[Alexander disease as an explanation for long-term unexplained psychiatric symptoms in a young girl]. / De ziekte van Alexander als verklaring voor langdurig onbegrepen psychiatrische klachten bij een jong meisje.

Alexander disease is a rare genetic disorder, usually characterized by leukodystrophy, causing progressive degeneration of white matter in the central nervous system. We describe a case of a 7-year-old girl with long term medically unexplained symptoms and suspicion of a psychiatric disorder, who was diagnosed with Alexander disease. Building on this case, a literature search was conducted for psychiatric symptoms..

Bergmann glia translocation: a new disease marker for vanishing white matter identifies therapeutic effects of Guanabenz treatment.

AIM: Vanishing White Matter (VWM) is a devastating leucoencephalopathy without effective treatment options. Patients have mutations in the EIF2B1-5 genes, encoding the five subunits of eIF2B, a guanine exchange factor that is an important regulator of protein translation. We recently developed mouse models for VWM that replicate the human disease. To study disease improvement after treatment in these mice, it is essential to have sensitive biomarkers related to disease stage. The Bergmann glia of the cerebellum, an astrocytic subpopulation, translocate into the molecular layer in symptomatic VWM mice and patients. This study looked at the prospects of using Bergmann glia pathology as an objective disease marker for VWM. METHODS: We defined a new quantitative measurement of Bergmann glia pathology in the cerebellum of VWM mice and patients. To test the sensitivity of this new marker for improvement, VWM mutant mice received long-term treatment with Guanabenz, an FDA-approved anti-hypertensive agent affecting eIF2B activity. RESULTS: Bergmann glia translocation was significantly higher in symptomatic VWM mice and VWM patients than in controls and worsened over the disease course. Both Bergmann glia pathology and cerebellar myelin pathology improved with Guanabenz treatment in mice, showing that Bergmann glia translocation is a sensitive measurement for improvement. CONCLUSIONS: Bergmann glia translocation can be used to objectively assess effects of treatment in VWM mice. Future treatment strategies involving compounds regulating eIF2 phosphorylation might benefit VWM patients.

Subunits of the translation initiation factor eIF2B are mutant in leukoencephalopathy with vanishing white matter.

Leukoencephalopathy with vanishing white matter (VWM) is an inherited brain disease that occurs mainly in children. The course is chronic-progressive with additional episodes of rapid deterioration following febrile infection or minor head trauma. We have identified mutations in EIF2B5 and EIF2B2, encoding the epsilon- and beta-subunits of the translation initiation factor eIF2B and located on chromosomes 3q27 and 14q24, respectively, as causing VWM. We found 16 different mutations in EIF2B5 in 29 patients from 23 families. We also found two distantly related individuals who were homozygous with respect to a missense mutation in EIF2B2, affecting a conserved amino acid. Three other patients also had mutations in EIF2B2. As eIF2B has an essential role in the regulation of translation under different conditions, including stress, this may explain the rapid deterioration of people with VWM under stress. Mutant translation initiation factors have not previously been implicated in disease.

Natural disease course and genotype-phenotype correlations in Complex I deficiency caused by nuclear gene defects: what we learned from 130 cases.

Mitochondrial complex I is the largest multi-protein enzyme complex of the oxidative phosphorylation system. Seven subunits of this complex are encoded by the mitochondrial and the remainder by the nuclear genome. We review the natural disease course and signs and symptoms of 130 patients (four new cases and 126 from literature) with mutations in nuclear genes encoding structural complex I proteins or those involved in its assembly. Complex I deficiency caused by a nuclear gene defect is usually a non-dysmorphic syndrome, characterized by severe multi-system organ involvement and a poor prognosis. Age at presentation may vary, but is generally within the first year of life. The most prevalent symptoms include hypotonia, nystagmus, respiratory abnormalities, pyramidal signs, dystonia, psychomotor retardation or regression, failure to thrive, and feeding problems. Characteristic symptoms include brainstem involvement, optic atrophy and Leigh syndrome on MRI, either or not in combination with internal organ involvement and lactic acidemia. Virtually all children ultimately develop Leigh syndrome or leukoencephalopathy. Twenty-five percent of the patients died before the age of six months, more than half before the age of two and 75 % before the age of ten years. Some patients showed recovery of certain skills or are still alive in their thirties . No clinical, biochemical, or genetic parameters indicating longer survival were found. No clear genotype-phenotype correlations were observed, however defects in some genes seem to be associated with a better or poorer prognosis, cardiomyopathy, Leigh syndrome or brainstem lesions.

