Fibroblast growth factor 21 and grow differentiation factor 15 are sensitive biomarkers of mitochondrial diseases due to mitochondrial transfer-RNA mutations and mitochondrial DNA deletions.

BACKGROUND: Diagnosis of mitochondrial diseases (MDs) is challenging, since they are multisystemic disorders, characterized by a heterogeneous symptomatology. Recently, an increase in serum levels of fibroblast growth factor 21 (FGF21) and growth differentiation factor 15 (GDF15) has been found in the majority of patients with MDs compared with healthy controls. On the other hand, the finding of low FGF21 and GDF15 levels in some patients with MDs suggests that different types of respiratory chain defects may lead to different profiles of these two proteins. OBJECTIVE: In this study, we aimed to validate the diagnostic reliability of FGF21 and GDF15 assays in MDs and to evaluate a possible correlation between serum levels of the two biomarkers with genotype of MD patients. Serum FGF21 and GDF15 levels were measured by a quantitative ELISA. RESULTS: Our results showed increased serum FGF21 and GDF15 levels in MD patients; however, GDF15 measurement seems to be more sensitive and specific for screening tests for MD than FGF21. Moreover, we showed a positive correlation with both FGF21 and GDF15 levels and the number of COX-negative fibers. CONCLUSION: Finally, we also demonstrated that the increase of FGF21 and GDF15 was related to MDs caused by mitochondrial translation defects, and multiple and single mtDNA deletions, but not to MDs due to mutations in the respiratory chain subunits.

HTRA1 expression profile and activity on TGF-ß signaling in HTRA1 mutation carriers.

High temperature requirement A1 (HTRA1) is a serine protease playing a modulatory role in various cell processes, particularly in the regulation of transforming growth factor-ß (TGF-ß) signaling. A deleterious role in late-onset cerebral small vessel diseases (CSVDs) of heterozygous HTRA1 mutations, otherwise causative in homozygosity of cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy, was recently suggested. However, the pathomechanism of these heterozygous mutations is still undefined. Our aim is to evaluate the expression profile and activity of HTRA1 on TGF-ß signaling in fibroblasts from four subjects carrying the HTRA1 heterozygous mutations-p.E42Dfs*173, p.A321T, p.G295R, and p.Q151K. We found a 50% reduction of HTRA1 expression in HTRA1 mutation carriers compared to the control. Moreover, we showed no changes in TGF-ß signaling pathway downstream intermediate, Phospho Smad2/3. However, we found overexpression of genes involved in the extracellular matrix formation in two heterozygous HTRA1 carriers. Our results suggest that each heterozygous HTRA1 missense mutation displays a different and peculiar HTRA1 expression pattern and that CSVD phenotype may also result from 50% of HTRA1 expression.

Primary familial brain calcification with a novel SLC20A2 mutation: Analysis of PiT-2 expression and localization.

Primary familial brain calcification (PFBC) is an autosomal dominant rare disorder characterized by bilateral and symmetric brain calcifications, and neuropsychiatric manifestations. Four genes have been linked to PFBC: SLC20A2, PDGFRB, PDGFB, and XPR1. In this study, we report molecular and clinical data of a PFBC patient carrying a novel SLC20A2 mutation and we investigate the impact of the mutation on PiT-2 expression and function. Sanger sequencing of SLC20A2, PDGFRB, PDGFB, XPR1 led to the identification of a novel duplication of twelve nucleotides (c.1876_1887dup/ p.Trp626_Thr629dup) in SLC20A2 gene. SLC20A2 encodes for a cell membrane transporter (PiT-2) involved in maintenance of inorganic phosphate homeostasis. We performed an analysis of expression and functionality of PiT-2 protein in patient primary cultured fibroblasts. In patient fibroblasts, the mutation does not affect PiT-2 expression but alter sub-cellular localization. The Pi-uptake assay revealed a less Pi depletion in patient than in control fibroblasts, suggesting that SLC20A2 duplication may impair Pi internalization. This is the first study reporting sub-cellular expression analysis of mutant PiT-2 in primary cultured fibroblasts from a PFBC patient, showing that p.Trp626_Thr629dup in SLC20A2 alters PiT-2 sub-cellular localization and reduces Pi-uptake, leading to onset of PFBC in our patient.

