health problems in a precise way and will allow them and their families to take a step forward in their lives.

Early-onset severe spinocerebellar ataxia 42 with neurodevelopmental deficits (SCA42ND): Case report, pharmacological trial, and literature review.

Early-onset severe spinocerebellar ataxia 42 with neurodevelopmental deficits (SCA42ND, MIM#604065) is an ultrarare autosomal dominant syndrome related to de novo CACNA1G gain-of-function pathogenic variants. All patients with SCA42ND show cerebellar atrophy and/or hypoplasia on neuroimaging and share common features such as dysmorphic features, global developmental delay, and axial hypotonia, all manifesting within the first year of life. To date, only 10 patients with SCA42ND have been reported with functionally confirmed gain-of-function variants, bearing either of two recurrent pathogenic variants. We describe a girl with congenital ataxia, without epilepsy, and a de novo p.Ala961Thr pathogenic variant in CACNA1G. We review the published subjects with the aim of better characterizing the dysmorphic features that may be crucial for clinical recognition of SCA42ND. Cerebellar atrophy, together with digital anomalies, particularly broad thumbs and/or halluces, should lead to clinical suspicion of this disease. We describe the first pharmacological attempt to treat a patient with SCA42ND using zonisamide, an antiepileptic drug with T-type channel blocker activity, in an off-label indication using an itemized study protocol. No efficacy was observed at the dose tested. However, without pharmacological treatment, she showed a positive evolution in neurodevelopment during the follow-up.

The Phenotype and Genotype of Congenital Myopathies Based on a Large Pediatric Cohort.

BACKGROUND: Congenital myopathies (CMs) are a clinically and genetically heterogeneous group of hereditary muscular disorders. The distribution of genetic and histologic subtypes has been addressed in only a few cohorts, and the relationship between phenotypes and genotypes is only partially understood. METHODS: This is a retrospective cross-sectional data collection study conducted at a single center. The clinical, histopathological, and molecular characterization of 104 patients with CM is reported. RESULTS: The most common histopathological subtype was core myopathy (42%). Patients with severe endomysial fibrosis were more commonly unable to walk than patients with only a mild-grade endomysial fibrosis (56% vs 16%). Inability to walk was also more prevalent in patients with severe fatty replacement (44% vs 19%). The genetic etiology was more frequently identified among those patients with "specific" histologic findings (74% vs 62%). A definite molecular diagnosis was reached in 65 of 104 patients (62%), with RYR1 (24/104) and TTN (8/104) being the most frequent causative genes. Neonatal onset occurred in 56%. Independent ambulation was achieved by 74%. Patients who walked late were more likely to become wheelchair-dependent. Respiratory support was needed in one of three patients. Gastrostomy placement was required in 15%. Cardiac involvement was observed in 3%, scoliosis in 43%, and intellectual disability in 6%. CONCLUSIONS: This study provides an updated picture of the clinical, histopathological, and molecular landscape of CMs. Independently of the causative gene, fibrosis and fatty replacement in muscle biopsy specimens are associated with clinical severity. Mutations in TTN are responsible for a higher proportion of cases than previously thought.

De Novo Variants in LMNB1 Cause Pronounced Syndromic Microcephaly and Disruption of Nuclear Envelope Integrity.

Lamin B1 plays an important role in the nuclear envelope stability, the regulation of gene expression, and neural development. Duplication of LMNB1, or missense mutations increasing LMNB1 expression, are associated with autosomal-dominant leukodystrophy. On the basis of its role in neurogenesis, it has been postulated that LMNB1 variants could cause microcephaly. Here, we confirm this hypothesis with the identification of de novo mutations in LMNB1 in seven individuals with pronounced primary microcephaly (ranging from -3.6 to -12 SD) associated with relative short stature and variable degree of intellectual disability and neurological features as the core symptoms. Simplified gyral pattern of the cortex and abnormal corpus callosum were noted on MRI of three individuals, and these individuals also presented with a more severe phenotype. Functional analysis of the three missense mutations showed impaired formation of the LMNB1 nuclear lamina. The two variants located within the head group of LMNB1 result in a decrease in the nuclear localization of the protein and an increase in misshapen nuclei. We further demonstrate that another mutation, located in the coil region, leads to increased frequency of condensed nuclei and lower steady-state levels of lamin B1 in proband lymphoblasts. Our findings collectively indicate that de novo mutations in LMNB1 result in a dominant and damaging effect on nuclear envelope formation that correlates with microcephaly in humans. This adds LMNB1 to the growing list of genes implicated in severe autosomal-dominant microcephaly and broadens the phenotypic spectrum of the laminopathies.