Expanding the spectrum of neonatal-onset AIFM1-associated disorders.

OBJECTIVES: Pathogenic variants in AIFM1 have been associated with a wide spectrum of disorders, spanning from CMT4X to mitochondrial encephalopathy. Here we present a novel phenotype and review the existing literature on AIFM1-related disorders. METHODS: We performed EEG recordings, brain MRI and MR Spectroscopy, metabolic screening, echocardiogram, clinical exome sequencing (CES) and family study. Effects of the variant were established on cultured fibroblasts from skin punch biopsy. RESULTS: The patient presented with drug-resistant, electro-clinical, multifocal seizures 6 h after birth. Brain MRI revealed prominent brain swelling of both hemispheres and widespread signal alteration in large part of the cortex and of the thalami, with sparing of the basal nuclei. CES analysis revealed the likely pathogenic variant c.5T>C; p.(Phe2Ser) in the AIFM1 gene. The affected amino acid residue is located in the mitochondrial targeting sequence. Functional studies on cultured fibroblast showed a clear reduction in AIFM1 protein amount and defective activities of respiratory chain complexes I, III and IV. No evidence of protein mislocalization or accumulation of precursor protein was observed. Riboflavin, Coenzyme Q10 and thiamine supplementation was therefore given. At 6 months of age, the patient exhibited microcephaly but did not experience any further deterioration. He is still fed orally and there is no evidence of muscle weakness or atrophy. INTERPRETATION: This is the first AIFM1 case associated with neonatal seizures and diffuse white matter involvement with relative sparing of basal ganglia, in the absence of clinical signs suggestive of myopathy or motor neuron disease.

Multimodal imaging evaluation of occult macular dystrophy associated with a novel RP1L1 variant.

Purpose: Occult Macular Dystrophy (OMD) is an autosomal dominant inherited retinal dystrophy caused by mutations in the retinitis pigmentosa 1-like 1 (RP1L1) gene. The present study describes a novel RP1L1 variant, identified for the first time in two Italian sisters diagnosed with OMD, along with multimodal imaging features, including Optical Coherence Tomography (OCT) Angiography. Methods: We performed multimodal imaging including spectral-domain OCT, blue light autofluorescence (BAF), infrared autofluorescence (IRAF), swept-source OCT Angiography (OCTA), full-field and multifocal electroretinography. Genetic analysis was performed using Next-Generation Sequencing. Pathogenic potential of nonsynonymous novel variants was scored with two in silico algorithms. Results: Proband 1 (P1) and proband 2 (P2) were two Italian sisters of 61 and 56 years old. Both reported a history of progressive visual loss without fundoscopic alterations. P1 reported a 4-year history of rapid visual function worsening, and her best-corrected visual acuity (BCVA) was counting fingers in both eyes. P2 reported a 20-year history of mild but progressive visual acuity loss, and her BCVA was 1/10 and 2/10 respectively in her right and left eye. Structural OCT displayed disorganization of outer retinal bands at the macula and foveal cavitation; loss of foveal photoreceptors was remarkably evident on en-face OCT slabs. OCTA quantitative analysis found that vessel density was reduced both at SCP and DCP while choriocapillaris blood flow was relatively spared. Genetic analysis found the same rare dominant c.2873G > C, p.Arg958Pro variant in the RP1L1 gene. The substitution was regarded as moderately radical according to Grantham score while PolyPhen2 classified the amino acidic substitution as probably damaging. Conclusions and importance: Our study expands the mutational spectrum of RP1L1 gene: the rare c.2873G > C, p.Arg958Pro missense variant may be considered a new pathogenic variant for OMD, the first to be identified exclusively in an Italian family. Moreover, our quantitative OCTA data suggest that OMD is characterized by a rarefaction of superficial and deep capillary plexus.

Lentiviral haematopoietic stem-cell gene therapy for early-onset metachromatic leukodystrophy: long-term results from a non-randomised, open-label, phase 1/2 trial and expanded access.

