Severe 5,10-methylenetetrahydrofolate reductase deficiency: a rare, treatable cause of complicated hereditary spastic paraplegia.

BACKGROUND AND PURPOSE: Juvenile- or adult-onset forms of severe 5,10-methylenetetrahydrofolate reductase (MTHFR) deficiency manifesting as complicated hereditary spastic paraplegia have rarely been described. METHODS: Two siblings with mental retardation developed a progressive spastic paraparesis in their late teens. Their diagnostic assessment included extensive neurophysiologic, neuroimaging and metabolic studies. RESULTS: Brain magnetic resonance imaging showed occipital white matter alterations, and electromyography documented a mixed polyneuropathy. Severe hyperhomocisteinemia (>150 µmol/L) associated with the characteristic amino acid profile suggested a diagnosis of severe MTHFR deficiency, confirmed by MTHFR direct sequencing. Treatment with betaine and vitamins benefitted patients' symptoms and diagnostic features. CONCLUSIONS: Severe MTHFR deficiency can be a rare, treatable cause of autosomal recessive complicated hereditary spastic paraplegia. Its screening should be part of the diagnostic flowchart for these disorders.

FLNA genomic rearrangements cause periventricular nodular heterotopia.

OBJECTIVE: To identify copy number variant (CNV) causes of periventricular nodular heterotopia (PNH) in patients for whom FLNA sequencing is negative. METHODS: Screening of 35 patients from 33 pedigrees on an Affymetrix 6.0 microarray led to the identification of one individual bearing a CNV that disrupted FLNA. FLNA-disrupting CNVs were also isolated in 2 other individuals by multiplex ligation probe amplification. These 3 cases were further characterized by high-resolution oligo array comparative genomic hybridization (CGH), and the precise junctional breakpoints of the rearrangements were identified by PCR amplification and sequencing. RESULTS: We report 3 cases of PNH caused by nonrecurrent genomic rearrangements that disrupt one copy of FLNA. The first individual carried a 113-kb deletion that removes all but the first exon of FLNA. A second patient harbored a complex rearrangement including a deletion of the 3' end of FLNA accompanied by a partial duplication event. A third patient bore a 39-kb deletion encompassing all of FLNA and the neighboring gene EMD. High-resolution oligo array CGH of the FLNA locus suggests distinct molecular mechanisms for each of these rearrangements, and implicates nearby low copy repeats in their pathogenesis. CONCLUSIONS: These results demonstrate that FLNA is prone to pathogenic rearrangements, and highlight the importance of screening for CNVs in individuals with PNH lacking FLNA point mutations.

Periventricular heterotopia, mental retardation, and epilepsy associated with 5q14.3-q15 deletion.

BACKGROUND: Periventricular heterotopia (PH) is an etiologically heterogeneous disorder characterized by nodules of neurons ectopically placed along the lateral ventricles. Most affected patients have seizures and their cognitive level varies from normal to severely impaired. At present, two genes have been identified to cause PH when mutated. Mutations in FLNA (Xq28) and ARFGEF2 (20q13) are responsible for X-linked bilateral PH and a rare autosomal recessive form of PH with microcephaly. Chromosomal rearrangements involving the 1p36, 5p15, and 7q11 regions have also been reported in association with PH but the genes implicated remain unknown. Fourteen additional distinct anatomoclinical PH syndromes have been described, but no genetic insights into their causes have been gleaned. METHODS: We report the clinical and imaging features of three unrelated patients with epilepsy, mental retardation, and bilateral PH in the walls of the temporal horns of the lateral ventricles, associated with a de novo deletion of the 5q14.3-15 region. We used microarray-based comparative genomic hybridization to define the boundaries of the deletions. RESULTS: The three patients shared a common deleted region spanning 5.8 Mb and containing 14 candidate genes. CONCLUSION: We identified a new syndrome featuring bilateral periventricular heterotopia (PH), mental retardation, and epilepsy, mapping to chromosome 5q14.3-q15. This observation reinforces the extreme clinical and genetic heterogeneity of PH. Array comparative genomic hybridization is a powerful diagnostic tool for characterizing causative chromosomal rearrangements of limited size, identifying potential candidate genes for, and improving genetic counseling in, malformations of cortical development.

High frequency of genomic deletions--and a duplication--in the LIS1 gene in lissencephaly: implications for molecular diagnosis.

