Andrea Mignarri

Mutations in SLC30A10 cause parkinsonism and dystonia with hypermanganesemia, polycythemia, and chronic liver disease.

Manganese is essential for several metabolic pathways but becomes toxic in excessive amounts. Manganese levels in the body are therefore tightly regulated, but the responsible protein(s) remain incompletely known. We studied two consanguineous families with neurologic disorders including juvenile-onset dystonia, adult-onset parkinsonism, severe hypermanganesemia, polycythemia, and chronic hepatic disease, including steatosis and cirrhosis. We localized the genetic defect by homozygosity mapping and then identified two different homozygous frameshift SLC30A10 mutations, segregating with disease. SLC30A10 is highly expressed in the liver and brain, including in the basal ganglia. Its encoded protein belongs to a large family of membrane transporters, mediating the efflux of divalent cations from the cytosol. We show the localization of SLC30A10 in normal human liver and nervous system, and its depletion in liver from one affected individual. Our in silico analyses suggest that SLC30A10 possesses substrate specificity different from its closest (zinc-transporting) homologs. We also show that the expression of SLC30A10 and the levels of the encoded protein are markedly induced by manganese in vitro. The phenotype associated with SLC30A10 mutations is broad, including neurologic, hepatic, and hematologic disturbances. Intrafamilial phenotypic variability is also present. Chelation therapy can normalize the manganesemia, leading to marked clinical improvements. In conclusion, we show that SLC30A10 mutations cause a treatable recessive disease with pleomorphic phenotype, and provide compelling evidence that SLC30A10 plays a pivotal role in manganese transport. This work has broad implications for understanding of the manganese biology and pathophysiology in multiple human organs.

Primary familial brain calcification: Genetic analysis and clinical spectrum

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.

Narcolepsy is a common phenotype in HSAN IE and ADCA-DN

We report on the extensive phenotypic characterization of five Italian patients from four unrelated families carrying dominant heterozygous DNMT1 mutations linked to two distinct autosomal dominant diseases: hereditary sensory and autonomic neuropathy with dementia and hearing loss type IE (HSAN IE) and autosomal dominant cerebellar ataxia, deafness and narcolepsy (ADCA-DN). Patients underwent genetic analysis of DNMT1 gene, neurophysiological tests investigating sleep, auditory functions and peripheral nervous system, ophthalmological studies including optical coherence tomography, lymphoscintigraphy, brain magnetic resonance and nuclear imaging, cerebrospinal fluid hypocretin-1, total tau, phosphorylated tau, amyloid-β1-42 and 14-3-3 proteins measurement, skin, muscular and sural nerve biopsies. Exome and direct sequencing studies disclosed two different point mutations affecting exon 21 of DNMT1 gene in patients with ADCA-DN, a novel heterozygous point mutation in exon 20 in two affected HSAN IE siblings, and a trinucleotide deletion in exon 20 in the latter patient with HSAN IE. Phenotypic characterization pinpoints that ADCA-DN and HSAN IE represent two discrete clinical entities belonging to the same disease spectrum, with variable degree of overlap. Remarkably, narcolepsy with or without cataplexy with low/intermediate or normal cerebrospinal fluid hypocretin-1 is present in both diseases. The human leukocyte antigen DQB1*06:02 was absent in all patients. Other common symptoms and features observed in our cases, involving the central and peripheral nervous system, include deafness, optic neuropathy-previously not reported in HSAN IE-large and small fibres polyneuropathy and lower limbs oedema. Overall, the two syndromes share more characteristics than previously recognized and narcolepsy is common to both. HSAN IE and ADCA-DN are two extreme phenotypic manifestations of a DNMT1 methylopathy.

Clinical relevance and neurophysiological correlates of spasticity in cerebrotendinous xanthomatosis.

