Identification and Characterization of Novel Founder Mutations in NDRG1: Refining the Genetic Landscape of Charcot-Marie-Tooth Disease Type 4D in Bulgaria.

Charcot-Marie-Tooth neuropathy type 4D (CMT4D) is a rare genetic disorder of the peripheral nervous system caused by biallelic mutations in the N-Myc Downstream Regulated 1 gene (NDRG1). Patients present with an early onset demyelinating peripheral neuropathy causing severe distal muscle weakness and sensory loss, leading to loss of ambulation and progressive sensorineural hearing loss. The disorder was initially described in the Roma community due to a common founder mutation, and only a handful of disease-causing variants have been described in this gene so far. Here, we present genetic and clinical findings from a large Bulgarian cohort of demyelinating CMT patients harboring recurrent and novel variants in the NDRG1 gene. Notably, two splice-site variants are exclusive to Bulgarian Muslims and reside in ancestral haplotypes, suggesting a founder effect. Functional characterization of these novel variants implicates a loss-of-function mechanism due to shorter gene products. Our findings contribute to a deeper understanding of the genetic and clinical heterogeneity of CMT4D and highlight novel founder mutations in the ethnic minority of Bulgarian Muslims.

Molecular defects in the motor adaptor BICD2 cause proximal spinal muscular atrophy with autosomal-dominant inheritance.

The most common form of spinal muscular atrophy (SMA) is a recessive disorder caused by deleterious SMN1 mutations in 5q13, whereas the genetic etiologies of non-5q SMA are very heterogeneous and largely remain to be elucidated. In a Bulgarian family affected by autosomal-dominant proximal SMA, we performed genome-wide linkage analysis and whole-exome sequencing and found a heterozygous de novo c.320C>T (p.Ser107Leu) mutation in bicaudal D homolog 2 (Drosophila) (BICD2). Further analysis of BICD2 in a cohort of 119 individuals with non-5q SMA identified a second de novo BICD2 mutation, c.2321A>G (p.Glu774Gly), in a simplex case. Detailed clinical and electrophysiological investigations revealed that both families are affected by a very similar disease course, characterized by early childhood onset, predominant involvement of lower extremities, and very slow disease progression. The amino acid substitutions are located in two interaction domains of BICD2, an adaptor protein linking the dynein molecular motor with its cargo. Our immunoprecipitation and localization experiments in HeLa and SH-SY5Y cells and affected individuals' lymphoblasts demonstrated that p.Ser107Leu causes increased dynein binding and thus leads to accumulation of BICD2 at the microtubule-organizing complex and Golgi fragmentation. In addition, the altered protein had a reduced colocalization with RAB6A, a regulator of vesicle trafficking between the Golgi and the endoplasmic reticulum. The interaction between p.Glu744Gly altered BICD2 and RAB6A was impaired, which also led to their reduced colocalization. Our study identifies BICD2 mutations as a cause of non-5q linked SMA and highlights the importance of dynein-mediated motility in motor neuron function in humans.

Phenotypic and molecular insights into spinal muscular atrophy due to mutations in BICD2.

