Homozygous Pathogenic Mutation Research Articles

Evidence for genetic susceptibility to the alcohol dependence syndrome from the thiamine transporter 2 gene solute carrier SLC19A3

The risk for developing the alcohol dependence syndrome (ADS) has a substantial genetic component. The human thiamine transporter protein 2 (hTHTR2) is encoded by the SLC19A3 gene, which is on chromosome 2q37. hTHTR2 is responsible for the cellular uptake of thiamine (B1), a water-soluble essential vitamin that plays a fundamental and ubiquitous role in carbohydrate metabolism. This gene was also found to be associated with biotin-responsive basal ganglia disease, an autosomal recessive metabolic disorder characterized by encephalopathy and ophthalmoplegia (Ozand et al., 1998; Zeng et al., 2005). Homozygous or compound heterozygous mutations in SLC19A3 cause two distinct clinical phenotypes: biotin-responsive basal ganglia disease and Wernicke’s-like encephalopathy. Biotin and/or thiamine are effective therapies for both diseases (Yamada et al., 2010). A missense mutation in exon 5 of the SLC19A3 was found in 18 cases of biotin/thiamine-responsive basal ganglion disease presenting with subacute encephalopathy and extrapyramidal signs (Alfadhel et al., 2013). Kono et al. (2009) described two Japanese brothers, who were both compound heterozygotes for the K44E and E320Q mutations in SLC19A3, who developed a syndrome of thiamine-responsive diplopia, ophthalmoplegia and ataxia, similar to Wernicke’s encephalopathy, despite normal serum thiamine levels (Kono et al., 2009). Yamada et al. (2010) reported a pathogenic homozygous mutation (c.958G>C, [p.E320Q]) in SLC19A3 in four patients from a single family. They report a wide variety of neurological signs in SLC19A3 mutation carriers. Our previous unpublished research found that four markers in the SLC19A3 gene showed significant allelic association with Wernicke–Korsakoff syndrome (WKS) in a sample of 120 cases when compared with normal controls. In the present study, the entire SLC19A3 gene was screened for DNA variation in a WKS subset (n=120) of a UK ADS case–control sample comprised of 1032 alcohol-dependent cases and 1022 controls. High resolution melting curve analysis, which is based on the melting characteristic of double-stranded DNA, was carried out using a LightCycler 480 Real-Time PCR System (Roche, Burgess Hill, UK). Genetic variation was validated with Sanger DNA sequencing. Thirteen single nucleotide variants were identified through high resolution melting analysis. Two exon 3 variants that were predicted to cause amino acid substitutions, 2:228563818T/C and rs148144444, were selected for genotyping in the entire ADS case–control sample using an allele-specific fluorescent PCR method (KasPar; LGC Genomics, Hoddesdon, UK). Statistical analysis was carried out on the previously unreported 2:228563818T/C change of a T to C substitution at position 228 563 818 on chromosome 2. This variant causes an R250G amino acid substitution in the largest cytoplasmic domain of the protein and it is, therefore, likely to affect post-translational function. rs148144444 causes the amino acid change G141S which is likely to exert an effect on protein phosphorylation and conformation because of the introduction of the aliphatic chain of serine. Neither the cases nor the controls in the present study had the SLC19A3 disease susceptibility variants that have been reported previously (Zeng et al., 2005; Kono et al., 2009). The minor allele of 2:228563818T/C was detected in five ADS cases, but was absent in the control samples (P=0.033). The minor allele of rs148144444 was detected in five ADS cases and in four controls and was not associated with ADS. Neither of these variants was present in the 120 WKS cases in our ADS sample. Our data suggest that genetic variation in the SLC19A3 thiamine transporter at 2:228563818T/C may make a modest contribution towards the genetic susceptibility to ADS.

