Clarissa Bueno

Screening of ARHSP-TCC patients expands the spectrum of SPG11 mutations and includes a large scale gene deletion.

Autosomal recessive spastic paraplegia with thinning of corpus callosum (ARHSP‐TCC) is a complex form of HSP initially described in Japan but subsequently reported to have a worldwide distribution with a particular high frequency in multiple families from the Mediterranean basin. We recently showed that ARHSP‐TCC is commonly associated with mutations in SPG11/KIAA1840 on chromosome 15q. We have now screened a collection of new patients mainly originating from Italy and Brazil, in order to further ascertain the spectrum of mutations in SPG11, enlarge the ethnic origin of SPG11 patients, determine the relative frequency at the level of single Countries (i.e., Italy), and establish whether there is one or more common mutation. In 25 index cases we identified 32 mutations; 22 are novel, including 9 nonsense, 3 small deletions, 4 insertions, 1 in/del, 1 small duplication, 1 missense, 2 splice‐site, and for the first time a large genomic rearrangement. This brings the total number of SPG11 mutated patients in the SPATAX collection to 111 cases in 44 families and in 17 isolated cases, from 16 Countries, all assessed using homogeneous clinical criteria. While expanding the spectrum of mutations in SPG11, this larger series also corroborated the notion that even within apparently homogeneous population a molecular diagnosis cannot be achieved without full gene sequencing.

Spastic Paraplegia, Optic Atrophy, and Neuropathy: New Observations, Locus Refinement, and Exclusion of Candidate Genes

SPOAN is an autosomal recessive neurodegenerative disorder which was recently characterized by our group in a large inbred Brazilian family with 25 affected individuals. This condition is clinically defined by: 1. congenital optic atrophy; 2. progressive spastic paraplegia with onset in infancy; and 3. progressive motor and sensory axonal neuropathy. Overall, we are now aware of 68 SPOAN patients (45 females and 23 males, with age ranging from 5 to 72 years), 44 of which are presented here for the first time. They were all born in the same geographic micro region. Those 68 patients belong to 43 sibships, 40 of which exhibit parental consanguinity. Sixty‐one patients were fully clinically evaluated and 64 were included in the genetic investigation. All molecularly studied patients are homozygotes for D11S1889 at 11q13. This enabled us to reduce the critical region for the SPOAN gene from 4.8 to 2.3 Mb, with a maximum two point lod score of 33.2 (with marker D11S987) and of 27.0 (with marker D11S1889). Three genes located in this newly defined critical region were sequenced, but no pathogenic mutation was detected. The gene responsible for SPOAN remains elusive.

A multi-exonic SPG4 duplication underlies sex-dependent penetrance of hereditary spastic paraplegia in a large Brazilian pedigree

SPG4 mutations are the most frequent cause of autosomal-dominant hereditary spastic paraplegia (HSP). SPG4 HSP is characterized by large inter- and intrafamilial variability in age at onset (AAO) and disease severity. The broad spectrum of SPG4 mutations has recently been further extended by the finding of large genomic deletions in SPG4-linked pedigrees negative for ‘small’ mutations. We had previously reported a very large pedigree, linked to the SPG4 locus with many affected members, which showed gender difference in clinical manifestation. Screening for copy number aberrations revealed the first case of a multi-exonic duplication (exon10_12dup) in the SPG4 gene. The mutation leads to a premature stop codon, suggesting that the protein product is not functional. The analysis of 30 individuals who carry the mutation showed that males have on average an earlier AAO and are more severely affected. The present family suggests that this HSP pathogenesis may be modulated by factors related to individual background and gender as observed for other autosomal dominant conditions, such as facio-scapulohumeral muscular dystrophy or amyloidosis. Understanding why some individuals, particularly women, are ‘partially protected’ from the effects of this and other pathogenic mutations is of utmost importance.

Reevaluation of a large family defines a new locus for X-linked recessive pure spastic paraplegia (SPG34) on chromosome Xq25