Spinal muscular atrophy is a disorder of lower motor neurons, most commonly caused by recessive mutations in SMN1 on chromosome 5q. Cases without SMN1 mutations are subclassified according to phenotype. Spinal muscular atrophy, lower extremity-predominant, is characterized by lower limb muscle weakness and wasting, associated with reduced numbers of lumbar motor neurons and is caused by mutations in DYNC1H1, which encodes a microtubule motor protein in the dynein-dynactin complex and one of its cargo adaptors, BICD2. We have now identified 32 patients with BICD2 mutations from nine different families, providing detailed insights into the clinical phenotype and natural history of BICD2 disease. BICD2 spinal muscular atrophy, lower extremity predominant most commonly presents with delayed motor milestones and ankle contractures. Additional features at presentation include arthrogryposis and congenital dislocation of the hips. In all affected individuals, weakness and wasting is lower-limb predominant, and typically involves both proximal and distal muscle groups. There is no evidence of sensory nerve involvement. Upper motor neuron signs are a prominent feature in a subset of individuals, including one family with exclusively adult-onset upper motor neuron features, consistent with a diagnosis of hereditary spastic paraplegia. In all cohort members, lower motor neuron features were static or only slowly progressive, and the majority remained ambulant throughout life. Muscle MRI in six individuals showed a common pattern of muscle involvement with fat deposition in most thigh muscles, but sparing of the adductors and semitendinosus. Muscle pathology findings were highly variable and included pseudomyopathic features, neuropathic features, and minimal change. The six causative mutations, including one not previously reported, result in amino acid changes within all three coiled-coil domains of the BICD2 protein, and include a possible 'hot spot' mutation, p.Ser107Leu present in four families. We used the recently solved crystal structure of a highly conserved region of the Drosophila orthologue of BICD2 to further-explore how the p.Glu774Gly substitution inhibits the binding of BICD2 to Rab6. Overall, the features of BICD2 spinal muscular atrophy, lower extremity predominant are consistent with a pathological process that preferentially affects lumbar lower motor neurons, with or without additional upper motor neuron involvement. Defining the phenotypic features in this, the largest BICD2 disease cohort reported to date, will facilitate focused genetic testing and filtering of next generation sequencing-derived variants in cases with similar features.

Novel mutations in the DYNC1H1 tail domain refine the genetic and clinical spectrum of dyneinopathies.

The heavy chain 1 of cytoplasmic dynein (DYNC1H1) is responsible for movement of the motor complex along microtubules and recruitment of dynein components. Mutations in DYNC1H1 are associated with spinal muscular atrophy (SMA), hereditary motor and sensory neuropathy (HMSN), cortical malformations, or a combination of these. Combining linkage analysis and whole-exome sequencing, we identified a novel dominant defect in the DYNC1H1 tail domain (c.1792C>T, p.Arg598Cys) causing axonal HMSN. Mutation analysis of the tail region in 355 patients identified a de novo mutation (c.791G>T, p.Arg264Leu) in an isolated SMA patient. Her phenotype was more severe than previously described, characterized by multiple congenital contractures and delayed motor milestones, without brain malformations. The mutations in DYNC1H1 increase the interaction with its adaptor BICD2. This relates to previous studies on BICD2 mutations causing a highly similar phenotype. Our findings broaden the genetic heterogeneity and refine the clinical spectrum of DYNC1H1, and have implications for molecular diagnostics of motor neuron diseases.

First cases of pyridoxine-dependent epilepsy in Bulgaria: novel mutation in the ALDH7A1 gene.

Pyridoxine-dependent epilepsy (PDE) is a rare autosomal recessive disorder characterized by intractable seizures in neonates and infants. The seizures cannot be controlled with antiepileptic medications but respond both clinically and electrographically to large daily supplements of pyridoxine (vitamin B6). PDE is caused by mutations in the ALDH7A1 gene. Molecular genetic analysis of the ALDH7A1 gene was performed in seven patients, referred with clinical diagnosis of PDE. Mutations were detected in a dizygotic twin pair and a non-related boy with classical form of PDE. Direct sequencing of the ALDH7A1 gene revealed one novel (c.297delG, p.Trp99*) and two already reported (c.328C>T, p.Arg110*; c.584A>G, p.Asn195Ser) mutations. Here, we report the first genetically proven cases of PDE in Bulgaria.

Essential components of an effective transition from paediatric to adult neurologist care for adolescents with Duchenne muscular dystrophy; a consensus derived using the Delphi methodology in Eastern Europe, Greece and Israel.

