Elena Parrini

Mutations in TUBG1, DYNC1H1, KIF5C and KIF2A cause malformations of cortical development and microcephaly

The genetic causes of malformations of cortical development (MCD) remain largely unknown. Here we report the discovery of multiple pathogenic missense mutations in TUBG1, DYNC1H1 and KIF2A, as well as a single germline mosaic mutation in KIF5C, in subjects with MCD. We found a frequent recurrence of mutations in DYNC1H1, implying that this gene is a major locus for unexplained MCD. We further show that the mutations in KIF5C, KIF2A and DYNC1H1 affect ATP hydrolysis, productive protein folding and microtubule binding, respectively. In addition, we show that suppression of mouse Tubg1 expression in vivo interferes with proper neuronal migration, whereas expression of altered γ-tubulin proteins in Saccharomyces cerevisiae disrupts normal microtubule behavior. Our data reinforce the importance of centrosomal and microtubule-related proteins in cortical development and strongly suggest that microtubule-dependent mitotic and postmitotic processes are major contributors to the pathogenesis of MCD.

Neuronal migration disorders.

Lissencephaly-pachygyria-severe band heterotopia are diffuse neuronal migration disorders (NMDs) causing severe, global neurological impairment. Abnormalities of the LIS1, DCX, ARX, TUBA1A and RELN genes have been associated with these malformations. NMDs only affecting subsets of neurons, such as mild subcortical band heterotopia and periventricular heterotopia, cause neurological and cognitive impairment that vary from severe to mild deficits. They have been associated with abnormalities of the DCX, FLN1A, and ARFGEF2 genes. Polymicrogyria results from abnormal late cortical organization and is inconstantly associated with abnormal neuronal migration. Localized polymicrogyria has been associated with anatomo-specific deficits, including disorders of language and higher cognition. Polymicrogyria is genetically heterogeneous and only in a small minority of patients a definite genetic cause has been identified. Mutations of the GPR56 and SRPX2 genes have been related to isolated polymicrogyria. Focal migration abnormalities associated with abnormal cell types, such as focal cortical dysplasia, are highly epileptogenic and variably influence the functioning of the affected cortex. The functional consequences of abnormal neuronal migration are still poorly understood. Conservation of function in the malformed cortex, its atypical representation, and relocation outside the malformed area are all possible. Localization of function based on anatomic landmarks may not be reliable.

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.

Somatic mutations in cerebral cortical malformations.

BACKGROUND Although there is increasing recognition of the role of somatic mutations in genetic disorders, the prevalence of somatic mutations in neurodevelopmental disease and the optimal techniques to detect somatic mosaicism have not been systematically evaluated. METHODS Using a customized panel of known and candidate genes associated with brain malformations, we applied targeted high-coverage sequencing (depth, ≥200×) to leukocyte-derived DNA samples from 158 persons with brain malformations, including the double-cortex syndrome (subcortical band heterotopia, 30 persons), polymicrogyria with megalencephaly (20), periventricular nodular heterotopia (61), and pachygyria (47). We validated candidate mutations with the use of Sanger sequencing and, for variants present at unequal read depths, subcloning followed by colony sequencing. RESULTS Validated, causal mutations were found in 27 persons (17%; range, 10 to 30% for each phenotype). Mutations were somatic in 8 of the 27 (30%), predominantly in persons with the double-cortex syndrome (in whom we found mutations in DCX and LIS1), persons with periventricular nodular heterotopia (FLNA), and persons with pachygyria (TUBB2B). Of the somatic mutations we detected, 5 (63%) were undetectable with the use of traditional Sanger sequencing but were validated through subcloning and subsequent sequencing of the subcloned DNA. We found potentially causal mutations in the candidate genes DYNC1H1, KIF5C, and other kinesin genes in persons with pachygyria. CONCLUSIONS Targeted sequencing was found to be useful for detecting somatic mutations in patients with brain malformations. High-coverage sequencing panels provide an important complement to whole-exome and whole-genome sequencing in the evaluation of somatic mutations in neuropsychiatric disease. (Funded by the National Institute of Neurological Disorders and Stroke and others.).