The homozygous p.V37I variant of GJB2 is associated with diverse hearing phenotypes

The homozygous p.V37I variant of GJB2 is frequent in East Asians and has been reported to have a pathogenic role in mild-to-moderate hearing impairment (HI). In this study, we investigated the prevalence and phenotypic spectrum of homozygous p.V37I in three Chinese Han cohorts with severe-to-profound HI (n = 857, Cohort S), mild-to-moderate HI (n = 88, Cohort M) and normal hearing (n = 1550, Cohort N). Sequencing of GJB2 showed that homozygous p.V37I was detected in 1.63% (14/857), 12.5% (11/88) and 0.32% (5/1550) of subjects in Cohorts S, M and N, respectively. It was strongly associated with both mild-to-moderate (p = 2.0 × 10(-11) ) and severe-to-profound (p = 0.001) HI, but was estimated to have a rather low penetrance (17%). Among the hearing impaired subjects with homozygous p.V37I, the onset of HI was congenital in 65% (11/17) and delayed in 35% (6/17). By targeted next-generation sequencing of 79 known deafness genes, we identified an additional homozygous pathogenic mutation of CDH23 in 1 of 14 p.V37I homozygous subjects from Cohort S. Our study suggested that homozygous p.V37I is associated with a broader spectrum of hearing phenotypes than previously revealed. Data presented in this study can be effectively applied to clinical evaluation and genetic counseling of people carrying this variant.

HIBCH mutations can cause Leigh-like disease with combined deficiency of multiple mitochondrial respiratory chain enzymes and pyruvate dehydrogenase

BackgroundDeficiency of 3-hydroxy-isobutyryl-CoA hydrolase (HIBCH) caused by HIBCH mutations is a rare cerebral organic aciduria caused by disturbance of valine catabolism. Multiple mitochondrial respiratory chain (RC) enzyme deficiencies can arise from a number of mechanisms, including defective maintenance or expression of mitochondrial DNA. Impaired biosynthesis of iron-sulphur clusters and lipoic acid can lead to pyruvate dehydrogenase complex (PDHc) deficiency in addition to multiple RC deficiencies, known as the multiple mitochondrial dysfunctions syndrome.MethodsTwo brothers born to distantly related Pakistani parents presenting in early infancy with a progressive neurodegenerative disorder, associated with basal ganglia changes on brain magnetic resonance imaging, were investigated for suspected Leigh-like mitochondrial disease. The index case had deficiencies of multiple RC enzymes and PDHc in skeletal muscle and fibroblasts respectively, but these were normal in his younger brother. The observation of persistently elevated hydroxy-C4-carnitine levels in the younger brother led to suspicion of HIBCH deficiency, which was investigated by biochemical assay in cultured skin fibroblasts and molecular genetic analysis.ResultsSpecific spectrophotometric enzyme assay revealed HIBCH activity to be below detectable limits in cultured skin fibroblasts from both brothers. Direct Sanger sequence analysis demonstrated a novel homozygous pathogenic missense mutation c.950G <A; p.Gly317Glu in the HIBCH gene, which segregated with infantile-onset neurodegeneration within the family.ConclusionsHIBCH deficiency, a disorder of valine catabolism, is a novel cause of the multiple mitochondrial dysfunctions syndrome, and should be considered in the differential diagnosis of patients presenting with multiple RC deficiencies and/or pyruvate dehydrogenase deficiency.

Disruption of the ATE1 and SLC12A1 Genes by Balanced Translocation in a Boy with Non-Syndromic Hearing Loss

We report on a boy with non-syndromic hearing loss and an apparently balanced translocation t(10;15)(q26.13;q21.1). The same translocation was found in the normally hearing brother, father and paternal grandfather; however, this does not exclude its involvement in disease pathogenesis, for example, by unmasking a second mutation. Breakpoint analysis via FISH with BAC clones and long-range PCR products revealed a disruption of the arginyltransferase 1 (ATE1) gene on translocation chromosome 10 and the solute carrier family 12, member 1 gene (SLC12A1) on translocation chromosome 15. SNP array analysis revealed neither loss nor gain of chromosomal regions in the affected child, and a targeted gene enrichment panel consisting of 130 known deafness genes was negative for pathogenic mutations. The expression patterns in zebrafish and humans did not provide evidence for ear-specific functions of the ATE1 and SLC12A1 genes. Sanger sequencing of the 2 genes in the boy and 180 GJB2 mutation-negative hearing-impaired individuals did not detect homozygous or compound heterozygous pathogenic mutations. Our study demonstrates the many difficulties in unraveling the molecular causes of a heterogeneous phenotype. We cannot directly implicate disruption of ATE1 and/or SLC12A1 to the abnormal hearing phenotype; however, mutations in these genes may have a role in polygenic or multifactorial forms of hearing impairment. On the other hand, it is conceivable that our patient carries a disease-causing mutation in a so far unidentified deafness gene. Evidently, disruption of ATE1 and/or SLC12A1 gene function alone does not have adverse effects.