Herein, we report a new locus, named SPG34, for a pure form of X-linked hereditary spastic paraplegia (HSP) in a large Brazilian family followed by our group since 1976 [1]. In 2002, a study of seven patients suggested linkage to Xq22.2 [2]. We now were able to perform a more comprehensive clinical and molecular evaluation, including five patients not previously ascertained, and reassigned the putative locus to Xq25. After Institutional Review Board approval, we genotyped 12 affected individuals (aged 24 to 79 years), one unaffected 60-year-old man, and 11 women (aged 40 to 81), seven of which were obligate carriers (Fig. 1). Neurological examination was performed in 11 of the 12 affected men and in all obligate women carriers. Age of onset varied from 12 to 25 years but was sometimes difficult to be determined. The clinical phenotype was stereotyped, and shuffling gait was the first recognized clinical sign. The disease was invariably progressive, and after two decades of onset, patients usually need support to walk; after three to four decades, they are usually wheelchair-bound. In upper limbs, strength was never affected, even late in life, but tendon reflexes were brisk. Lower limbs spasticity was progressive and debilitating. Babinski sign, ankle clonus, and brisk reflexes were frequently present. Lower limb vibratory sensibility was commonly reduced after the sixth decade of life; spontaneous lower limb pain was also a common complaint. No urinary or bowel sphincter dysfunction were ever reported. The SPG34 locus encompasses a 14 cM region at Xq24– q25, in which 69 genes and bona fide transcripts have been assigned. Using a candidate gene approach to try to identify the causative sequence abnormality of SPG34, we fully sequenced AIFM1, which encodes a mitochondrial flavoprotein essential for apoptotic nuclear disassembly [3], but no mutation was detected. Therefore, the molecular basis for SPG34 remains unknown and we will sequence additional candidate genes that are highly expressed in the central nervous system. The calculated logarithm of the odds score of 4.13 at marker DXS8057 was much higher than in the previous study [2], and additional markers definitely excluded Xq22.2. No other X-linked HSP have been so far assigned to this region, which defines the fourth locus for X-linked HSP. Differently from the other two well-characterized X-linked HSP, SPG1 and SPG2 [4, 5], SPG34 is not associated to mental retardation. Evidences for the SPG16 [6] locus, located in a large region at Xq11.2–q23 overlapping SPG2 locus, are weaker and based only in two small families. In short, we identified in a large multigenerational family with pure spastic paraplegia a new locus (named SPG34) at Xq25 for an X-linked pure form of HSP, with onset in the Neurogenetics (2008) 9:225–226 DOI 10.1007/s10048-008-0130-8

Further diffusion tensor imaging contribution in horizontal gaze palsy and progressive scoliosis

In two siblings with clinical diagnosis of horizontal gaze palsy associated with progressive scoliosis (HGPPS) we could demonstrate by diffusion tensor imaging: (1) An anterior displacement of the transverse pontine fibers; (2) Posterior clumping of the corticospinal, medial lemniscus and central tegmental tracts and of the medial and dorsal longitudinal fasciculi complex; (3) Absent decussation of superior cerebellar peduncle. Those findings can contribute as surrogate markers for the diagnosis.

Thrombotic microangiopathy caused by methionine synthase deficiency: diagnosis and treatment pitfalls

BackgroundInborn errors of cobalamin (Cbl) metabolism form a large group of rare diseases. One of these, Cbl deficiency type C (CblC), is a well-known cause of thrombotic microangiopathy (TMA), especially in infants. However, there has only been a single published case of TMA associated to Cbl deficiency type G (CblG), also known as methionine synthase deficiency (MSD).Case diagnosis/treatmentA 21-month-old boy presented with pallor and oral ulcers during episodes of upper respiratory infection (URI). Further examination revealed signs of TMA, and the patient progressed to acute renal failure (ARF). Renal biopsy showed TMA. Evaluation for infection and autoantibodies were negative. The C3 and C4 complement fractions were normal. Analysis of the bone marrow aspirate suggested megaloblastic anemia and signs of hematopoiesis activation (secondary to peripheral hemolysis). Although the serum vitamin B12 level was normal, the patient was treated with cyanocobalamin, with no improvement. The ARF and hematologic parameters improved with conservative treatment. A severe relapse occurred during the follow-up, with normal ADAMTS13 activity. The presumed diagnosis was atypical hemolytic uremic syndrome, and the patient was started on eculizumab, but his response was poor, even when the dosage was increased. At this point it was also recognized that his developmental speech was delayed. Based on these findings, whole exome sequencing was performed, leading to the detection of two novel deleterious variants in the gene coding for methionine synthase, confirming the diagnosis of MSD. Subsequent treatment consisted of elevating the patient’s serum homocysteine level and starting him on hydroxicobalamin, with normalization of all hematologic parameters although the microalbuminuria remained.ConclusionsMethionine synthase deficiency is very rare and characterized by megaloblastic anemia and neurological symptoms. We report the second case of MSD associated to TMA previously diagnosed as aHUS in which the patient had a poor response to eculizumab.

Development of sleep/wake, activity and temperature rhythms in newborns maintained in a neonatal intensive care unit and the impact of feeding schedules.