PURPOSE: An increasing number of patients with Duchenne muscular dystrophy (DMD) now have access to improved standard of care and disease modifying treatments, which improve the clinical course of DMD and extend life expectancy beyond 30 years of age. A key issue for adolescent DMD patients is the transition from paediatric- to adult-oriented healthcare. Adolescents and adults with DMD have unique but highly complex healthcare needs associated with long-term steroid use, orthopaedic, respiratory, cardiac, psychological, and gastrointestinal problems meaning that a comprehensive transition process is required. A sub-optimal transition into adult care can have disruptive and deleterious consequences for a patient's long-term care. This paper details the results of a consensus amongst clinicians on transitioning adolescent DMD patients from paediatric to adult neurologists that can act as a guide to best practice to ensure patients have continuous comprehensive care at every stage of their journey. METHODS: The consensus was derived using the Delphi methodology. Fifty-three statements were developed by a Steering Group (the authors of this paper) covering seven topics: Define the goals of transition, Preparing the patient, carers/parents and the adult centre, The transition process at the paediatric centre, The multidisciplinary transition summary - Principles, The multidisciplinary transition summary - Content, First visit in the adult centre, Evaluation of transition. The statements were shared with paediatric and adult neurologists across Central Eastern Europe (CEE) as a survey requesting their level of agreement with each statement. RESULTS: Data from 60 responders (54 full responses and six partial responses) were included in the data set analysis. A consensus was agreed across 100% of the statements. CONCLUSIONS: It is hoped that the findings of this survey which sets out agreed best practice statements, and the transfer template documents developed, will be widely used and so facilitate an effective transition from paediatric to adult care for adolescents with DMD.

Ethnicity-related DMD Genotype Landscapes in European and Non-European Countries.

OBJECTIVE: Genetic diagnosis and mutation identification are now compulsory for Duchenne (DMD) and Becker muscular dystrophies (BMD), which are due to dystrophin (DMD) gene mutations, either for disease prevention or personalized therapies. To evaluate the ethnic-related genetic assortments of DMD mutations, which may impact on DMD genetic diagnosis pipelines, we studied 328 patients with DMD and BMD from non-European countries. METHODS: We performed a full DMD mutation detection in 328 patients from 10 Eastern European countries (Poland, Hungary, Lithuania, Romania, Serbia, Croatia, Bosnia, Bulgaria, Ukraine, and Russia) and 2 non-European countries (Cyprus and Algeria). We used both conventional methods (multiplex ligation-dependent probe amplification [MLPA] followed by gene-specific sequencing) and whole-exome sequencing (WES) as a pivotal study ran in 28 patients where DMD mutations were already identified by standard techniques. WES output was also interrogated for DMD gene modifiers. RESULTS: We identified DMD gene mutations in 222 male patients. We identified a remarkable allele heterogeneity among different populations with a mutation landscape often country specific. We also showed that WES is effective for picking up all DMD deletions and small mutations and its adoption could allow a detection rate close to 90% of all occurring mutations. Gene modifiers haplotypes were identified with some ethnic-specific configurations. CONCLUSIONS: Our data provide unreported mutation landscapes in different countries, suggesting that ethnicity may orient genetic diagnosis flowchart, which can be adjusted depending on the mutation type frequency, with impact in drug eligibility.

SCN1A mutation spectrum in a cohort of Bulgarian patients with GEFS+ phenotype.

BACKGROUND: Dravet syndrome (DS) is the most severe form of Generalized Epilepsy with Febrile Seizures plus (GEFS+) syndrome with a clear genetic component in 85% of the cases. It is characterized by fever-provoked seizure onset around six months of age and subsequent developmental deterioration later in life. METHODS: In the current study, 60 patients with fever-provoked seizures and suspicion either of GEFS+ (50 patients) or of DS (10 patients) were referred for SCN1A gene sequence analysis. RESULTS: SCN1A gene sequencing revealed clinically significant variants in 11 patients (18.3%); seven pathogenic (11.7%) and four likely pathogenic (6.7%). Five of these variants have not been reported previously. Among the preselected group of ten DS patients, five had pathogenic SCN1A variants which confirmed diagnosis of DS. In four patients with preliminary diagnosis GEFS+, the detected SCN1A variant enabled us to specify the diagnosis of DS in these patients. Thus, SCN1A sequencing led to confirmation of the genetic diagnosis in 50% (5/10) of DS patients, as well as clarification of the diagnosis of DS in 8% of GEFS+ patients (4/50). In this study, four patients with truncating mutations had refractory seizures and additional psychomotor abnormalities. Additionally, pathogenic missense mutations were detected in three children with comparable phenotypes, which support the observations that missense mutations in critical channel function regions can cause a devastating epileptic condition. CONCLUSIONS: This is the first systematic screening of SCN1A gene in our country, which expands the spectrum of SCN1A variants with five novel variants from Bulgaria and demonstrates the clinical utility of confirmatory SCN1A testing, which helps clinicians make early and precise diagnoses. It is important for a better followup, choice of proper treatment, avoidance of development of refractory seizures and neuropsychological complications. Identification of pathogenic variants in SCN1A in the milder GEFS+ and severe DS cases, will help to offer adequate prenatal diagnosis and improve the genetic counselling provided to affected families.