Whole-exome sequencing links caspase recruitment domain 11 (CARD11) inactivation to severe combined immunodeficiency

Primary immunodeficiencies represent model diseases for the mechanistic understanding of the human innate and adaptive immune response. They are clinically highly relevant per se because in patients with severe combined immunodeficiency (SCID), infections caused by opportunistic pathogens are typically life-threatening early in life. We aimed at defining and functionally characterizing a novel form of SCID in an infant of consanguineous parents who presented with life-threatening Pneumocystis jirovecii pneumonia using a comprehensive immunologic and whole-exome genetic diagnostic strategy. Analysis of leukocyte subpopulations was performed by using multicolor flow cytometry and was combined with stimulation tests for T-cell function. The search for a disease-causing mutation was performed with diagnostic whole-exome sequencing and systematic variant categorization. Reconstitution assays were used for validating the loss-of-function mutation. The novel entity of SCID was characterized by agammaglobulinemia and profoundly deficient T-cell function despite quantitatively normal T and B lymphocytes. Genetic analysis revealed a single pathogenic homozygous nonsense mutation of the caspase recruitment domain 11 (CARD11) gene. In reconstitution assays we demonstrated that the patient-derived truncated CARD11 protein is defective in antigen receptor signaling and nuclear factor κB activation. We show that an inactivating CARD11 mutation links defective nuclear factor κB signaling to a novel cause of autosomal recessive SCID.

Bilateral External Ophthalmoplegia in Biotin-Responsive Basal Ganglia Disease

A 10-year-old girl with a 4-month history of abnormal gait and dysarthria was admitted to the hospital with altered mental status that had progressed over the previous 3 days in the context of febrile illness. The patient's family reported that she did not use drugs, and the family history is remarkable only for consanguinity. On physical examination, she was confused, having generalized dystonia and bilateral external ophthalmoplegia, and she was anarthric. The results of toxicologic and autoimmune, cerebrospinal fluid, thyroid, bacterial and viral, and metabolic studies, including tandem mass spectrometry, urine for organic acids, and levels of lactic acid, ammonia, acylcarnitines, copper, ceruloplasmin, biotin, and thiamine, were all negative. An initial magnetic resonance imaging (MRI) of the brain showed abnormal signal intensity alterations bilaterally involving the cerebellum, basal ganglia, medial nucleus of the thalamus with a characteristic “bat-wing” appearance, and cerebral cortex (Figure). The affected areas showed increased signal consistent with vasogenic edema on diffusion-weighted images and apparent diffusion coefficient map (not shown). The MRI findings suggested biotin-responsive basal ganglia disease. Sequencing of the SLC19A3 gene identified a pathogenic homozygous mutation c.1264A>G (p.Thr422Ala). The patient was administered thiamine and biotin, achieving marked improvement in her confusion, extraocular movements, and ambulation during the next 4 days. Three months later, she exhibited only mild dysarthria. Repeated brain MRI showed resolution of the vasogenic edema with residual atrophy and gliosis in the basal ganglia (Figure). The physical exam finding of acute to subacute, bilateral external ophthalmoplegia is telling for an energy breakdown, as occurs with biotin-responsive basal ganglia disease, thiamine deficiency, and mitochondrial defects. That exam finding would lead one to examine the midbrain, thalamus, and basal ganglia on brain MRI. Biotin-responsive basal ganglia disease is an autosomal recessive, underdiagnosed pan-ethnic treatable condition, related to thiamine transporter-2 deficiency. It needs to be included in the differential diagnosis for unexplained acute dystonia, bilateral external ophthalmoplegia and encephalopathy, and neuroimaging showing vasogenic edema in the bilateral putamen and caudate nuclei, infratentorial and supratentorial cortex, and brainstem, because a therapeutic trial with thiamine and biotin can be lifesaving.1Ozand P.T. Gascon G.G. Al Essa M. Joshi S. Al Jishi E. Bakheet S. et al.Biotin-responsive basal ganglia disease: a novel entity.Brain. 1998; 121: 1267-1279Crossref PubMed Scopus (136) Google Scholar, 2Serrano M. Rebollo M. Depienne C. Rastetter A. Fernández-Álvarez E. Muchart J. et al.Reversible generalized dystonia and encephalopathy from thiamine transporter 2 deficiency.Mov Disord. 2012; 27: 1295-1298Crossref PubMed Scopus (35) Google Scholar

