Overlapping SETBP1 gain-of-function mutations in Schinzel-Giedion syndrome and hematologic malignancies.

Schinzel-Giedion syndrome (SGS) is a rare developmental disorder characterized by multiple malformations, severe neurological alterations and increased risk of malignancy. SGS is caused by de novo germline mutations clustering to a 12bp hotspot in exon 4 of SETBP1. Mutations in this hotspot disrupt a degron, a signal for the regulation of protein degradation, and lead to the accumulation of SETBP1 protein. Overlapping SETBP1 hotspot mutations have been observed recurrently as somatic events in leukemia. We collected clinical information of 47 SGS patients (including 26 novel cases) with germline SETBP1 mutations and of four individuals with a milder phenotype caused by de novo germline mutations adjacent to the SETBP1 hotspot. Different mutations within and around the SETBP1 hotspot have varying effects on SETBP1 stability and protein levels in vitro and in in silico modeling. Substitutions in SETBP1 residue I871 result in a weak increase in protein levels and mutations affecting this residue are significantly more frequent in SGS than in leukemia. On the other hand, substitutions in residue D868 lead to the largest increase in protein levels. Individuals with germline mutations affecting D868 have enhanced cell proliferation in vitro and higher incidence of cancer compared to patients with other germline SETBP1 mutations. Our findings substantiate that, despite their overlap, somatic SETBP1 mutations driving malignancy are more disruptive to the degron than germline SETBP1 mutations causing SGS. Additionally, this suggests that the functional threshold for the development of cancer driven by the disruption of the SETBP1 degron is higher than for the alteration in prenatal development in SGS. Drawing on previous studies of somatic SETBP1 mutations in leukemia, our results reveal a genotype-phenotype correlation in germline SETBP1 mutations spanning a molecular, cellular and clinical phenotype.

KIAA0586 is Mutated in Joubert Syndrome.

Joubert syndrome (JS) is a recessive neurodevelopmental disorder characterized by a distinctive mid-hindbrain malformation. JS is part of a group of disorders called ciliopathies based on their overlapping phenotypes and common underlying pathophysiology linked to primary cilium dysfunction. Biallelic mutations in one of 28 genes, all encoding proteins localizing to the primary cilium or basal body, can cause JS. Despite this large number of genes, the genetic cause can currently be determined in about 62% of individuals with JS. To identify novel JS genes, we performed whole exome sequencing on 35 individuals with JS and found biallelic rare deleterious variants (RDVs) in KIAA0586, encoding a centrosomal protein required for ciliogenesis, in one individual. Targeted next-generation sequencing in a large JS cohort identified biallelic RDVs in eight additional families for an estimated prevalence of 2.5% (9/366 JS families). All affected individuals displayed JS phenotypes toward the mild end of the spectrum.

Analysis of LMNB1 duplications in autosomal dominant leukodystrophy provides insights into duplication mechanisms and allele-specific expression.

Autosomal dominant leukodystrophy (ADLD) is an adult onset demyelinating disorder that is caused by duplications of the lamin B1 (LMNB1) gene. However, as only a few cases have been analyzed in detail, the mechanisms underlying LMNB1 duplications are unclear. We report the detailed molecular analysis of the largest collection of ADLD families studied, to date. We have identified the minimal duplicated region necessary for the disease, defined all the duplication junctions at the nucleotide level and identified the first inverted LMNB1 duplication. We have demonstrated that the duplications are not recurrent; patients with identical duplications share the same haplotype, likely inherited from a common founder and that the duplications originated from intrachromosomal events. The duplication junction sequences indicated that nonhomologous end joining or replication-based mechanisms such fork stalling and template switching or microhomology-mediated break induced repair are likely to be involved. LMNB1 expression was increased in patients' fibroblasts both at mRNA and protein levels and the three LMNB1 alleles in ADLD patients show equal expression, suggesting that regulatory regions are maintained within the rearranged segment. These results have allowed us to elucidate duplication mechanisms and provide insights into allele-specific LMNB1 expression levels.

Morquio-like dysostosis multiplex presenting with neuronopathic features is a distinct GLB1-related phenotype.

BACKGROUND: Morquio B disease (MBD) is a distinct GLB1-related dysostosis multiplex presenting a mild phenocopy of GALNS-related Morquio A disease. Previously reported cases from European countries carry the W273L variant on at least one GLB1 allele and exhibit a pure skeletal phenotype (pure MBD). Only a minority of MBD cases have been described with additional neuronopathic findings (MBD plus). OBJECTIVES AND METHODS: With the aim to further describe patterns of MBD-related dysostosis multiplex, we analyzed clinical, biochemical, and genetic features in 17 cases with GLB1-related dysostosis multiplex living and diagnosed in Brazil. RESULTS: About 14 of the 17 individuals had three or more skeletal findings characteristic of Morquio syndrome. Two had no additional neuronopathic features (pure MBD) and 12 exhibited additional neuronopathic features (MBD plus). Three of the 17 cases had mild dysostosis without distinct features of MBD. Seven of the 12 MBD plus patients had signs of spinal cord compression (SCC), as a result of progressive spinal vertebral dysostosis. There was an age-dependent increase in the number of skeletal findings and in the severity of growth impairment. GLB1 mutation analysis was completed in 10 of the 14 MBD patients. T500A occurred in compound heterozygosity in 8 of the 19 alleles. CONCLUSION: Our study extends the phenotypic spectrum of GLB1-related conditions by describing a cohort of patients with MBD and GM1-gangliosidosis (MBD plus). Targeting the progressive nature of the skeletal manifestations in the development of new therapies for GLB1-related conditions is warranted.

