Immune Sensing of Synthetic, Bacterial, and Protozoan RNA by Toll-like Receptor 8 Requires Coordinated Processing by RNase T2 and RNase 2.

Human toll-like receptor 8 (TLR8) activation induces a potent T helper-1 (Th1) cell response critical for defense against intracellular pathogens, including protozoa. The receptor harbors two distinct binding sites, uridine and di- and/or trinucleotides, but the RNases upstream of TLR8 remain poorly characterized. We identified two endolysosomal endoribonucleases, RNase T2 and RNase 2, that act synergistically to release uridine from oligoribonucleotides. RNase T2 cleaves preferentially before, and RNase 2 after, uridines. Live bacteria, P. falciparum-infected red blood cells, purified pathogen RNA, and synthetic oligoribonucleotides all required RNase 2 and T2 processing to activate TLR8. Uridine supplementation restored RNA recognition in RNASE2-/- or RNASET2-/- but not RNASE2-/-RNASET2-/- cells. Primary immune cells from RNase T2-hypomorphic patients lacked a response to bacterial RNA but responded robustly to small-molecule TLR8 ligands. Our data identify an essential function of RNase T2 and RNase 2 upstream of TLR8 and provide insight into TLR8 activation.

Heterogeneity of PNPT1 neuroimaging: mitochondriopathy, interferonopathy or both?

BACKGROUND: Biallelic variants in PNPT1 cause a mitochondrial disease of variable severity. PNPT1 (polynucleotide phosphorylase) is a mitochondrial protein involved in RNA processing where it has a dual role in the import of small RNAs into mitochondria and in preventing the formation and release of mitochondrial double-stranded RNA into the cytoplasm. This, in turn, prevents the activation of type I interferon response. Detailed neuroimaging findings in PNPT1-related disease are lacking with only a few patients reported with basal ganglia lesions (Leigh syndrome) or non-specific signs. OBJECTIVE AND METHODS: To document neuroimaging data in six patients with PNPT1 highlighting novel findings. RESULTS: Two patients exhibited striatal lesions compatible with Leigh syndrome; one patient exhibited leukoencephalopathy and one patient had a normal brain MRI. Interestingly, two unrelated patients exhibited cystic leukoencephalopathy resembling RNASET2-deficient patients, patients with Aicardi-Goutières syndrome (AGS) or congenital CMV infection. CONCLUSION: We suggest that similar to RNASET2, PNPT1 be searched for in the setting of cystic leukoencephalopathy. These findings are in line with activation of type I interferon response observed in AGS, PNPT1 and RNASET2 deficiencies, suggesting a common pathophysiological pathway and linking mitochondrial diseases, interferonopathies and immune dysregulations.

Bi-allelic Mutations in NDUFA6 Establish Its Role in Early-Onset Isolated Mitochondrial Complex I Deficiency.

Isolated complex I deficiency is a common biochemical phenotype observed in pediatric mitochondrial disease and often arises as a consequence of pathogenic variants affecting one of the ∼65 genes encoding the complex I structural subunits or assembly factors. Such genetic heterogeneity means that application of next-generation sequencing technologies to undiagnosed cohorts has been a catalyst for genetic diagnosis and gene-disease associations. We describe the clinical and molecular genetic investigations of four unrelated children who presented with neuroradiological findings and/or elevated lactate levels, highly suggestive of an underlying mitochondrial diagnosis. Next-generation sequencing identified bi-allelic variants in NDUFA6, encoding a 15 kDa LYR-motif-containing complex I subunit that forms part of the Q-module. Functional investigations using subjects' fibroblast cell lines demonstrated complex I assembly defects, which were characterized in detail by mass-spectrometry-based complexome profiling. This confirmed a marked reduction in incorporated NDUFA6 and a concomitant reduction in other Q-module subunits, including NDUFAB1, NDUFA7, and NDUFA12. Lentiviral transduction of subjects' fibroblasts showed normalization of complex I. These data also support supercomplex formation, whereby the ∼830 kDa complex I intermediate (consisting of the P- and Q-modules) is in complex with assembled complex III and IV holoenzymes despite lacking the N-module. Interestingly, RNA-sequencing data provided evidence that the consensus RefSeq accession number does not correspond to the predominant transcript in clinically relevant tissues, prompting revision of the NDUFA6 RefSeq transcript and highlighting not only the importance of thorough variant interpretation but also the assessment of appropriate transcripts for analysis.

The failure of microglia to digest developmental apoptotic cells contributes to the pathology of RNASET2-deficient leukoencephalopathy.