Three cases of paralytic poliomyelitis associated with type 3 vaccine poliovirus strains in Bulgaria.

Oral poliovirus vaccine (OPV) can cause, in extremely rare cases vaccine-associated paralytic poliomyelitis in recipients, or contacts of vaccinees. Three cases of vaccine-associated paralytic poliomyelitis (two contacts and one recipient) occurred in the Bourgas region of Bulgaria in the spring of 2006. The first two cases, notified as acute flaccid paralysis, were 55 days old unvaccinated twin brothers, having been in contact with vaccinees. The third case concerned a 4-month-old infant who had received the first OPV dose 37 days prior to the onset of illness. Complete clinical, epidemiological, virological, serological and molecular investigations of the children with paralysis and their contacts were undertaken. In all the three cases type 3 polioviruses were isolated from fecal samples and characterized as Sabin-like poliovirus strains. Type 3 polioviruses isolated from the twin brothers demonstrated by sequence analysis U-to-C back mutation at nt 472 of the 5' UTR, known to correlate with neurovirulence, and mutation in the VP1 region. Type 3 poliovirus isolated from the third child demonstrated in the 3D sequenced region a recombination with Sabin type 1 poliovirus. In the latter region, three silent mutations and one, resulting in amino acid substitution, were also observed. The clinical, epidemiological and virological data and the neurological sequelae observed 60 days following the onset of paralysis, confirmed the diagnosis of vaccine-associated paralytic poliomyelitis in all the three patients.

Hydrocarbon molecular markers in the Holocene bottom sediments of the Barents Sea as indicators of natural and anthropogenic impacts.

The recent intensification of energy resource exploration and human activities in the Barents Sea (BS) requires a more thorough assessment of the natural and anthropogenic impact of hydrocarbons on the environment. We analyzed a wide set of sensitive indicators, including hydrocarbon molecular markers and organic matter (OM) maturity parameters in the Holocene sediments from three regions of the BS: the Kola-Kanin Monocline (KKM), the Svalbard shelf, and the Shtokman gas-condensate field (GCF). An increase in pyrogenic polycyclic aromatic hydrocarbons toward the core surface traces the intensification of anthropogenic contamination in the KKM region during last century. An input of highly mature OM from the eroded coal rocks of Barentsburg were confirmed by comparison of biomarker distribution in sediments and coals. An increase in biogenic hopanes and hopenes content down-core, and a crude-oil stage of OM maturity in surface sediments of the Shtokman GCF attests to hydrocarbons migration from subsurface strata.

Chromosomal microarray analysis of Bulgarian patients with epilepsy and intellectual disability.