UNLABELLED Biological rhythms in infants are described as evolving from an ultradian to a circadian pattern along the first months of life. Recently, the use of actigraphy and thermistors with memory has contributed to the understanding of temporal relations of different variables along development. The aim of this study was to describe and compare the development of the rhythmic pattern of wrist temperature, activity/rest cycle, sleep/wake and feeding behavior in term and preterm newborns maintained in a neonatal intensive care unit (NICU). METHODS Nineteen healthy preterm and seven fullterm newborns had the following variables monitored continuously while they were in the NICU: activity recorded by actigraphy, wrist temperature recorded with a thermistor and observed sleep and feeding behavior recorded by the NICU staff with diaries. Subjects were divided in 3 groups according to their gestational age at birth and rhythmic parameters were compared. RESULTS A dominant daily rhythm was observed for wrist temperature since the first two weeks of life and no age relation was demonstrated. Otherwise, a daily pattern in activity/rest cycle was observed for most preterm newborns since 35 weeks of postconceptional age and was more robust in term babies. Feeding and sleep/wake data showed an almost exclusive 3h rhythm, probably related to a masking effect of feeding schedules. CONCLUSIONS We found that wrist temperature develops a daily pattern as soon as previously reported for rectal temperature, and with acrophase profile similar to adults. Moreover, we were able to find a daily rhythm in activity/rest cycle earlier than previously reported in literature. We also suggest that sleep/wake rhythm and feeding behavior follow independent developmental courses, being more suitable to masking effects.

Haploidentical bone marrow transplantation with post transplant cyclophosphamide for patients with X-linked adrenoleukodystrophy: a suitable choice in an urgent situation

Allogeneic hematopoietic stem cell transplantation (HSCT) is the only treatment that enhances survival and stabilizes neurologic symptoms in X-linked adrenoleukodystrophy (X-ALD) with cerebral involvement, a severe demyelinating disease of childhood. Patients with X-ALD who lack a well-matched HLA donor need a rapid alternative. Haploidentical HSCT using post transplant cyclophosphamide (PT/Cy) has been performed in patients with malignant and nonmalignant diseases showing similar outcomes compared to other alternative sources. We describe the outcomes of transplants performed for nine X-ALD patients using haploidentical donors and PT/Cy. Patients received conditioning regimen with fludarabine 150 mg/m2, cyclophosphamide 29 mg/kg and 2 Gy total body irradiation (TBI) with or without antithymocyte globulin. Graft-vs.-host disease prophylaxis consisted of cyclophosphamide 50 mg/kg/day on days +3 and +4, tacrolimus or cyclosporine A and mycophenolate mofetil. One patient had a primary graft failure and was not eligible for a second transplant. Three patients had secondary graft failure and were successfully rescued with second haploidentical transplants. Trying to improve engraftment, conditioning regimen was changed, substituting 2 Gy TBI for 4 Gy total lymphoid irradiation. Eight patients are alive and engrafted (17–37 months after transplant). Haploidentical HSCT with PT/Cy is a feasible alternative for X-ALD patients lacking a suitable matched donor. Graft failure has to be addressed in further studies.

Breastfeeding, sleep and wake circadian rhythms show distinct temporal emerging patterns

The emerging patterns of breastfeeding, sleep and wake circadian rhythms in an infant and the breastfeeding emergence pattern of his elder sister are presented. Both children were raised under regular contact with photic and non-photic Zeitgebers. Data are related to the first four months of life of the infants, which correspond to the exclusive and ad libitum breastfeeding stage of their lives. Discrimination is made of “fasting-associated-wakefulness” (FAW) which is a wake state without feeding. Our data show that while FAW episodes are concentrated in the diurnal phase of the day since the first week of life, breastfeeding rhythm takes longer to show statistically significant circadian periodicity (four weeks) and to become monophasic, concentrated in the diurnal phase of the day (three/four months). This precedence of the consolidation of FAW rhythm indicates tight association between nocturnal sleep fragmentation and the drive to feed, in the first months of life of infants.

Melanopsin system dysfunction in Smith-Magenis syndrome patients

Purpose Smith-Magenis syndrome (SMS) causes sleep disturbance that is related to an abnormal melatonin profile. It is not clear how the genomic disorder leads to a disturbed synchronization of the sleep/wake rhythm in SMS patients. To evaluate the integrity of the intrinsically photosensitive retinal ganglion cell (ipRGC)/melanopsin system, the transducers of the light-inhibitory effect on pineal melatonin synthesis, we recorded pupillary light responses (PLR) in SMS patients. Methods Subjects were SMS patients (n = 5), with molecular diagnosis and melatonin levels measured for 24 hours and healthy controls (n = 4). Visual stimuli were 1-second red light flashes (640 nm; insignificant direct ipRGC activation), followed by a 470-nm blue light, near the melanopsin peak absorption region (direct ipRGC activation). Blue flashes produce a sustained pupillary constriction (ipRGC driven) followed by baseline return, while red flashes produce faster recovery. Results Pupillary light responses to 640-nm red flash were normal in SMS patients. In response to 470-nm blue flash, SMS patients had altered sustained responses shown by faster recovery to baseline. SMS patients showed impairment in the expected melatonin production suppression during the day, confirming previous reports. Conclusions SMS patients show dysfunction in the sustained component of the PLR to blue light. It could explain their well-known abnormal melatonin profile and elevated circulating melatonin levels during the day. Synchronization of daily melatonin profile and its photoinhibition are dependent on the activation of melanopsin. This retinal dysfunction might be related to a deficit in melanopsin-based photoreception, but a deficit in rod function is also possible.