Early-onset LBSL: how severe does it get?

AIM: Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is known as a relatively mild leukoencephalopathy. We investigated the occurrence of severe variants of LBSL with extensive brain magnetic resonance imaging (MRI) abnormalities. METHOD: MRIs of approximately 3,000 patients with an unknown leukoencephalopathy were retrospectively reviewed for extensive signal abnormalities of the cerebral and cerebellar white matter, posterior limb of the internal capsule, cerebellar peduncles, pyramids, and medial lemniscus. Clinical data were retrospectively collected. RESULTS: Eleven patients fulfilled the MRI criteria (six males); six had DARS2 mutations. Clinical and laboratory findings did not distinguish between patients with and without DARS2 mutations, but MRI did. Patients with DARS2 mutations more often had involvement of structures typically affected in LBSL, including decussatio of the medial lemniscus, anterior spinocerebellar tracts, and superior and inferior cerebellar peduncles. Also, involvement of the globus pallidus was associated with DARS2 mutations. Earliest disease onset was neonatal; earliest death at 20 months. INTERPRETATION: This study confirms the occurrence of early infantile, severe LBSL, extending the known phenotypic range of LBSL. Abnormality of specific brainstem tracts and cerebellar peduncles are MRI findings that point to the correct diagnosis.

Unclassified white matter disorders: A diagnostic journey requiring close collaboration between clinical and laboratory services.

BACKGROUND: Next generation sequencing studies have revealed an ever-increasing number of causes for genetic disorders of central nervous system white matter. A substantial number of disorders are identifiable from their specific pattern of biochemical and/or imaging findings for which single gene testing may be indicated. Beyond this group, the causes of genetic white matter disorders are unclear and a broader approach to genomic testing is recommended. AIM: This study aimed to identify the genetic causes for a group of individuals with unclassified white matter disorders with suspected genetic aetiology and highlight the investigations required when the initial testing is non-diagnostic. METHODS: Twenty-six individuals from 22 families with unclassified white matter disorders underwent deep phenotyping and genome sequencing performed on trio, or larger, family groups. Functional studies and transcriptomics were used to resolve variants of uncertain significance with potential clinical relevance. RESULTS: Causative or candidate variants were identified in 15/22 (68.2%) families. Six of the 15 implicated genes had been previously associated with white matter disease (COL4A1, NDUFV1, SLC17A5, TUBB4A, BOLA3, DARS2). Patients with variants in the latter two presented with an atypical phenotype. The other nine genes had not been specifically associated with white matter disease at the time of diagnosis and included genes associated with monogenic syndromes, developmental disorders, and developmental and epileptic encephalopathies (STAG2, LSS, FIG4, GLS, PMPCA, SPTBN1, AGO2, SCN2A, SCN8A). Consequently, only 46% of the diagnoses would have been made via a current leukodystrophy gene panel test. DISCUSSION: These results confirm the importance of broad genomic testing for patients with white matter disorders. The high diagnostic yield reflects the integration of deep phenotyping, whole genome sequencing, trio analysis, functional studies, and transcriptomic analyses. CONCLUSIONS: Genetic white matter disorders are genetically and phenotypically heterogeneous. Deep phenotyping together with a range of genomic technologies underpin the identification of causes of unclassified white matter disease. A molecular diagnosis is essential for prognostication, appropriate management, and accurate reproductive counseling.

Membrane lysis by gramicidin S visualized in red blood cells and giant vesicles.

The cationic amphiphilic antimicrobial peptide gramicidin S (GS) is an effective antibiotic. Its applicability is however restricted to topical infections due to its hemolytic activity. In this study, the process of GS induced hemolysis was investigated in detail for the first time. The morphological changes of red blood cells (RBCs) inflicted by GS were visualized and explained in terms of a physical model. The observed fast rupture events were further investigated with giant unilamellar vesicles (GUVs) as model systems for RBCs. Measurements of membrane fluctuations in GUVs revealed that the membrane surface tension was increased after incubation with GS. These findings are in agreement with the hypothesis that amphiphilic peptides induce membrane rupture by an increase in membrane tension.

Promoter mutation is a common variant in GJC2-associated Pelizaeus-Merzbacher-like disease.