Whole-Exome Sequencing Links CARD11 Inactivation with SCID

AbstractAbstract 258Primary immunodeficiencies represent model diseases for the mechanistic understanding of the human innate and the adaptive immune response and are per se clinically highly relevant, because in SCID patients infections by opportunistic pathogens are typically life-threatening early in life. We identified an infant of consanguineous parents suffering from a novel form of SCID, who presented with a life-threatening Pneumocystis jirovecii pneumonia. This entity was characterized by agammaglobulinemia and profoundly deficient T-cell function despite quantitatively normal T- and B-lymphocytes. Lymphocyte proliferation was strongly inhibited after stimulation of PBMCs with T-cell mitogens such as PHA, Con A, or anti-CD3 monoclonal antibody. The expression of several T-cell response associated cytokines upon stimulation with PMA/ionomycin was dramatically reduced in comparison to normal controls. By contrast, proliferation induced by the classical B-cell mitogen PWM was almost comparable to healthy controls. Immunophenotyping revealed a predominantly naïve phenotype (CD45RA+ CCR7+) in CD4+ and CD8+ T-lymphocytes, whereas central memory T-lymphocytes (CD45RA− CCR7+) were nearly absent. B-lymphocytes from peripheral blood were mainly naïve B-cells (CD27−) with a uniformly immature transitional B-lymphocyte phenotype (CD24++, CD38++). Patient B-lymphocytes retained the ability to proliferate and differentiate in response to BCR-independent stimuli, while their response to BCR activation was defective. Our findings thus revealed a combined defect of TCR-mediated T-lymphocyte functions and BCR-mediated B-lymphocyte functions but did not enable us to link the immunological phenotype with one of the known molecularly defined categories of SCID. Diagnostic whole-exome sequencing and systematic variant categorization revealed a single pathogenic homozygous nonsense mutation of the caspase recruitment domain 11 (CARD11) gene. CARD11 is a scaffold protein that is known to be required for the assembly and activation of the NF-kB complex. In reconstitution assays we demonstrated that the patient derived truncated CARD11 protein is defective in antigen receptor signaling and NF-kB activation. Several lines of evidence substantiate the involvement of the identified CARD11 mutation in the new form of SCID that we report here. First, PCR and Sanger re-sequencing validated the truncating CARD11 mutation to be homozygous in the patient and heterozygous in the parents, in agreement with the recessive transmission of the mutation through the healthy consanguineous parents. Second, CARD11 is a scaffold protein required for TCR- and BCR-induced NF-kB activation as well as lymphocyte activation and proliferation, which is specifically expressed in hematopoietic cells, consistent with a causative role of CARD11 mutations in the context of an immune disorder. Third, the GUK domain of CARD11, which is missing in the mutated form of CARD11 due to truncation, was previously reported to be necessary for NF-kB activation by PMA/ionomycin treatment, further supporting the presumed damaging nature of the homozygous CARD11 mutation observed in the female patient reported here. Finally, the immunological findings in this patient are compatible with the phenotype of a previously described Card11 −/− k.o. mouse, which shows a selective defect in NF-κB activation leading to diminished antigen receptor or PKC mediated proliferation and defective cytokine production in T-cells and B-cells. Thus, we have identified an inactivating CARD11 mutation linking defective NF-kB signaling with a novel cause of autosomal recessive SCID, which must be considered in the diagnostic assessment of patients with suspected SCID but with quantitatively normal T-cells. Disclosures:No relevant conflicts of interest to declare.