Cerebrotendinous xanthomatosis (CTX) is a rare neurometabolic disease due to defective activity of sterol 27-hydroxylase, with plasma and tissue cholestanol storage. Clinical phenotype is characterized by both systemic manifestations and neurological signs. Therapy with chenodeoxycholic acid (CDCA) suppresses abnormal bile acid synthesis. The purpose of the study was to assess the frequency and clinical relevance of spasticity in the CTX phenotype and to study the usefulness of transcranial magnetic stimulation (TMS) in detecting corticospinal tract damage and monitoring the effects of replacement therapy. Twenty-four CTX patients underwent clinical evaluation including general disability scores, pyramidal and cerebellar function scales, assessment of serum cholestanol and TMS. Nine patients who started CDCA therapy at baseline received clinical and neurophysiological follow up. All patients showed signs of pyramidal damage which were relevant for clinical disability in 18 out of 24 cases (75%), resulting in spastic paraparesis. TMS revealed corticospinal alterations even in subjects with mild clinical signs of corticospinal tract involvement. After CDCA treatment, serum cholestanol decreased to normal concentrations in all patients. Clinical picture was unchanged in seven out of nine cases; in two others pyramidal signs disappeared. A reduction in abnormal neurophysiological parameters was found. Spastic paraparesis is the most frequent and relevant neurological feature in CTX patients. Replacement treatment with CDCA can prevent the progression of pyramidal damage, especially if started early in the course of the disease. TMS represents a sensitive indicator of corticospinal tract dysfunction and subclinical improvements in pyramidal function after CDCA therapy.

Four novel CYP27A1 mutations in seven Italian patients with CTX

Background and purpose:  Cerebrotendinous xanthomatosis (CTX) is a rare autosomal recessive disease, because of sterol 27‐hydroxylase deficiency. Clinical manifestations of CTX are tendon xanthomas, juvenile cataracts, osteoporosis, diarrhoea and multiple progressive neurological dysfunctions. More than 300 patients with CTX have been reported to date worldwide and about fifty different mutations identified in CYP27A1 gene. This study describes the clinical and laboratory findings of seven new patients.

Two-year follow-up after chelating therapy in a patient with adult-onset parkinsonism and hypermanganesaemia due to SLC30A10 mutations

A new inherited metal storage disease characterized by hypermanganesaemia, polycythaemia, dystonia or parkinsonism, and liver disease has been recently described as a result of SLC30A10 mutations [1, 2]. This autosomal recessive syndrome delineates a primary manganese metabolism disorder and suggests that the causative gene is a critical regulator of manganese transport. In relationship to the primary metabolic disturbance, the efficacy of chelation therapy has been reported in two previous studies [3, 4]. Here we report a two-year followup of a 60-year-old Italian subject carrying SLC30A10 mutations (patient SIE-03 of our previous article) [1]. At baseline, neurological examination showed hypomimia, monotone speech, global bradykinesia, mild rigidity, widebased gate with freezing, and starting hesitation with an unified Parkinson’s disease rating scale-part III (UPDRSIII) score of 27/108. Laboratory investigations revealed increased manganese concentrations in blood and urine [106 mcg/l, normal range (n.r.) 3–8; 16 mcg/24 h, n.r. 0.5–4], polycythaemia, thrombocytopenia, increased blood transferrin and decreased ferritin. Abdominal ultrasound showed hepatomegaly with liver steatosis, nevertheless, liver function parameters were within the normal range. The MRI showed T1-weighted hyperintensities in the basal ganglia and cerebellum, characteristic of manganese deposition. We also quantified the signal intensity of the globus pallidus by calculating the pallidal index (PI) with Krieger’s formula [5]. At baseline PI was 168. The patient received chelation therapy with infusions of CaNa2-EDTA for five consecutive days every two months (20 mg/kg per dose). Before and after each cycle, we assessed blood and urinary Mn levels as well as haemochrome, creatinine, ferritin, serum iron and transferrin. The neurological course was followed using the UPDRS-III score at the end of each cycle. The first CaNa2-EDTA infusion resulted in a marked increase in 24 h urinary manganese excretion, while hypermanganesaemia, polycythaemia and iron metabolism alterations persisted (Fig. 1). The UPDRS-III was sharply reduced from 27/108 to 13/108. A marked decrease in blood manganese levels was observed after the fourth cycle, while high urinary manganese excretion persisted. From the fifth cycle onward, a stabilization of UPDRS-III score (4–6/108) was observed. After 18 months of regular treatment, the progression of manganese accumulation had been prevented, and the patient’s neurological examination was substantially unchanged. At month 16, oral ferrous sulphate was added to limit intestinal dietary manganese absorption, but we discontinued it after one month due to gastrointestinal disturbances (dyspepsia and diarrhea). Abdominal ultrasound showed improvement of steatosis, while brain MRI did not demonstrate substantial variations L. Di Toro Mammarella (&) A. Mignarri C. Battisti A. Federico Department of Medicine, Surgery and Neuroscience, University of Siena, 53100 Siena, Italy e-mail: laraditorom@libero.it

Parkinsonism as neurological presentation of late-onset cerebrotendinous xanthomatosis