Pelizaeus-Merzbacher-like disease (PMLD) is a clinically and genetically heterogeneous neurological disorder of cerebral hypomyelination. It is clinically characterised by early onset (usually infantile) nystagmus, impaired motor development, ataxia, choreoathetoid movements, dysarthria and progressive limb spasticity. We undertook autozygosity mapping studies in a large consanguineous family of Pakistani origin in which affected children had progressive lower limb spasticity and features of cerebral hypomyelination on MR brain imaging. SNP microarray and microsatellite marker analysis demonstrated linkage to chromosome 1q42.13-1q42.2. Direct sequencing of the gap junction protein gamma-2 gene, GJC2, identified a promoter region mutation (c.-167A>G) in the non-coding exon 1. The c.-167A>G promoter mutation was identified in a further 4 individuals from two families (who were also of Pakistani origin) with clinical and radiological features of PMLD in whom previous routine diagnostic screening of GJC2 had been reported as negative. A common haplotype was identified at the GJC2 locus in the three mutation-positive families, consistent with a common origin for the mutation and likely founder effect. This promoter mutation has only recently been reported in GJC2-PMLD but it has been postulated to affect the binding of the transcription factor SOX10 and appears to be a prevalent mutation, accounting for ~29% of reported patients with GJC2-PMLD. We propose that diagnostic screening of GJC2 should include sequence analysis of the non-coding exon 1, as well as the coding regions to avoid misdiagnosis or diagnostic delay in suspected PMLD.

Clinical features and X-inactivation in females heterozygous for creatine transporter defect.

The creatine transporter defect is an X-linked cause of mental retardation. We investigated the clinical features and pattern of X-inactivation in a Dutch cohort of eight female heterozygotes. We show that symptoms of the creatine transporter defect (mental retardation, learning difficulties, and constipation) can be present in female heterozygotes. We further show that the diagnosis in females is not straightforward: (i) The creatine/creatinine ratio in urine was elevated only in three of eight females. (ii) Although as a group the females had a significantly decreased cerebral creatine concentration, individual females had creatine concentrations overlapping with normal controls. (iii) Skewed X-inactivation was found in the cultured fibroblasts, in favour of either the mutated or the wild-type allele, leading to either deficient or normal results in the creatine uptake studies in fibroblasts. Thus, screening by these tests is unreliable for the diagnosis. In addition, we found no consistent skewing of the X-inactivation in peripheral tissues indicating that there is no selection against the creatine transporter defect. We conclude that testing for creatine transporter defect should be considered in females with (mild) mental retardation. Screening by DNA analysis of the SLC6A8 gene is recommended.

Fetal origin of brain damage in 2 infants with a COL4A1 mutation: fetal and neonatal MRI.

Mutations in the gene COL4A1, encoding collagen IV A1, are associated with familial porencephaly. Previously, COL4A1 mutation-associated antenatal hemorrhages have been suggested by early post-natal imaging. We describe 2 children with fetal intracerebral hemorrhages and a COL4A1 mutation. There was also extensive hemispheric tissue loss in both infants and loss of cerebellar tissue in one infant. This paper show prenatal evidence of fetal hemorrhage in association with a COL4A1 mutation.

N-acetylaspartylglutamate in CNS hypomyelination.

CSF N-acetylaspartylglutamate (NAAG) has been found to be elevated in some hypomyelinating disorders. This study addressed the question whether it could be used as a marker for hypomyelination and as a means to distinguish between hypomyelinating disorders biochemically. We have measured CSF NAAG in a cohort of 28 patients with hypomyelination with known and unknown aetiology. NAAG was found to be elevated in 7 patients, but was normal in the majority, including patients with defined hypomyelinating disorders. CSF NAAG is not a universal marker of hypomyelination, and the mechanism of its elevation remains poorly understood.

Familial hemophagocytic lymphohistiocytosis in a pediatric patient diagnosed by brain magnetic resonance imaging.