A missense founder mutation in VLDLR is associated with Dysequilibrium Syndrome without quadrupedal locomotion

BackgroundDysequilibrium syndrome is a genetically heterogeneous condition that combines autosomal recessive, nonprogressive cerebellar ataxia with mental retardation. The condition has been classified into cerebellar ataxia, mental retardation and disequilibrium syndrome types 1 (CAMRQ1), 2 (CAMRQ2) and 3 (CAMRQ3) and attributed to mutations in VLDLR, CA8 and WDR81 genes, respectively. Quadrupedal locomotion in this syndrome has been reported in association with mutations in all three genes.MethodsSNP mapping and candidate gene sequencing in one consanguineous Omani family from the United Arab Emirates with cerebellar hypoplasia, moderate mental retardation, delayed ambulation and truncal ataxia was used to identify the mutation. In a second unrelated consanguineous Omani family, massively parallel exonic sequencing was used.ResultsWe identified a homozygous missense mutation (c.2117 G > T, p.C706F) in the VLDLR gene in both families on a shared affected haplotype block.This is the first reported homozygous missense mutation in VLDLR and it occurs in a highly conserved residue and predicted to be damaging to protein function.ConclusionsWe have delineated the phenotype associated with dysequilibrium syndrome in two Omani families and identified the first homozygous missense pathogenic mutation in VLDLR gene with likely founder effect in the southeastern part of the Arabian Peninsula.

G.P.26 DOK7 limb girdle myasthenic syndrome mimicking congenital muscular dystrophy

Congenital myasthenic syndromes show a wide clinical heterogeneity, however the unusual pattern of muscle weakness and the presence of variable degrees of muscle pathology, together with only subtle electrophysiological abnormalities and lack of circadian variability of symptoms may complicate its recognition. We have previously reported a Palestinian family with suspected congenital muscular dystrophy, who later showed linkage to chromosome 4p16.3. As the DOK7 gene is located in this genetic interval, we considered it a potential candidate for this condition. All patients showed a homozygous DOK7 pathogenic mutation (c.957delC). Therefore, we believe it is important to report this data in order to clarify that this family does not have congenital muscular dystrophy (CMD) and the locus associated with this family should not be classified as a CMD locus anymore. It also highlights the fact that patients with limb-girdle myasthenic syndrome could be easily misdiagnosed as congenital muscular dystrophy if fatigability is lacking or subtle. Following the diagnosis, patients were treated with salbutamol for 8months with significant improvement in their muscle strength and function.

DOK7 limb-girdle myasthenic syndrome mimicking congenital muscular dystrophy

Congenital myasthenic syndrome shows a wide clinical heterogeneity. However, the unusual pattern of muscle weakness and the presence of variable degree of muscle pathology, subtle electrophysiological abnormalities and lack of circadian variability of symptoms may complicate its recognition. We have previously reported a Palestinian family with suspected congenital muscular dystrophy and linkage to chromosome 4p16.3. As the DOK7 gene is located in this genetic interval, we considered it a potential candidate for this condition. Patients showed a homozygous DOK7 pathogenic mutation (c.957delC). We have re-examined six patients and found permanent limb-girdle weakness, but also episodic crises without clear precipitating factors. Following the revised diagnosis, patients were treated with salbutamol for 8 months with significant improvement in their muscle strength and function. This family needs to be reclassified as congenital myasthenic syndrome rather than congenital muscular dystrophy.

A wide spectrum of clinical and brain MRI findings in patients with SLC19A3mutations

BackgroundSLC19A3 (solute carrier family 19, member 3) is a thiamin transporter with 12 transmembrane domains. Homozygous or compound heterozygous mutations in SLC19A3 cause two distinct clinical phenotypes, biotin-responsive basal ganglia disease and Wernicke's-like encephalopathy. Biotin and/or thiamin are effective therapies for both diseases.MethodsWe conducted on the detailed clinical, brain MRI and molecular genetic analysis of four Japanese patients in a Japanese pedigree who presented with epileptic spasms in early infancy, severe psychomotor retardation, and characteristic brain MRI findings of progressive brain atrophy and bilateral thalami and basal ganglia lesions.ResultsGenome-wide linkage analysis revealed a disease locus at chromosome 2q35-37, which enabled identification of the causative mutation in the gene SLC19A3. A pathogenic homozygous mutation (c.958G > C, [p.E320Q]) in SLC19A3 was identified in all four patients and their parents were heterozygous for the mutation. Administration of a high dose of biotin for one year improved neither the neurological symptoms nor the brain MRI findings in one patient.ConclusionOur cases broaden the phenotypic spectrum of disorders associated with SLC19A3 mutations and highlight the potential benefit of biotin and/or thiamin treatments and the need to assess the clinical efficacy of these treatments.