BACKGROUND: Effective treatment for metachromatic leukodystrophy (MLD) remains a substantial unmet medical need. In this study we investigated the safety and efficacy of atidarsagene autotemcel (arsa-cel) in patients with MLD. METHODS: This study is an integrated analysis of results from a prospective, non-randomised, phase 1/2 clinical study and expanded-access frameworks. 29 paediatric patients with pre-symptomatic or early-symptomatic early-onset MLD with biochemical and molecular confirmation of diagnosis were treated with arsa-cel, a gene therapy containing an autologous haematopoietic stem and progenitor cell (HSPC) population transduced ex vivo with a lentiviral vector encoding human arylsulfatase A (ARSA) cDNA, and compared with an untreated natural history (NHx) cohort of 31 patients with early-onset MLD, matched by age and disease subtype. Patients were treated and followed up at Ospedale San Raffaele, Milan, Italy. The coprimary efficacy endpoints were an improvement of more than 10% in total gross motor function measure score at 2 years after treatment in treated patients compared with controls, and change from baseline of total peripheral blood mononuclear cell (PBMC) ARSA activity at 2 years after treatment compared with values before treatment. This phase 1/2 study is registered with ClinicalTrials.gov, NCT01560182. FINDINGS: At the time of analyses, 26 patients treated with arsa-cel were alive with median follow-up of 3·16 years (range 0·64-7·51). Two patients died due to disease progression and one due to a sudden event deemed unlikely to be related to treatment. After busulfan conditioning, all arsa-cel treated patients showed sustained multilineage engraftment of genetically modified HSPCs. ARSA activity in PBMCs was significantly increased above baseline 2 years after treatment by a mean 18·7-fold (95% CI 8·3-42·2; p<0·0001) in patients with the late-infantile variant and 5·7-fold (2·6-12·4; p<0·0001) in patients with the early-juvenile variant. Mean differences in total scores for gross motor function measure between treated patients and age-matched and disease subtype-matched NHx patients 2 years after treatment were significant for both patients with late-infantile MLD (66% [95% CI 48·9-82·3]) and early-juvenile MLD (42% [12·3-71·8]). Most treated patients progressively acquired motor skills within the predicted range of healthy children or had stabilised motor performance (maintaining the ability to walk). Further, most displayed normal cognitive development and prevention or delay of central and peripheral demyelination and brain atrophy throughout follow-up; treatment benefits were particularly apparent in patients treated before symptom onset. The infusion was well tolerated and there was no evidence of abnormal clonal proliferation or replication-competent lentivirus. All patients had at least one grade 3 or higher adverse event; most were related to conditioning or to background disease. The only adverse event related to arsa-cel was the transient development of anti-ARSA antibodies in four patients, which did not affect clinical outcomes. INTERPRETATION: Treatment with arsa-cel resulted in sustained, clinically relevant benefits in children with early-onset MLD by preserving cognitive function and motor development in most patients, and slowing demyelination and brain atrophy. FUNDING: Orchard Therapeutics, Fondazione Telethon, and GlaxoSmithKline.

Analyzing histopathological features of rare charcot-marie-tooth neuropathies to unravel their pathogenesis.

BACKGROUND: Charcot-Marie-Tooth (CMT) neuropathies are very heterogeneous disorders from both a clinical and genetic point of view. The CMT genes identified so far encode different proteins that are variably involved in regulating Schwann cells and/or axonal functions. However, the function of most of these proteins still remains to be elucidated. OBJECTIVE: To characterize a large cohort of patients with demyelinating, axonal, and intermediate forms of CMT neuropathy. DESIGN: A cohort of 131 unrelated patients were screened for mutations in 12 genes responsible for CMT neuropathies. Demyelinating, axonal, and intermediate forms of CMT neuropathy were initially distinguished as usual on the basis of electrophysiological criteria and clinical evaluation. A sural nerve biopsy was also performed for selected cases. Accordingly, patients underwent first-level analysis of the genes most frequently mutated in each clinical form of CMT neuropathy. RESULTS: Although our cohort had a particularly high percentage of cases of rare axonal and intermediate CMT neuropathies, we found mutations in 40% of patients. Among identified changes, 7 represented new mutations occurring in the MPZ, GJB1, EGR2, MFN2, NEFL, and HSBP1/HSP27 genes. Histopathological analysis performed in selected cases revealed morphological features, which correlated with the molecular diagnosis and provided evidence of the underlying pathogenetic mechanism. CONCLUSION: Clinical and pathological analysis of patients with CMT neuropathies contributes to our understanding of the molecular mechanisms of CMT neuropathies.

High resolution melting for the identification of mutations in the iron responsive element of the ferritin light chain gene.

BACKGROUND: Among the causes of hyperferritinemia, hereditary hyperferritinemia cataract syndrome (HHCS) is an autosomal dominant disease characterized by distinctive cataracts and high serum ferritin. It is caused by mutations in the iron responsive element (IRE) of the ferritin light chain gene (FTL). METHODS: To speed up and simplify mutational scanning in this genomic region, we developed a protocol based on high-resolution melting (HRM) analysis. RESULTS: Validation was carried out using 18 wild-type and 14 DNA samples carrying different mutations, each analyzed in replicates of 20. The method allowed for correct identification and genotyping of all mutant samples, and each variant generated a specific profile distinguishable from the wild type. A 5.5% proportion of false positive results were obtained. In addition, in two patients with HHCS, two new mutations were identified by HRM based on an altered melting profile. These mutations were subsequently characterized by direct sequencing (7C>G+40A>G and 49A>C). CONCLUSIONS: The high reliability of HRM in detecting known and new DNA variations indicate that this could be an effective and sensitive method for molecular scanning of mutations in the IRE of the FTL gene in patients presenting with either HHCS or unexplained hyperferritinemia.