Familial hemophagocytic lymphohistiocytosis (fHLH) is an autosomal recessive disorder characterized by proliferation and infiltration of several organs by activated lymphocytes and macrophages. Without allogeneic stem cell transplantation, fHLH is fatal. We describe a previously healthy 11-month-old boy with a rapidly progressive encephalopathy. An older brother died at 8 months following a subacute encephalopathy diagnosed as meningoencephalitis. The family history led to the suspicion of a metabolic disease, but metabolic studies were unrevealing. MRI showed multiple inhomogeneous signal abnormalities in the cortex and white matter, most prominent in the cerebral hemispheres and around the dentate nucleus. Gadolinium-enhanced T1-weighted images showed a multitude of enhancing foci, suggestive of perivascular enhancement. Based on MRI pattern with multiple lesions, perivascular enhancement and family history, fHLH was suspected. DNA analysis showed that the patient was compound-heterozygous for the c.445 G>A (p.Gly149Ser) mutation in exon 1 and the c.757 G>A (p.Glu253Lys) mutation in exon 2 of the perforin 1 gene. The patient was treated according to the international HLH-2004 protocol (dexamethasone, etoposide, cyclosporine, intrathecal methotrexate and prednisolone) followed by allogeneic cord blood transplantation. He showed a significant neurological and radiological improvement. The reported case demonstrates that MRI pattern recognition can lead to early diagnosis of fHLH, with subsequent adequate treatment.

COL4A1 mutations associated with a characteristic pattern of intracranial calcification.

Intracranial calcification (ICC) is a relatively common radiological finding in children undergoing investigation for neurological disorders. Many causes are recognised, and ICC is often regarded as a non-specific sign.From an ongoing study of ICC, we identified 5 patients with characteristic radiological features, in whom a mutation in the COL4A1 gene was found.All patients had CT and MR imaging. MR images demonstrated features of periventricular leukomalacia with irregular dilatation of the lateral ventricles with or without porencephaly, loss of hemispheric white matter volume, and high signal on T2 and FLAIR sequences within periventricular and deep white matter. Calcification was apparent on MR in 4 patients. CT scans demonstrated spot and linear calcification in the subependymal region and around areas of porencephaly. Calcification was also visible in the deep cerebral white matter and basal ganglia. 1 patient showed calcification in the central pons.ICC occurs in COL4A1-related disease. The radiological features are distinct from other conditions demonstrating recognisable patterns of ICC, such as congenital cytomegalovirus infection and Aicardi-Goutiéres syndrome. In the absence of a known risk factor for periventricular leukomalacia, the presence of these radio-logical findings should suggest the possibility of COL4A1-related disease.

Imaging Patterns Characterizing Mitochondrial Leukodystrophies.

BACKGROUND AND PURPOSE: Achieving a specific diagnosis in leukodystrophies is often difficult due to clinical and genetic heterogeneity. Mitochondrial defects cause 5%-10% of leukodystrophies. Our objective was to define MR imaging features commonly shared by mitochondrial leukodystrophies and to distinguish MR imaging patterns related to specific genetic defects. MATERIALS AND METHODS: One hundred thirty-two patients with a mitochondrial leukodystrophy with known genetic defects were identified in the data base of the Amsterdam Leukodystrophy Center. Numerous anatomic structures were systematically assessed on brain MR imaging. Additionally, lesion characteristics were scored. Statistical group analysis was performed for 57 MR imaging features by hierarchic testing on clustered genetic subgroups. RESULTS: MR imaging features indicative of mitochondrial disease that were frequently found included white matter rarefaction (n = 50 patients), well-delineated cysts (n = 20 patients), T2 hyperintensity of the middle blade of the corpus callosum (n = 85 patients), and symmetric abnormalities in deep gray matter structures (n = 42 patients). Several disorders or clusters of disorders had characteristic features. The combination of T2 hyperintensity in the brain stem, middle cerebellar peduncles, and thalami was associated with complex 2 deficiency. Predominantly periventricular localization of T2 hyperintensities and cystic lesions with a distinct border was associated with defects in complexes 3 and 4. T2-hyperintense signal of the cerebellar cortex was specifically associated with variants in the gene NUBPL. T2 hyperintensities predominantly affecting the directly subcortical cerebral white matter, globus pallidus, and substantia nigra were associated with Kearns-Sayre syndrome. CONCLUSIONS: In a large group of patients with a mitochondrial leukodystrophy, general MR imaging features suggestive of mitochondrial disease were found. Additionally, we identified several MR imaging patterns correlating with specific genotypes. Recognition of these patterns facilitates the diagnosis in future patients.

Translation initiation factor eIF2Bε promotes Wnt-mediated clonogenicity and global translation in intestinal epithelial cells.