BACKGROUND: LIS1 is the main gene causing classical (isolated) lissencephaly predominating in the posterior brain regions (p>a). However, about 40% of patients with this malformation pattern show no abnormality after fluorescence in situ hybridisation (FISH) analysis of the 17p13.3 region and LIS1 sequencing. To investigate whether alternative gene(s) or genomic deletions/duplications of LIS1 may account for the high percentage of individuals who show no abnormalities on FISH and sequencing, we performed multiplex ligation dependent probe amplification assay (MLPA) in a series of patients. METHODS: We initially performed DNA sequencing in 45 patients with isolated lissencephaly with a p>a gradient, in whom FISH had revealed normal results. We subsequently performed MLPA in those who were mutation negative, and long range polymerase chain reaction (PCR) to characterise the breakpoint regions in patients in whom the deletions were small enough. RESULTS: We found LIS1 mutations in 44% of patients (20/45) of the whole sample and small genomic deletions/duplications in 76% of the remaining (19/25). Deletions were much more frequent than duplications (18 vs 1). Overall, small genomic deletions/duplications represented 49% (19/39) of all LIS1 alterations and brought to 87% (39/45) the number of patients in whom any involvement of LIS1 could be demonstrated. Breakpoint characterisation, performed in 5 patients, suggests that Alu mediated recombination is a major molecular mechanism underlying LIS1 deletions. CONCLUSIONS: LIS1 is highly specific for isolated p>a lissencephaly. The high frequency of genomic deletions/duplications of LIS1 is in keeping with the over representation of Alu elements in the 17p13.3 region. MLPA has a high diagnostic yield and should be used as first line molecular diagnosis for p>a lissencephaly.

Multiplex ligation-dependent probe amplification detects DCX gene deletions in band heterotopia.

BACKGROUND: Subcortical band heterotopia (SBH, or double cortex syndrome) is a neuronal migration disorder consisting of heterotopic bands of gray matter located between the cortex and the ventricular surface, with or without concomitant pachygyria. Most cases show diffuse or anteriorly predominant (A>P) migration abnormality. All familial and 53% to 84% of sporadic cases with diffuse or A>P SBH harbor a mutation of the DCX gene, leaving the genetic causes unexplained, and genetic counseling problematic, in the remaining patients. Our purpose was to verify the extent to which exonic deletions or duplications of the DCX gene would account for sporadic SBH with A>P gradient but normal gene sequencing. METHODS: We identified 23 patients (22 women, 1 man) with sporadic, diffuse, or anteriorly predominant SBH. After sequencing the DCX gene and finding mutations in 12 (11 women, 1 man), we used multiplex ligation-dependent probe amplification (MLPA) to search for whole-exon deletions or duplications in the 11 remaining women. We used semiquantitative fluorescent multiplex PCR (SQF-PCR) and Southern blot to confirm MLPA findings. RESULTS: MLPA assay uncovered two deletions encompassing exons 3 to 5, and one involving exon 6, in 3 of 11 women (27%) and raised the percentage of DCX mutations from 52% to 65% in our series. SQF-PCR performed in all three women and Southern blot analysis performed in two confirmed the deletions. CONCLUSIONS: MLPA uncovers large genomic deletions of the DCX gene in a subset of patients with SBH in whom no mutations are found after gene sequencing. Deletions of DCX are an underascertained cause of SBH.

Periventricular heterotopia: phenotypic heterogeneity and correlation with Filamin A mutations.

Periventricular heterotopia (PH) occurs when collections of neurons lay along the lateral ventricles or just beneath. Human Filamin A gene (FLNA) mutations are associated with classical X-linked bilateral periventricular nodular heterotopia (PNH), featuring contiguous heterotopic nodules, mega cisterna magna, cardiovascular malformations and epilepsy. FLNA encodes an F-actin-binding cytoplasmic phosphoprotein and is involved in early brain neurogenesis and neuronal migration. A rare, recessive form of bilateral PNH with microcephaly and severe delay is associated with mutations of the ADP-ribosylation factor guanine nucleotide-exchange factor-2 (ARFGEF2) gene, required for vesicle and membrane trafficking from the trans-Golgi. However, PH is a heterogeneous disorder. We studied clinical and brain MRI of 182 patients with PH and, based on its anatomic distribution and associated birth defects, identified 15 subtypes. Classical bilateral PNH represented the largest group (98 patients: 54%). The 14 additional phenotypes (84 patients: 46%) included PNH with Ehlers-Danlos syndrome (EDS), temporo-occipital PNH with hippocampal malformation and cerebellar hypoplasia, PNH with fronto-perisylvian or temporo-occipital polymicrogyria, posterior PNH with hydrocephalus, PNH with microcephaly, PNH with frontonasal dysplasia, PNH with limb abnormalities, PNH with fragile-X syndrome, PNH with ambiguous genitalia, micronodular PH, unilateral PNH, laminar ribbon-like and linear PH. We performed mutation analysis of FLNA in 120 patients, of whom 72 (60%) had classical bilateral PNH and 48 (40%) other PH phenotypes, and identified 25 mutations in 40 individuals. Sixteen mutations had not been reported previously. Mutations were found in 35 patients with classical bilateral PNH, in three with PNH with EDS and in two with unilateral PNH. Twenty one mutations were nonsense and frame-shift and four missense. The high prevalence of mutations causing protein truncations confirms that loss of function is the major cause of the disorder. FLNA mutations were found in 100% of familial cases with X-linked PNH (10 families: 8 with classical bilateral PNH, 1 with EDS and 1 with unilateral PH) and in 26% of sporadic patients with classical bilateral PNH. Overall, mutations occurred in 49% of individuals with classical bilateral PNH irrespective of their being familial or sporadic. However, the chances of finding a mutation were exceedingly gender biased with 93% of mutations occurring in females and 7% in males. The probability of finding FLNA mutations in other phenotypes was 4% but was limited to the minor variants of PNH with EDS and unilateral PNH. Statistical analysis considering all 42 mutations described so far identifies a hotspot region for PNH in the actin-binding domain (P < 0.05).