827 Slc25A19 Mutation is a Novel Cause of Neuropathy and Bilateral Striatal Necrosis

Acute basal ganglia necrosis in the pediatric age group is a neurological disorder characterized by symmetrical degeneration of the caudate nucleus, putamen, and occasionally the globus pallidus. The outcome is less favorable for the familial cases where the course is relentlessly progressive or punctuated by recurrent episodes with involvement of additional brain regions and significant residual neurological damage. The differential diagnosis of the latter group includes mitochondrial respiratory chain defects organic acid disorders, Wilson disease, juvenile Huntington's chorea, pantothenate kinaseassociated neurodegeneration, biotin responsive basal ganglia disease, and the striatal necrosis associated with NUP62 mutation.In this study we report on four siblings from a consanguineous Arab Muslim family who suffered from recurrent episodes of flaccid paralysis and encephalopathy and chronic progressive polyneuropathy associated with bilateral striatal necrosis. Using homozygosity mapping the pathogenic homozygous missense mutation c.373G>A was identified. This mutation alters a highly conserved glycine residue at position 125 to serine in the SLC25A19 gene which encodes the mitochondrial thiamine pyrophosphate transporter. SLC25A19 mutation was previously reported only in the Amish congenital lethal microcephaly but our patients' phenotype is markedly different with normal head circumference, early childhood development, and age-appropriate cognitive skills. Based on the role of SLC25A19 in mitochondrial thiamine transport, supplementation with high dose thiamine was initiated in all patients. Preliminary results suggest a favorable effect.Our report suggests an allelic phenotype in SLC25A19 gene defects. Determination of the SLC25A19 sequence may be warranted in patients with bilateral striatal necrosis or progressive polyneuropathy.

Clinical Value of NPHS2 Analysis in Early- and Adult-Onset Steroid-Resistant Nephrotic Syndrome

To date, very few cases with adult-onset focal segmental glomerulosclerosis (FSGS) carrying NPHS2 variants have been described, all of them being compound heterozygous for the p.R229Q variant and one pathogenic mutation. Mutation analysis was performed in 148 unrelated Spanish patients, of whom 50 presented with FSGS after 18 years of age. Pathogenicity of amino acid substitutions was evaluated through an in silico scoring system. Haplotype analysis was carried out using NPHS2 single nucleotide polymorphism and microsatellite markers. Compound heterozygous or homozygous NPHS2 pathogenic mutations were identified in seven childhood-onset steroid-resistant nephrotic syndrome (SRNS) cases. Six additional cases with late childhood- and adult-onset SRNS were compound heterozygotes for p.R229Q and one pathogenic mutation, mostly p.A284V. p.R229Q was more frequent among SRNS cases relative to controls (odds ratio=2.65; P=0.02). Significantly higher age at onset of the disease and slower progression to ESRD were found in patients with one pathogenic mutation plus the p.R229Q variant in respect to patients with two NPHS2 pathogenic mutations. NPHS2 analysis has a clinical value in both childhood- and adult-onset SRNS patients. For adult-onset patients, the first step should be screening for p.R229Q and, if positive, for p.A284V. These alleles are present in conserved haplotypes, suggesting a common origin for these substitutions. Patients carrying this specific NPHS2 allele combination did not respond to corticoids or immunosuppressors and showed FSGS, average 8-year progression to ESRD, and low risk for recurrence of FSGS after kidney transplant.