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.

Epilepsy in Rett syndrome, and CDKL5‐ and FOXG1‐gene–related encephalopathies

Rett syndrome is an X‐linked neurodevelopmental disorder that manifests in early childhood with developmental stagnation, and loss of spoken language and hand use, with the development of distinctive hand stereotypies, severe cognitive impairment, and autistic features. About 60% of patients have epilepsy. Seizure onset before the age of 3 years is unlikely, and onset after age 20 is rare. Diagnosis of Rett syndrome is based on key clinical elements that identify “typical” Rett syndrome but also “variant” or “atypical” forms. Diagnostic criteria have been modified only slightly over time, even after discovering that MECP2 gene alterations are present in >90% of patients with typical Rett syndrome but only in 50–70% of atypical cases. Over the last several years, intragenic or genomic alterations of the CDKL5 and FOXG1 genes have been associated with severe cognitive impairment, early onset epilepsy and, often, dyskinetic movement disorders, which have variably been defined as Rett variants. It is now clearly emerging that epilepsy has distinctive characteristics in typical Rett syndrome and in the different syndromes caused by CDKL5 and FOXG1 gene alterations. The progressive parting of CDKL5‐ and FOXG1‐gene–related encephalopathies from the core Rett syndrome is reflected by the effort to produce clearer diagnostic criteria for typical and atypical Rett syndrome. Efforts to characterize the molecular pathology underlying these developmental encephalopathies are pointing to abnormalities of telencephalic development, neuronal morphogenesis, maturation and maintenance, and dendritic arborization.

Nonsyndromic mental retardation and cryptogenic epilepsy in women with doublecortin gene mutations

DCX mutations cause mental retardation in male subjects with lissencephalypachygyria and in female subjects with subcortical band heterotopia (SBH). We observed four families in which carrier women had normal brain magnetic resonance imaging (MRI) and mild mental retardation, with or without epilepsy. Affected male subjects had SBH or pachygyria‐SBH. In two families, the phenotype was mild in both genders. In the first family, we found a tyr138his mutation that is predicted to result in abnormal folding in the small hinge region. In the second family, we found an arg178cys mutation at the initial portion of R2, in the putative β‐sheet structure. Carrier female subjects with normal MRI showed no somatic mosaicism or altered X‐inactivation in lymphocytes, suggesting a correlation between mild mutations and phenotypes. In the two other families, with severely affected boys, we found arg76ser and arg56gly mutations within the R1 region that are predicted to affect DCX folding, severely modifying its activity. Both carrier mothers showed skewed X‐inactivation, possibly explaining their mild phenotypes. Missense DCX mutations may manifest as non‐syndromic mental retardation with cryptogenic epilepsy in female subjects and SBH in boys. Mutation analysis in mothers of affected children is mandatory, even when brain MRI is normal. Ann Neurol 2003

Xp22.3 genomic deletions involving the CDKL5 gene in girls with early onset epileptic encephalopathy

Purpose:  Mutations of the X‐linked gene cyclin‐dependent kinase‐like 5 (CDKL5) cause an X‐linked encephalopathy with early onset intractable epilepsy, including infantile spasms and other seizure types, and a Rett syndrome (RTT)–like phenotype. Very limited information is available on the frequency and phenotypic spectrum associated with CDKL5 deletions/duplications. We investigated the role of CDKL5 deletions/duplications in causing early onset intractable epilepsy of unknown etiology in girls.

Generalized Epilepsy with Febrile Seizures Plus (GEFS+): Clinical Spectrum in Seven Italian Families Unrelated to SCN1A, SCN1B, and GABRG2 Gene Mutations

Summary:  Purpose: We describe seven Italian families with generalized epilepsy with febrile seizures plus (GEFS+), in which mutations of SCN1A, SCN1B, and GABRG2 genes were excluded and compare their clinical spectrum with that of previously reported GEFS+ with known mutations.

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.