Heterozygous mutations of HTRA1 gene in patients with familial cerebral small vessel disease.

AIMS: Cerebral small vessel disease (SVD) is the leading cause of vascular dementia. Although the most of cases are sporadic, familial monogenic causes have been identified in a growing minority of patients. CADASIL, due to mutations of NOTCH3 gene, is the most common genetic SVD, and CARASIL, linked to HTRA1 gene mutations, is a rare but well known autosomal recessive SVD. Recently, also heterozygous HTRA1 mutations have been described in patients with familial SVD. To detect a genetic cause of familial SVD, we performed mutational analysis of HTRA1 gene in a large cohort of Italian NOTCH3-negative patients. METHODS: We recruited 142 NOTCH3-negative patients and 160 healthy age-matched controls. Additional control data were obtained from five pathogenicity prediction software. RESULTS: Five different HTRA1 heterozygous mutations were detected in nine patients from five unrelated families. Clinical phenotype was typical of SVD, and the onset was presenile. Brain magnetic resonance imaging (MRI) showed a subcortical leukoencephalopathy, with involvement of the external and internal capsule, corpus callosum, and multiple lacunar infarcts. Cerebral microbleeds were also seen, while anterior temporal lobes involvement was not present. CONCLUSION: Our observation further supports the pathogenic role of the heterozygous HTRA1 mutations in familial SVD.

Hereditary diffuse leukoencephalopathy with axonal spheroids (HDLS): update on molecular genetics.

Hereditary diffuse leukoencephalopathy with spheroids (HDLS) is a rare autosomal dominant disease characterized by giant neuroaxonal swellings (spheroids) within the cerebral white matter (WM). Symptoms are variable and can include cognitive, mental and motor dysfunctions. Patients carry mutations in the protein kinase domain of the colony-stimulating factor 1 receptor (CSF1R) which is a tyrosine kinase receptor essential for microglia development. To date, more than 50 pathogenic variants have been reported in patients with HDLS, including missense, frameshift and non-sense mutations, but also deletions and splice-site mutations, all located in the intracellular tyrosine kinase domain, encoded by exons 12-22. The aim of this paper is to review the literature data about the molecular genetic pattern of HDLS.

A Novel CSF1R Mutation in a Patient with Clinical and Neuroradiological Features of Hereditary Diffuse Leukoencephalopathy with Axonal Spheroids.

Hereditary diffuse leukoencephalopathy with axonal spheroids (HDLS) is an autosomal dominant cerebral white matter degeneration leading to progressive cognitive and motor dysfunction. The peripheral nervous system is generally spared. Recently, mutations in the colony-stimulating factor-1 receptor (CSF1R) gene have been shown to be associated with HDLS. Here we report a new case of HDLS, carrying a mutation in CSF1R and manifesting rapidly progressive dementia and peripheral neuropathy.

Sporadic PEO caused by a novel POLG variation and a Twinkle mutation: digenic inheritance?

Progressive external ophthalmoplegia (PEO) with multiple deletions of mitochondrial DNA (mtDNA) is associated with several mutations in nuclear genes. They include POLG, POLG2, ANT1, C10orf2/Twinkle, and OPA1. However, digenic inheritance in mitochondrial disorders has been documented in a few cases over the years. Here we describe an 80-year-old man with sporadic PEO associated with mtDNA deletions. Sequencing of the POLG revealed a novel heterozygous mutation (c.2831A>G; p.Glu944Gly), predicted in silico as damaging, in the patient who also carried a heterozygous mutation in C10orf2/Twinkle (c.1142T>C; p.Leu381Pro). This case provides a second report of a PEO with different mutations in the POLG and C10orf2/Twinkle genes, supporting the hypothesis that the PEO phenotype can be determined by the co-existence of two abnormalities in separate genes, both involved in the maintenance and stability of mtDNA. Finally, this study expands the spectrum of POLG mutations and highlights the need to sequence the whole set of nuclear genes associated with PEO and multiple mtDNA deletions.

Primary familial brain calcification: update on molecular genetics.