Neuropathology in Succinic Semialdehyde Dehydrogenase Deficiency

Reported here is the novel finding of neuropathology in a patient with succinic semialdehyde dehydrogenase deficiency, an inherited disorder of gamma-aminobutyric acid metabolism characterized by intellectual deficiency, hypotonia, and epilepsy, with 4-hydroxybutyric aciduria and abnormalities of the globus pallidus on neuroimaging. A 19-year-old woman of European origin with a neurodevelopmental disorder and epilepsy died unexpectedly in 1998. A postmortem examination was performed, with a final diagnosis of sudden unexpected death in epilepsy patients. Eight years later, her sister with a neurodevelopmental disorder presented at 13 years of age with seizures and was diagnosed with succinic semialdehyde dehydrogenase deficiency. In the decedent, succinic semialdehyde dehydrogenase deficiency was established at the molecular level, 10 years after her death, using genomic DNA from brain tissue specimens. The neuropathologic findings revealed striking discoloration of the globi pallidi, leptomeningeal congestion, and a scar in the frontal cortex. After detection of the pathogenic homozygous mutation c.1226G>A, p.Gly409Asp in the living sister, it was confirmed in the decedent. An underlying metabolic disease may be an additional risk factor for sudden unexpected death in epilepsy patients.

P102 Fatal acute rhabdomyolysis in a young child due to pathogenic homozygous mutation in LPIN1 gene

P102 Fatal acute rhabdomyolysis in a young child due to pathogenic homozygous mutation in LPIN1 gene

Frontorhiny, a Distinctive Presentation of Frontonasal Dysplasia Caused by Recessive Mutations in the ALX3 Homeobox Gene

We describe a recessively inherited frontonasal malformation characterized by a distinctive facial appearance, with hypertelorism, wide nasal bridge, short nasal ridge, bifid nasal tip, broad columella, widely separated slit-like nares, long philtrum with prominent bilateral swellings, and midline notch in the upper lip and alveolus. Additional recurrent features present in a minority of individuals have been upper eyelid ptosis and midline dermoid cysts of craniofacial structures. Assuming recessive inheritance, we mapped the locus in three families to chromosome 1 and identified mutations in ALX3, which is located at band 1p13.3 and encodes the aristaless-related ALX homeobox 3 transcription factor. In total, we identified seven different homozygous pathogenic mutations in seven families. These mutations comprise missense substitutions at critical positions within the conserved homeodomain as well as nonsense, frameshift, and splice-site mutations, all predicting severe or complete loss of function. Our findings contrast with previous studies of the orthologous murine gene, which showed no phenotype in Alx3(-/-) homozygotes, apparently as a result of functional redundancy with the paralogous Alx4 gene. We conclude that ALX3 is essential for normal facial development in humans and that deficiency causes a clinically recognizable phenotype, which we term frontorhiny.

Paraplegin mutations in sporadic adult-onset upper motor neuron syndromes

To investigate the frequency of autosomal recessive paraplegin mutations in patients with sporadic adult-onset upper motor neuron (UMN) syndromes. We analyzed the paraplegin gene in 98 Dutch patients with a sporadic adult-onset UMN syndrome. Inclusion criteria were a progressive UMN syndrome, adult onset, duration >6 months, and negative family history. Exclusion criteria were clinical or electrophysiologic evidence of lower motor neuron loss and evidence of other causes using a predefined set of laboratory tests, including analysis of the spastin gene. Seven patients had homozygous or compound heterozygous pathogenic paraplegin mutations: six patients had UMN symptoms restricted to the legs and one had UMN symptoms in legs and arms. No mutations were found in the 33 patients with UMN involvement of the bulbar region. Age at onset was lower in the seven patients with paraplegin mutations (37 years, range 34-42) than in the 91 patients without mutations (51 years, range 18-77, p = 0.001). Three of the seven patients with paraplegin mutations and none of the patients without mutations developed cerebellar signs during follow-up. Paraplegin mutations are a frequent cause of sporadic spastic paraparesis.