High resolution chromosomal microarray analysis (CMA) has facilitated the identification of small chromosomal rearrangements throughout the genome, associated with various neurodevelopmental phenotypes, including ID/DD. Recently, it became evident that intellectual disability (ID)/developmental delay (DD) can occur with associated co-morbidities like epileptic seizures, autism and additional congenital anomalies. These observations require whole genome approach in order to detect the genetic causes of these complex disorders. In this study, we examined 92 patients of Bulgarian origin at age between 1 and 22 years with ID, generalized epilepsy, autistic signs and congenital anomalies. CMA was carried out using SurePrint G3 Human CGH Microarray Kit, 4 × 180 K and SurePrint G3 Unrestricted CGH ISCA v2, 4 × 180 K oligo platforms. Referral indications for selection of the patients were the presence of generalized refractory seizures disorders and co-morbid ID. Clearly pathogenic copy number variations (CNVs) were detected in eight patients (8.7%) from our cohort. Additionally, possibly pathogenic rearrangements of unclear clinical significance were detected in six individuals (6.5%), which make for an overall diagnostic yield of 15.2% among our cohort of patients. We report here the patients with clearly pathogenic CNVs, discuss the potential causality of the possibly pathogenic CNVs and make genotype - phenotype correlations. One novel possibly pathogenic heterozygous deletion in 15q22.31 region was detected in a case with ID/DD. Additionally, whole APBA2 gene duplication in 15q13.1 was found in three generations of a family with epilepsy, ID and psychiatric abnormalities. The results from this study allow us to define the genetic diagnosis in a subset of Bulgarian patients and improve the genetic counseling of the affected families. To our knowledge, this is the first aCGH evaluation of a Bulgarian cohort of children with epilepsy and ID so far.

Three novel SLC2A1 mutations in Bulgarian patients with different forms of genetic generalized epilepsy reflecting the clinical and genetic diversity of GLUT1-deficiency syndrome.

PURPOSE: GLUT1-deficiency syndrome (GLUT1-DS) is a metabolic brain disorder with a great clinical heterogeneity underlined by various mutations in the SLC2A1 gene which make the clinical and genetic diagnosis complicated. The purpose of our study is to investigate the genetic defects affecting the SLC2A1 gene in a group of Bulgarian patients with genetic generalized epilepsy (GGE), and to bring new insights into the molecular pathology of GLUT1-DS that would strengthen the genotype-phenotype correlations and improve the diagnostic procedure. METHODS: We have performed sequencing analysis of the SLC2A1 gene in thirty-eight Bulgarian patients with different forms of GGE having emerged in childhood followed by array comparative genome (aCGH) hybridization in patients with severe forms of GLUT1-DS who display extraneurological features. RESULTS: We have detected three novel SLC2A1 gene mutations that are predicted to have different impacts on the GLUT1 protein structure and function - one being to cause the amino acid substitution p.H160Q, another leading to the truncation p.Q360*, and also a 1p34.2 microdeletion. The overall frequency of the SLC2A1 mutations in the studied group is 8.1%. They have been found in clinical cases that differ notably by their severity. CONCLUSION: Our study enriches the mutation spectrum of the SLC2A1 gene by 3 novel cases that reflect the genetic and phenotypic diversity of GLUT1-DS and brings new insights into the molecular pathology of that disorder. The clinical data showed that the SLC2A1 genetic defects should be considered equally in the entire range of the clinical manifestations of GGE paying attention to the extraneurological features. The aCGH analysis should be considered as an ultimate step during the diagnostic procedure of GLUT1-DS in patients with a complex clinical picture of intractable epilepsy involving neuropsychological impairments and accompanied by extraneurological features.

Hereditary spastic paraplegia 3A associated with axonal neuropathy.

OBJECTIVE: To study the frequency and distribution of mutations in SPG3A in a large cohort of patients with hereditary spastic paraplegia. DESIGN: We screened a large cohort of 182 families and isolated cases with pure or complex hereditary spastic paraplegia phenotypes, which were negative for mutations in SPG4. RESULTS: In 12 probands (6.6%), we identified 12 different SPG3A mutations (11 missense and 1 insertion/frameshift) of which 7 were novel and 3 were de novo. We found incomplete penetrance in 1 family (G482V). In most cases, SPG3A mutations were associated with an early age at onset (mean, 3 y); however, in 1 family (R495W mutation), symptoms started later (mean, 14 y) with clear intrafamilial variability (8-28 y). Six patients with an SPG3A mutation (F151S, Q191R, M408T, G469A, R495W) originating from 5 unrelated families presented with a complex form of hereditary spastic paraplegia associated with a neuropathy (17%). Our electrophysiological and pathological findings confirmed an axonal sensory-motor neuropathy. There was no correlation between the genotype and the presence of a neuropathy. CONCLUSIONS: We conclude that mutations in SPG3A represent an important cause of patients in the overall hereditary spastic paraplegia population. SPG3A is more often associated with a neuropathy than previously assumed. Therefore, patients with a bipyramidal syndrome and a neuropathy should be screened for mutations in SPG3A.