Modulation of global mRNA translation, which is essential for intestinal stem cell function, is controlled by Wnt signaling. Loss of tumor supressor APC in stem cells drives adenoma formation through hyperactivion of Wnt signaling and dysregulated translational control. It is unclear whether factors that coordinate global translation in the intestinal epithelium are needed for APC-driven malignant transformation. Here we identified nucleotide exchange factor eIF2Bε as a translation initiation factor involved in Wnt-mediated intestinal epithelial stemness. Using eIF2BεArg191His mice with a homozygous point mutation that leads to dysfunction in the enzymatic activity, we demonstrate that eIF2Bε is involved in small intestinal crypt formation, stemness marker expression, and secreted Paneth cell-derived granule formation. Wnt hyperactivation in ex vivo eIF2BεArg191His organoids, using a GSK3ß inhibitor to mimic Apc driven transformation, shows that eIF2Bε is essential for Wnt-mediated clonogenicity and associated increase of the global translational capacity. Finally, we observe high eIF2Bε expression in human colonic adenoma tissues, exposing eIF2Bε as a potential target of CRC stem cells with aberrant Wnt signaling.

Development and implementation of a novel assay for L-2-hydroxyglutarate dehydrogenase (L-2-HGDH) in cell lysates: L-2-HGDH deficiency in 15 patients with L-2-hydroxyglutaric aciduria.

L-2-hydroxyglutaric aciduria (L-2-HGA) is a rare inherited autosomal recessive neurometabolic disorder caused by mutations in the gene encoding L-2-hydroxyglutarate dehydrogenase. An assay to evaluate L-2-hydroxyglutarate dehydrogenase (L-2-HGDH) activity in fibroblast, lymphoblast and/or lymphocyte lysates has hitherto been unavailable. We developed an L-2-HGDH enzyme assay in cell lysates based on the conversion of stable-isotope-labelled L-2-hydroxyglutarate to 2-ketoglutarate, which is converted into L-glutamate in situ. The formation of stable isotope labelled L-glutamate is therefore a direct measure of L-2-HGDH activity, and this product is detected by liquid chromatography-tandem mass spectrometry. A deficiency of L-2-HGDH activity was detected in cell lysates from 15 out of 15 L-2-HGA patients. Therefore, this specific assay confirmed the diagnosis unambiguously affirming the relationship between molecular and biochemical observations. Residual activity was detected in cells derived from one L-2-HGA patient. The L-2-HGDH assay will be valuable for examining in vitro riboflavin/FAD therapy to rescue L-2-HGDH activity.

Quantitative MR imaging and spectroscopy in congenital cytomegalovirus infection and periventricular leukomalacia suggests a comparable neuropathological substrate of the cerebral white matter lesions.

Congenital CYTOMEGALOVIRUS (CMV) infection and periventricular leukomalacia (PVL) both lead to static cerebral white matter lesions. In contrast to PVL, the neuropathologicAL substrate of these lesions in congenital CMV is not clear. By comparing changes in quantitative magnetic resonance (MR) parameters and MR spectroscopy metabolite concentrations we wanted to determine whether the nature of the white matter pathology in congenital CMV infection could be similar to the known pathology of PVL. Diffusion parameters, apparent diffusion coefficient (ADC) and fractional anisotropy (FA), magnetization transfer ratio (MTR) and MR spectroscopy concentrations were studied in white matter lesions in five patients with a congenital CMV infection and six patients with PVL. In both groups ADC values were increased, FA and MTR values were reduced, concentrations of total N-acetylaspartate and choline-containing compounds were reduced; and MYO-inositol concentrations were slightly increased. No differences were found between the two groups, suggesting that the pathology of the white matter lesions in congenital CMV infections is similar to that of PVL and also characterized by axonal losses, lack of myelin deposition due to oligodendrocytic losses, and astrogliosis. Congenital CMV infection and PVL affect the cerebral white matter in the same developmental period when immature oligodendrocytes are particularly vulnerable.

Severe Leukoencephalopathy with Clinical Recovery Caused by Recessive BOLA3 Mutations.