Filamin A mutations cause periventricular heterotopia with Ehlers-Danlos syndrome.

OBJECTIVE: To define the clinical, radiologic, and genetic features of periventricular heterotopia (PH) with Ehlers-Danlos syndrome (EDS). METHODS: Exonic sequencing and single stranded conformational polymorphism (SSCP) analysis was performed on affected individuals. Linkage analysis using microsatellite markers on the X-chromosome was performed on a single pedigree. Western blotting evaluated for loss of filamin A (FLNA) protein and Southern blotting assessed for any potential chromosome rearrangement in this region. RESULTS: The authors report two familial cases and nine additional sporadic cases of the EDS-variant form of PH, which is characterized by nodular brain heterotopia, joint hypermobility, and development of aortic dilatation in early adulthood. MRI typically demonstrated bilateral nodular PH, indistinguishable from PH due to FLNA mutations. Exonic sequencing or SSCP analyses of FLNA revealed a 2762 delG single base pair deletion in one affected female. Another affected female harbored a C116 single point mutation, resulting in an A39G change. A third affected female had a 4147 delG single base pair deletion. One pedigree with no detectable exonic mutation demonstrated positive linkage to the FLNA locus Xq28, an affected individual in this family also had no detectable FLNA protein, but no chromosomal rearrangement was detected. CONCLUSION: These results suggest that the Ehlers-Danlos variant of periventricular heterotopia (PH), in part, represents an overlapping syndrome with X-linked dominant PH due to filamin A mutations.

Germline and mosaic mutations of FLN1 in men with periventricular heterotopia.

OBJECTIVE: To describe the phenotypic spectrum and genetics of periventricular nodular heterotopia (PNH) caused by FLN1 mutations in four men. BACKGROUND: X-linked PNH caused by FLN1 mutations (MIM #300049) implies prenatal or early postnatal lethality in boys and 50% recurrence risk in daughters of affected women. METHODS: Clinical examination, cognitive testing, MRI, and mutation analysis (denaturing high-performance liquid chromatography and direct sequencing) on blood lymphocytes and single hair roots were performed for nine affected individuals, including three men. Neuropathologic study of the brain was performed for an affected boy. RESULTS: In two families, missense mutations were transmitted from mother to son (Met102Val) and from father to daughter (Ser149Phe), causing mild phenotypes in both genders, including unilateral PNH. In a third family, a man was mosaic for an A>G substitution (intron 11 acceptor splice site) on leukocyte DNA and hair roots (mutant = 42% and 69%). Single hair root analysis confirmed that the mutation was not present in all ectodermal derivative cells. A healthy daughter had inherited the X chromosome from her father's wild-type germinal cell population. In the fourth family, an eight-base deletion (AGGAGGTG, intron 25 donor splice site) led to early deaths of boys. Postmortem study in a newborn boy revealed PNH and cardiovascular, genitourinary, and gut malformations. CONCLUSIONS: Periventricular nodular heterotopia caused by FLN1 mutations in men has a wide clinical spectrum and is caused by different genetic mechanisms, including somatic mosaicism. Mutation analysis of FLN1 should support genetic counseling in men with periventricular nodular heterotopia.

[Effect of sex steroids in vivo and in vitro on the binding of uric acid to plasma proteins]. / Influenza degli steroidi sessuali in vivo ed in vitro sul legame acido urico-proteine plasmatiche.

Modifications on the binding of uric acid to human plasma proteins have been studied in regularly menstruating females aged 25-30 years with a normal cycle, in comparison with a group of healthy age-matched males and with a group of post-menopausal females. The binding of uric acid to plasma proteins was estimated using micropartition system Amicon. The results obtained demonstrate a significant increase of uric acid binding during ovulatory and mid-luteal phase of menstrual cycle. No modifications are shown in post-menopausal females and in healthy males. No modifications have been shown with the same experiments performed in vitro.