Early clinical and electrophysiologic features of the two most common autosomal recessive forms of Charcot-Marie-Tooth disease in the Roma (Gypsies).

Our recent studies of the genetic epidemiology of neuromuscular disorders in Gypsies in Bulgaria have revealed that two private disorders, hereditary motor and sensory neuropathy type Lom and hereditary motor and sensory neuropathy type Russe, account for most cases of Charcot-Marie-Tooth disease in this population. In this study, we examined the clinical and electrophysiologic manifestations of the two disorders in childhood, aiming to identify the distinctive features that allow early differential diagnosis. The study included 13 patients, aged between 2 and 15 years. The childhood clinical manifestations of both neuropathies were similar, although they tended to be more severe in hereditary motor and sensory neuropathy type Lom. The nerve conduction velocities in hereditary motor and sensory neuropathy type Lom were lower than in hereditary motor and sensory neuropathy type Russe. Brainstem auditory evoked potentials were abnormal in hereditary motor and sensory neuropathy type Lom, even at an early age, and normal in hereditary motor and sensory neuropathy type Russe. Although electrophysiologic data provide a more reliable differentiation than clinical data, the definitive diagnosis should rely on genetic testing. (J Child Neurol 2006;21:20-25).

Myotonia congenita type Becker in Bulgaria: First genetically proven cases and mutation screening of two presumable endemic regions.

Myotonia congenita type Becker is an autosomal recessive nondystrophic skeletal muscle disorder, caused by mutations in the CLCN1 gene. The disease is characterized by muscle stiffness and an inability of the muscle to relax after voluntary contraction. Here we report the results from molecular genetic testing of 6 families, referred for sequencing of the CLCN1 gene. The disease causing mutations were detected in 5 of the cases, representing diverse type of nucleotide changes: nonsense (p.Arg894*), splice-site (c.1471+1G>A), missense (p.Val273Met; p.Tyr524Cys). Two additional changes were detected in an asymptomatic individual (c.2284+5C>T and p.Phe167Leu). Two of the detected mutations are interesting from population point of view. The novel missense mutation p.Tyr524Cys was found in a large Bulgarian family with affected individuals in both vertical and horizontal pedigree directions, all of them carrying the mutation in homozygous form. They populate a village located in the northwest part of the country. Endogamous marriages are very unusual for the Bulgarian population, supposing a high carrier frequency in this subpopulation. Screening of 154 residents of the corresponding region showed a significant carrier frequency for the p.Tyr524Cys mutation of about 0.65% (1/154). The second interesting region in the context of Myotonia congenita type Becker is the southwest part of the country, where we found a large family of Bulgarian Turkish origin. The disease causing missense mutation p.Val273Met was again present in homozygous state. Surprisingly, the genetic testing of newborns from southwest Bulgaria showed an even higher carrier status of about 2.6% (3/116), disproving our initial hypothesis of endogamous marriages (traditionally common in this subpopulation) being the cause of the disease in these patients. However the probability of consanguineous marriages being the cause for further exaggeration of the anyway very high carrier frequency cannot be excluded.

Clinical, neurophysiological and morphological study of dominant intermediate Charcot-Marie-Tooth type C neuropathy.