Next Generation Molecular Diagnosis of Hereditary Spastic Paraplegias: An Italian Cross-Sectional Study.

Hereditary spastic paraplegia (HSP) refers to a group of genetically heterogeneous neurodegenerative motor neuron disorders characterized by progressive age-dependent loss of corticospinal motor tract function, lower limb spasticity, and weakness. Recent clinical use of next generation sequencing (NGS) methodologies suggests that they facilitate the diagnostic approach to HSP, but the power of NGS as a first-tier diagnostic procedure is unclear. The larger-than-expected genetic heterogeneity-there are over 80 potential disease-associated genes-and frequent overlap with other clinical conditions affecting the motor system make a molecular diagnosis in HSP cumbersome and time consuming. In a single-center, cross-sectional study, spanning 4 years, 239 subjects with a clinical diagnosis of HSP underwent molecular screening of a large set of genes, using two different customized NGS panels. The latest version of our targeted sequencing panel (SpastiSure3.0) comprises 118 genes known to be associated with HSP. Using an in-house validated bioinformatics pipeline and several in silico tools to predict mutation pathogenicity, we obtained a positive diagnostic yield of 29% (70/239), whereas variants of unknown significance (VUS) were found in 86 patients (36%), and 83 cases remained unsolved. This study is among the largest screenings of consecutive HSP index cases enrolled in real-life clinical-diagnostic settings. Its results corroborate NGS as a modern, first-step procedure for molecular diagnosis of HSP. It also disclosed a significant number of new mutations in ultra-rare genes, expanding the clinical spectrum, and genetic landscape of HSP, at least in Italy.

Plasma lysosphingomyelin demonstrates great potential as a diagnostic biomarker for Niemann-Pick disease type C in a retrospective study.

Niemann-Pick disease type C (NP-C) is a devastating, neurovisceral lysosomal storage disorder which is characterised by variable manifestation of visceral signs, progressive neuropsychiatric deterioration and premature death, caused by mutations in the NPC1 and NPC2 genes. Due to the complexity of diagnosis and the availability of an approved therapy in the EU, improved detection of NP-C may have a huge impact on future disease management. At the cellular level dysfunction or deficiency of either the NPC1 or NPC2 protein leads to a complex intracellular endosomal/lysosomal trafficking defect, and organ specific patterns of sphingolipid accumulation. Lysosphingolipids have been shown to be excellent biomarkers of sphingolipidosis in several enzyme deficient lysosomal storage disorders. Additionally, in a recent study the lysosphingolipids, lysosphingomyelin (SPC) and glucosylsphingosine (GlcSph), appeared to be elevated in the plasma of three adult NP-C patients. In order to investigate the clinical utility of SPC and GlcSph as diagnostic markers, an in-depth fit for purpose biomarker assay validation for measurement of these biomarkers in plasma by liquid chromatography-tandem mass spectrometry was performed. Plasma SPC and GlcSph are stable and can be measured accurately, precisely and reproducibly. In a retrospective analysis of 57 NP-C patients and 70 control subjects, median plasma SPC and GlcSph were significantly elevated in NP-C by 2.8-fold and 1.4-fold respectively. For miglustat-naïve NP-C patients, aged 2-50 years, the area under the ROC curve was 0.999 for SPC and 0.776 for GlcSph. Plasma GlcSph did not correlate with SPC levels in NP-C patients. The data indicate excellent potential for the use of lysosphingomyelin in NP-C diagnosis, where it could be used to identify NP-C patients for confirmatory genetic testing.

Phenotypic variability in ARCA2 and identification of a core ataxic phenotype with slow progression.

Autosomal recessive cerebellar ataxia 2 (ARCA2) is a recently identified recessive ataxia due to ubiquinone deficiency and biallelic mutations in the ADCK3 gene. The phenotype of the twenty-one patients reported worldwide varies greatly. Thus, it is difficult to decide which ataxic patients are good candidates for ADCK3 screening without evidence of ubiquinone deficiency. We report here the clinical and molecular data of 10 newly diagnosed patients from seven families and update the disease history of four additional patients reported in previous articles to delineate the clinical spectrum of ARCA2 phenotype and to provide a guide to the molecular diagnosis. First signs occurred before adulthood in all 14 patients. Cerebellar atrophy appeared in all instances. The progressivity and severity of ataxia varied greatly, but no patients had the typical inexorable ataxic course that characterizes other childhood-onset recessive ataxias. The ataxia was frequently associated with other neurological signs. Importantly, stroke-like episodes contributed to significant deterioration of the neurological status in two patients. Ubidecarenone therapy markedly improved the movement disorders, including ataxia, in two other patients. The 7 novel ADCK3 mutations found in the 10 new patients were two missense and five truncating mutations. There was no apparent correlation between the genotype and the phenotype. Our series reveals that the clinical spectrum of ARCA2 encompasses a range of ataxic phenotypes. On one end, it may manifest as a pure ataxia with very slow progressivity and, on the other end, as a severe infantile encephalopathy with cerebellar atrophy. The phenotype of most patients, however, lies in between. It is characterized by a very slowly progressive or apparently stable ataxia associated with other signs of central nervous system involvement. We suggest undergoing the molecular analysis of ADCK3 in patients with this phenotype and in those with cerebellar atrophy and a stroke-like episode. The diagnosis of patients with a severe ARCA2 phenotype may also be performed on the basis of biological data, i.e. low ubiquinone level or functional evidence of ubiquinone deficiency. This diagnosis is crucial since the neurological status of some patients may be improved by ubiquinone therapy.