DNA analysis of AHI1, NPHP1 and CYCLIN D1 in Joubert syndrome patients from the Netherlands

Joubert syndrome (JBS) is a clinically variable and genetically heterogeneous developmental brain disorder with autosomal recessive inheritance. Five genes, AHI1, NPHP1, CEP290, MKS3, and RPGRIP1L, and two additional loci on chromosome 9 and 11 have been identified so far. The relative contributions of AHI1 mutations and NPHP1 deletions have not yet been determined in a population-based JBS patient cohort. We therefore undertook a nationwide survey of JBS in the Netherlands and performed DNA analysis of the AHI1 and NPHP1 genes, as well as a new candidate gene CYCLIN D1. We obtained clinical data and DNA samples of 25 Dutch JBS patients. DNA analysis of AHI1 revealed pathogenic homozygous or compound heterozygous AHI1 mutations in four patients (16%). Based on the birth prevalence of about 1 in 100,000 for JBS in the Netherlands, we estimated a carrier frequency of AHI1 mutations of approximately 1 in 400. In another two patients, the AHI1 mutation Arg830Trp was identified (homozygously and heterozygously), a possible low penetrance allele. No deletions of NPHP1 or CYCLIN D1 mutations were detected in these 25 patients. In the four patients with AHI1 mutations, retinal disease (Leber congenital amaurosis or retinal dystrophy) was present in two, whereas none had renal disease. Pooling our data and data from the literature, retinal disease seems to occur in 75% of AHI1-associated JBS patients. Renal disease is present in 10% at most. We conclude that AHI1 mutations are an important cause of JBS in Dutch patients, and should always be looked for in patients suspected of JBS, especially when retinal dystrophy is present. Patients with AHI1 mutations should be regularly checked for retinal and renal disease up until adolescence.

Mutations in STRA6 Cause a Broad Spectrum of Malformations Including Anophthalmia, Congenital Heart Defects, Diaphragmatic Hernia, Alveolar Capillary Dysplasia, Lung Hypoplasia, and Mental Retardation

We observed two unrelated consanguineous families with malformation syndromes sharing anophthalmia and distinct eyebrows as common signs, but differing for alveolar capillary dysplasia or complex congenital heart defect in one and diaphragmatic hernia in the other family. Homozygosity mapping revealed linkage to a common locus on chromosome 15, and pathogenic homozygous mutations were identified in STRA6, a member of a large group of "stimulated by retinoic acid" genes encoding novel transmembrane proteins, transcription factors, and secreted signaling molecules or proteins of largely unknown function. Subsequently, homozygous STRA6 mutations were also demonstrated in 3 of 13 patients chosen on the basis of significant phenotypic overlap to the original cases. While a homozygous deletion generating a premature stop codon (p.G50AfsX22) led to absence of the immunoreactive protein in patient's fibroblast culture, structural analysis of three missense mutations (P90L, P293L, and T321P) suggested significant effects on the geometry of the loops connecting the transmembrane helices of STRA6. Two further variations in the C-terminus (T644M and R655C) alter specific functional sites, an SH2-binding motif and a phosphorylation site, respectively. STRA6 mutations thus define a pleiotropic malformation syndrome representing the first human phenotype associated with mutations in a gene from the "STRA" group.

Early-onset parkinsonism associated with PINK1 mutations

To assess the prevalence, nature, and associated phenotypes of PINK1 gene mutations in a large series of patients with early-onset (<50 years) parkinsonism. The authors studied 134 patients (116 sporadic and 18 familial; 77% Italian) and 90 Italian controls. The whole PINK1 coding region was sequenced from genomic DNA; cDNA was analyzed in selected cases. Homozygous pathogenic mutations were identified in 4 of 90 Italian sporadic cases, including the novel Gln456Stop mutation; single heterozygous truncating or missense mutations were found in another 4 Italian sporadic cases, including two novel mutations, Pro196Leu and Gln456Stop. Pathogenic mutations were not identified in the familial cases. Novel (Gln115Leu) and known polymorphisms were identified with similar frequency in cases and controls. In cases carrying single heterozygous mutation, cDNA analysis detected no additional mutations, and revealed a major pathogenic effect at mRNA level for the mutant C1366T/Gln456Stop allele. All patients with homozygous mutations had very early disease onset, slow progression, and excellent response to l-dopa, including, in some, symmetric onset, dystonia at onset, and sleep benefit, resembling parkin-related disease. Phenotype in patients with single heterozygous mutation was similar, but onset was later. PINK1 homozygous mutations are a relevant cause of disease among Italian sporadic patients with early-onset parkinsonism. The role of mutations found in single heterozygous state is difficult to interpret. Our study suggests that, at least in some patients, these mutations are disease causing, in combination with additional, still unknown factors.