Cerebrotendinous xanthomatosis (CTX) is an inherited multisystem lipid storage disease due to mutations of CYP27A1 gene resulting in sterol-27-hydroxylase enzyme deficiency and increased concentration of plasma and tissue cholestanol. The phenotype is characterized by bilateral cataract and diarrhea in infancy, whereas tendon xanthomas, osteoporosis and neurological involvement including behavioural and cognitive impairment, spastic paraparesis and cerebellar ataxia usually present in early adulthood [1]. Signal abnormalities of the dentate nuclei on magnetic resonance imaging (MRI) of the brain are considered a characteristic feature of the disease [2]. Replacement therapy with chenodeoxycholic acid (CDCA) suppresses abnormal bile acid synthesis and stabilizes clinical and laboratory parameters. Parkinsonism has been described in a few CTX cases. In these subjects, extrapyramidal signs were invariably associated with other neurological manifestations such as limb spasticity, cerebellar ataxia, neurobehavioral changes and epilepsy [3]. Here we report a patient with parkinsonism as the only neurological manifestation of CTX, presenting during the seventh decade of life. A 67 year-old Italian female patient was recently referred to our Unit for a five-year history of bradykinesia, gait unsteadiness and resting tremor, associated with mild cognitive impairment and depression. Long-term treatment with high-dose oral levodopa (600 mg/day for six months) had failed to improve clinical manifestations. Family history was unremarkable. She had worked as an employee up to the age of retirement. Severe osteoporosis and multiple bone fractureswere reported over the last tenyears. Moreover, small sized Achilles tendon xanthomas were observed since her fifties. Otherwise, clinical history was unremarkable. On admission, neurological examination showed mild bradykinesia, slight hypomimia, monotone speech, asymmetric resting tremor of hand and foot (right> left) with moderate amplitude and low frequency (5–7 Hz), resting tremor of the head with mild amplitude and low frequency (3–5 Hz), moderate impairment of hand movements andfinger taps (right> left). Unified Parkinson’s disease rating scale (UPDRS)motorexamination (part III) scorewas27.Minimental state examination (MMSE) score was 24/30. No visual impairment was reported. However, ophthalmological evaluation uncovered initial cataract bilaterally. Serum cholestanol level (0.618 mg/dl) was slightly higher than normal (0.337 0.155 mg/dl). Brain MRI revealed the presence of diffuse white matter lesions, cortical and cerebellar atrophy, and signal abnormalities of the substantia nigra bilaterally (Fig. 1). MR signal of the dentate nuclei was normal (Fig. 1). Single photon emission computed tomography (SPECT) of

The spectrum of magnetic resonance findings in cerebrotendinous xanthomatosis: redefinition and evidence of new markers of disease progression

Cerebrotendinous xanthomatosis (CTX) is a metabolic disease characterized by systemic signs and neurological impairment, which can be prevented if chenodeoxycholic acid (CDCA) treatment is started early. Despite brain MRI represents an essential diagnostic tool, the spectrum of findings is worth to be reappraised, and follow-up data are needed. We performed clinical evaluation and brain MRI in 38 CTX patients. Sixteen of them who were untreated at baseline examination underwent clinical and MRI follow-up after long-term treatment with CDCA. Brain MRI abnormalities included cortical and cerebellar atrophy, and T2W/FLAIR hyperintensity involving subcortical, periventricular, and cerebellar white matter, the brainstem and the dentate nuclei. Regarding the dentate nuclei, we also observed T1W/FLAIR hypointensity consistent with cerebellar vacuolation and T1W/FLAIR/SW hypointense alterations compatibly with calcification in a subgroup of patients. Long-term follow-up showed that clinical and neuroradiological stability or progression were almost invariably associated. In patients with cerebellar vacuolation at baseline, a worsening over time was observed, while subjects lacking vacuoles were clinically and neuroradiologically stable at follow-up. The brains of CTX patients very often show both supratentorial and infratentorial abnormalities at MRI, the latter being related to clinical disability and including a wide spectrum of dentate nuclei alterations. The presence of cerebellar vacuolation may be regarded as a useful biomarker of disease progression and unsatisfactory response to therapy. On the other hand, the absence of dentate nuclei signal alteration should be considered an indicator of better prognosis.