Primary familial brain calcification is a neuropsychiatric disorder with calcium deposits in the brain, especially in basal ganglia, cerebellum and subcortical white matter. The disease is characterized by a clinical heterogeneity, with a various combination of symptoms that include movement disorders and psychiatric disturbances; asymptomatic patients have been also reported. To date, three causative genes have been found: SLC20A2, PDGFRB and PDGFB. SLC20A2 gene codes for the 'sodium-dependent phosphate transporter 2' (PiT-2), a cell membrane transporters of inorganic phosphate, involved in Pi uptake by cells and maintenance of Pi body levels. Over 40 pathogenic variants of SLC20A2 have been reported, affecting the regulation of Pi homeostasis. It was hypothesized that SLC20A2 mutations cause brain calcification most likely through haploinsufficiency. PDGFRB encodes for the platelet-derived growth factor receptor-ß (PDGFRß), a cell-surface tyrosine-kinase (RTK) receptor that regulates cell proliferation, migration, survival and differentiation. PDGFB encodes for the 'platelet-derived growth factor beta' (PDGFß), the ligand of PDGFRß. The loss of function of PDGFRß and PDGFß could lead to the impairment of the pericytes function and blood brain barrier integrity, causing vascular and perivascular calcium accumulation. SLC20A2 accounts for about 40 % of familial form and 14 % of sporadic cases, while PDGFRB and PDGFB mutations are likely rare. However, approximately 50 % of patients are not genetically defined and there should be at least another causative gene.

Primary familial brain calcification: Genetic analysis and clinical spectrum.

BACKGROUND: Primary familial brain calcification (PFBC) is a rare autosomal dominant disorder with bilateral calcification of basal ganglia and other cerebral regions, movement disorders, and neuropsychiatric disturbances. So far, three causative genes have been discovered: SLC20A2, PDGFRB and PDGFB, accounting for approximately 50% of cases. METHODS: Seven unrelated families with primary brain calcification were recruited to undergo clinical and genetic analysis, including Sanger sequencing of SLC20A2, PDGFRB, and PDGFB, and copy number analysis of SLC20A2. RESULTS: Mutations in SLC20A2 have been detected in three families: p.Glu368Glyfs*46, p.Ser434Trp, and p.Thr595Met. Intrafamilial phenotype variability has been observed. In spite of this, we found similar neuroimaging pattern among members of the same family. CONCLUSIONS: This molecular analysis expands the mutational spectrum of SLC20A2, which remains the major causative gene of primary familial brain calcification, and suggests the existence of disease-causing mutations in at least another, still unknown gene.

Hereditary diffuse leukoencephalopathy with axonal spheroids: three patients with stroke-like presentation carrying new mutations in the CSF1R gene.

Hereditary diffuse leukoencephalopathy with axonal spheroids (HDLS) is an autosomal dominant disorder characterized by white matter neurodegeneration, progressive cognitive decline, and motor symptoms. Histologically, it is characterized by axonal swellings ("spheroids"). To date, over 20 different mutations affecting the tyrosine kinase domain of the protein have been identified in the colony stimulating factor 1 receptor (CSF1R) gene. Our goal is to describe three unrelated Italian patients affected by HDLS and carrying new CSF1R mutations, thus expanding the mutational spectrum and phenotypic presentation. CSF1R gene analysis was performed in 15 patients (age range 25-83 years) with undefined leukoencephalopathy and progressive cognitive decline. In three patients (two males and one female, aged 58, 37, and 48 years, respectively), new heterozygous missense mutations affecting the protein tyrosine kinase domain of the CSF1R gene were detected. In all of these patients, behavioural and cognitive changes were preceded by an ischemic stroke-like episode. A positive family history was present in only one case.

Hereditary cerebral small vessel diseases: a review.

Cerebral microangiopathies are responsible of a great number of strokes. In the recent years advances in molecular genetics identified several monogenic conditions involving cerebral small vessels and predisposing to ischemic and/or hemorrhagic stroke and diffuse white matter disease leading to vascular dementia. Clinical features and diagnostic clues of these conditions, [cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL), COL4A1-related cerebral small vessel diseases, autosomal dominant retinal vasculopathy with cerebral leukodystrophy (AD-RVLC), and Fabry's disease] are here reviewed. Albeit with variable phenotypes and with different defective genes, all these disorders produce arteriopathy and microvascular disintegration with changes in brain functions. Specific diagnostic tools are recommended, genetic analysis being the gold standard for the diagnosis.