AIM: To identify the genetic aetiology of a distinct leukoencephalopathy causing acute neurological regression in infancy with apparently complete clinical recovery. METHODS: We performed trio whole genome sequencing (WGS) to determine the genetic basis of the disorder. Mitochondrial function analysis in cultured patient fibroblasts was undertaken to confirm the pathogenicity of candidate variants. RESULTS: The patient presented at 18 months with acute hemiplegia and cognitive regression without obvious trigger. This was followed by clinical recovery over 4 years. MRI at disease onset revealed bilateral T2 hyperintensity involving the periventricular and deep white matter and MR spectroscopy of frontal white matter demonstrated a lactate doublet. Lactate levels and mitochondrial respiratory chain enzyme activity in muscle, liver and fibroblasts were normal. Plasma glycine was elevated. The MRI abnormalities improved. WGS identified compound heterozygous variants in BOLA3: one previously reported (c.136C>T, p.Arg46*) and one novel variant (c.176G>A, p.Cys59Tyr). Analysis of cultured patient fibroblasts demonstrated deficient pyruvate dehydrogenase (PDH) activity and reduced quantity of protein subunits of mitochondrial complexes I and II, consistent with BOLA3 dysfunction. Previously reported cases of multiple mitochondrial dysfunctions syndrome 2 (MMDS2) with hyperglycinaemia caused by BOLA3 mutations have leukodystrophy with severe, progressive neurological and multisystem disease. CONCLUSIONS: We report a novel phenotype for MMDS2 associated with apparently complete clinical recovery and partial resolution of MRI abnormalities. We have identified a novel disease-causing variant in BOLA3 validated by functional cellular studies. Our patient's clinical course broadens the phenotypic spectrum of MMDS2 and highlights the potential for some genetic leukoencephalopathies to spontaneously improve.

Exon skipping mutations in collagen VI are common and are predictive for severity and inheritance.

Mutations in the genes encoding collagen VI (COL6A1, COL6A2, and COL6A3) cause Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD), two related conditions of differing severity. BM is a relatively mild dominantly inherited disorder characterized by proximal weakness and distal joint contractures. UCMD was originally regarded as an exclusively autosomal recessive condition causing severe muscle weakness with proximal joint contractures and distal hyperlaxity. We and others have subsequently modified this model when we described UCMD patients with heterozygous in-frame deletions acting in a dominant-negative way. Here we report 10 unrelated patients with a UCMD clinical phenotype and de novo dominant negative heterozygous splice mutations in COL6A1, COL6A2, and COL6A3 and contrast our findings with four UCMD patients with recessively acting splice mutations and two BM patients with heterozygous splice mutations. We find that the location of the skipped exon relative to the molecular structure of the collagen chain strongly correlates with the clinical phenotype. Analysis by immunohistochemical staining of muscle biopsies and dermal fibroblast cultures, as well as immunoprecipitation to study protein biosynthesis and assembly, suggests different mechanisms each for exon skipping mutations underlying dominant UCMD, dominant BM, and recessive UCMD. We provide further evidence that de novo dominant mutations in severe UCMD occur relatively frequently in all three collagen VI chains and offer biochemical insight into genotype-phenotype correlations within the collagen VI-related disorders by showing that severity of the phenotype depends on the ability of mutant chains to be incorporated in the multimeric structure of collagen VI.

Refining the phenotype of alpha-1a Tubulin (TUBA1A) mutation in patients with classical lissencephaly.

Mutations in the alpha-1a Tubulin (TUBA1A) gene have recently been found to cause cortical malformations resemblant of classical lissencephaly but with a specific combination of features. To date, TUBA1A mutations have been described in five patients and three foetuses. Our aims were to establish how common TUBA1A mutations are in patients with lissencephaly and to contribute to defining the phenotype associated with TUBA1A mutation. We performed mutation analysis in the TUBA1A gene in 46 patients with classical lissencephaly. In 44 of the patients, mutations in the LIS1 and/or DCX genes had previously been excluded; in 2 patients, mutation analysis was only performed in TUBA1A based on magnetic resonance imaging (MRI) findings. We identified three new mutations and one recurrent mutation in five patients with variable patterns of lissencephaly on brain MRI. Four of the five patients had congenital microcephaly, and all had dysgenesis of the corpus callosum and cerebellar hypoplasia, and variable cortical malformations, including subtle subcortical band heterotopia and absence or hypoplasia of the anterior limb of the internal capsule. We estimate the frequency of mutation in TUBA1A gene in patients with classical lissencephaly to be approximately 4%, and although not as common as mutations in the LIS1 or DCX genes, mutation analysis in TUBA1A should be included in the molecular genetic diagnosis of classical lissencephaly, particularly in patients with the combination of features highlighted in this paper.