Cerebrotendinous xanthomatosis with progressive cerebellar vacuolation

Dear Sir, Cerebrotendinous xanthomatosis (CTX) is a rare autosomal recessive lipid storage disease with deposition of cholestanol in many tissues causing both systemic and neurological involvement. CTX is due to mutations in the CYP27A1 gene leading to defective activity of sterol 27-hydroxylase. Therapy with chenodeoxycholic acid (CDCA) normalizes bile acids synthesis and prevents clinical deterioration. Brain magnetic resonance imaging (MRI) typically includes dentate nuclei hyperintensities on T2-weighted and FLAIR sequences, and has been reported to be substantially stable after treatment with CDCA [1, 2]. We report on a 33-year-old woman with gait unsteadiness since the age of 30 years. Her history revealed mental retardation, diarrhea and cataracts. Clinical examination disclosed dysarthria and ataxia. Tendon xanthomas were absent. Biochemical analysis revealed elevated plasma levels of cholestanol and 7α-hydroxy-4-cholesten-3-one, and decreased 27-hydroxycholesterol (27OHC) concentration. CYP27A1 gene analysis uncovered the 752C>A homozygous mutation. Therapy with CDCA was started and the patient periodically re-evaluated. Tendon xanthomas never emerged, and diarrhea disappeared. By contrast, ataxia worsened over time (Fig. 1a). A sharp reduction of cholestanol and 7α-hydroxy-4-cholesten-3-one serum levels was found (Fig. 1b, c), while 27OHC still showed very low concentrations without substantial changes from baseline. Brain MRI at 1.5 T was performed every 2 years until the age of 39 years together with clinical and biochemical follow-up. At baseline, T2-weighted and FLAIR hyperintensities in the dentate nuclei and surrounding white matter (WM) were evident. Areas of hypointensity on both T1-weighted and FLAIR images were found within cerebellar lesions. Computed tomography, and susceptibility weighted and T2*-weighted MRI ruled out the presence of calcifications. Six-year MRI follow-up showed on FLAIR images increase of the hypointense areas, which replaced hyperintensities suggesting vacuolation (Fig. 1d). MR spectroscopy (MRS) on the cerebellar hemispheres (Fig. 2) revealed the following: i) diffuse decrease of N-acetylaspartate (NAA)/creatine (Cr)01.58 (n.v. 2.27±0.29) and choline (Cho)/Cr00.53 (n.v. 0.78±0.04), and increased myoinositol (mI)/Cr ratio, mI/Cr00.43 (n.v. 0.27±0.03) in normal appearing areas; ii) lactate (Lac) and lipids (Lip) peaks in the voxels of the FLAIRhyperintense areas surrounding the FLAIR-hypointense lesions; and iii) very high Lac and Lip peaks and marked NAA/Cr decrease in the FLAIR-hypointense lesions. The serial evaluation of clinical, biochemical and MRI findings in this patient revealed that CDCA therapy was effective in decreasing serum levels of bile acids intermediates and stabilizing systemic manifestations, but failed to prevent worsening of neurological disturbances and brain lesions. The lack of correspondence between neurological and biochemical data is a crucial point, which remains open to a wide range of interpretations. A. Mignarri :M. T. Dotti (*) :G. N. Gallus :A. Giorgio Department of Neurological and Behavioural Sciences, University of Siena, Viale Bracci 2, 53100 Siena, Italy e-mail: dotti@unisi.it

Mitochondrial dysfunction in hereditary spastic paraparesis with mutations in DDHD1/SPG28

Mutations in DDHD1 cause the SPG28 subtype of hereditary spastic paraplegia (HSP). Recent studies suggested that mitochondrial dysfunction occurs in SPG28. Here we describe two siblings with SPG28, and report evidence of mitochondrial impairment in skeletal muscle and skin fibroblasts. Patient 1 (Pt1) was a 35-year-old man with spastic paraparesis and urinary incontinence, while his 25-year-old brother (Pt2) had gait spasticity and motor axonal neuropathy. In these patients we identified the novel homozygous c.1429C>T/p.R477* mutation in DDHD1, using a next-generation sequencing (NGS) approach. Histochemical analyses in muscle showed mitochondrial alterations, and multiple mitochondrial DNA (mtDNA) deletions were evident. In Pt1, respiratory chain enzyme activities were altered in skeletal muscle, mitochondrial ATP levels reduced, and analysis of skin fibroblasts revealed mitochondrial fragmentation. It seems possible that the novel nonsense mutation identified abolishes DDHD1 protein function thus altering oxidative metabolism. Qualitative alterations of mtDNA could have a pathogenetic significance. We suggest to perform DDHD1 analysis in patients with multiple mtDNA deletions.