Dominant intermediate Charcot-Marie-Tooth neuropathy subtype C (DI-CMTC) was associated with mutations in the YARS gene, encoding tyrosyl-tRNA synthetase, in two large unrelated Bulgarian and US pedigrees and one sporadic case. Here for the first time we describe the clinical, neurophysiological and histopathological features, and phenotypic differences between these two DI-CMTC families. Twenty-one affected individuals from the US family and 27 from the Bulgarian family were evaluated. The mean age of onset in US subjects was 10.7 years in men and 7.3 years in women, while in the Bulgarian participants it was 18.2 years in men and 33.7 years in women. The course was slowly progressive. Extensor digitorum brevis atrophy was uniform. Atrophy and/or weakness of upper and lower limb muscles were found in over 50 % of the subjects. Nerve conduction studies (NCS) were abnormal in all US adults and five of six children and all Bulgarian patients except one asymptomatic 25-year-old man. Median motor NCS were in the range of 29.5-45.6 m/s in the US family and 24.7-57.8 m/s in the Bulgarian family. Sural sensory nerve action potentials were absent in 14/21 and 4/12 NCS from adult US and Bulgarian participants, respectively. Analysis of sural nerve biopsies from US patients revealed age-dependent morphological changes of axonal degeneration, absence of onion bulbs, and <10 % fibers with segmental remyelination. Our findings provide further insights into the diagnosis and pathology of intermediate CMT. They also extend the phenotypic spectrum of peripheral neuropathies associated with aminoacyl-tRNA synthetase mutations.

Spinal muscular atrophy among the Roma (Gypsies) in Bulgaria and Hungary.

Spinal muscular atrophy is one of the most common autosomal recessive disorders, classified into three major clinical forms. It is caused mainly by deletions or gene conversions of the telomeric survival motor neuron gene (SMN1) on human chromosome 5. We have conducted molecular studies of the disorder in genetically isolated Romani (Gypsy) communities in Bulgaria and Hungary, where spinal muscular atrophy appears to have different prevalence and both mild and severe spinal muscular atrophy phenotypes have been diagnosed. We have observed three distinct genetic defects which, in different combinations, lead to different forms of the disease. The similar chromosomal background on which the different mutations occur suggests a common origin and founder effect, with rearrangements of a single ancestral chromosome resulting in a diversity of molecular defects.

One novel and one recurrent mutation in IGHMBP2 gene, causing severe spinal muscular atrophy respiratory distress 1 with onset soon after birth.

A family with 2 siblings with severe spinal muscular atrophy with respiratory distress 1 (SMARD1) was genetically proved to be caused by mutations in IGHMBP2 gene. Both patients developed progressive muscular weakness and respiratory distress and died before 6 months of age. One novel deletion, c.780delG;p.(Gln260Hisfs*24), inherited from the father and a nonsense mutation, c.1488C>A;p.(Cys496*), inherited from the mother were detected. An attempt was made to correlate the genetic-clinical data available in the literature. The clinical case presented in this study might be considered as the most severe form of spinal muscular atrophy respiratory distress 1 reported so far, presumably because of the total absence of IGHMBP2 enzyme activity.

Sources of polycyclic aromatic hydrocarbons in marine sediments from southern and northern areas of the Norwegian continental shelf.

Variability in levels and sources of polycyclic aromatic hydrocarbons (PAH) in sediments from one large sea area off the coast of northern Norway ("North area", NA) have been compared to similar data from another large area off the coast of southern Norway ("South area", SA). Samples from NA were collected at the Norwegian continental shelf in south-western Barents Sea and north-eastern Norwegian Sea. Samples from SA were from the Norwegian Trench and the Skagerrak. Sediment cores have been dated, characterised by grain size distribution (GS) and organic carbon content (TOC), and the composition of PAH and geochemical biomarkers (alkanes and triterpanes) studied to provide an insight into the different sources of PAH. Generally, PAH levels are higher in sediments from SA compared to NA. A mixture of pyrogenic and petrogenic sources contribute to PAH levels in SA, while the contents of petrogenic PAH is negligible in surface sediments in NA. At some locations in NA, petrogenic PAH levels are elevated in the deepest sediment layers from pre-industrial times, indicating a natural input of petroleum through seepage. Occurrence of elevated levels of microbial hopanoids (hopenes) in the deepest sediment layers at some locations both in the north and the south indicate the presence of petroleum.