The contribution of microglia in neurological disorders is emerging as a leading disease driver rather than a consequence of pathology. RNAseT2-deficient leukoencephalopathy is a severe childhood white matter disorder affecting patients in their first year of life and mimicking a cytomegalovirus brain infection. The early onset and resemblance of the symptoms to a viral infection suggest an inflammatory and embryonic origin of the pathology. There are no treatments available for this disease as our understanding of the cellular drivers of the pathology are still unknown. In this study, using a zebrafish mutant for the orthologous rnaset2 gene, we have identified an inflammatory signature in early development and an antiviral immune response in mature adult brains. Using the optical transparency and the ex utero development of the zebrafish larvae we studied immune cell behavior during brain development and identified abnormal microglia as an early marker of pathology. Live imaging and electron microscopy identified that mutant microglia displayed an engorged morphology and were filled with undigested apoptotic cells and undigested substrate. Using microglia-specific depletion and rescue experiments, we identified microglia as drivers of this embryonic phenotype and potential key cellular player in the pathology of RNAseT2-deficient leukoencephalopathy. Our zebrafish model also presented with reduced survival and locomotor defects, therefore recapitulating many aspects of the human disease. Our study therefore placed our rnaset2 mutant at the forefront of leukodystrophy preclinical models and highlighted tissue-specific approaches as future therapeutic avenues.

Opening New Horizons in the Treatment of Childhood Onset Leukodystrophies.

Leukodystrophies (LDs) predominantly affect the white matter of the central nervous system and its main component, the myelin. The majority of LDs manifests in infancy with progressive neurodegeneration. Main clinical signs are intellectual and motor function losses of already attained developmental skills. Classical LDs include lysosomal storage disorders like metachromatic leukodystrophy (MLD), peroxisomal disorders like X-linked adrenoleukodystrophy (X-ALD), disorders of mitochondrial dysfunction, and myelin protein defects like Pelizaeus-Merzbacher disease. So far, there are only single LD disorders with effective treatment options in an early stage of disease. The increasing number of patients diagnosed with LDs emphasizes the need for novel therapeutic options. Impressive advances in biotechnology have not only led to the continuous identification of new disease genes for so far unknown LDs but also led to new effective neuroprotective and disease-modifying therapeutic approaches. This review summarizes ongoing and novel innovative treatment options for LD patients and their challenges. It includes in vitro and in vivo approaches with focus on stem cell and gene therapies, intrathecal substrate or enzyme replacement, and genome editing.

Complex genomic rearrangements at the PLP1 locus include triplication and quadruplication.

Inverted repeats (IRs) can facilitate structural variation as crucibles of genomic rearrangement. Complex duplication-inverted triplication-duplication (DUP-TRP/INV-DUP) rearrangements that contain breakpoint junctions within IRs have been recently associated with both MECP2 duplication syndrome (MIM#300260) and Pelizaeus-Merzbacher disease (PMD, MIM#312080). We investigated 17 unrelated PMD subjects with copy number gains at the PLP1 locus including triplication and quadruplication of specific genomic intervals-16/17 were found to have a DUP-TRP/INV-DUP rearrangement product. An IR distal to PLP1 facilitates DUP-TRP/INV-DUP formation as well as an inversion structural variation found frequently amongst normal individuals. We show that a homology-or homeology-driven replicative mechanism of DNA repair can apparently mediate template switches within stretches of microhomology. Moreover, we provide evidence that quadruplication and potentially higher order amplification of a genomic interval can occur in a manner consistent with rolling circle amplification as predicted by the microhomology-mediated break induced replication (MMBIR) model.

Compound heterozygous variants in PGAP1 causing severe psychomotor retardation, brain atrophy, recurrent apneas and delayed myelination: a case report and literature review.

BACKGROUND: Mutations in proteins involved in the glycosylphosphatidylinositol anchor biosynthesis and remodeling pathway are associated with autosomal recessive forms of intellectual disability. Recently mutations in the PGAP1 gene that codes for PGAP1, a protein localized in the endoplasmic reticulum responsible for the first step of the remodeling of glycosylphosphatidylinositol was linked to a disorder characterized by psychomotor retardation and facial dysmorphism. Whole exome sequencing (WES) was performed in siblings with severely delayed myelination and psychomotor retardation. Mutations in PGAP1 were confirmed by Sanger sequencing and RNA analysis. A literature search was performed to describe the emerging phenotype of PGAP1 related disease. CASE PRESENTATION: WES resulted in the detection of two novel compound heterozygous mutations in PGAP1, one base pair insertion leading to a frame shift c.334_335InsA (p.A112fs) and a splice site mutation leading to exon skipping c.G1173C (p.L391L). A symptom not described in PGAP1 related disorder before but prominent in the siblings were recurrent apnea especially during sleep that persisted at least until age 2 years. Sequential cerebral MRI at age one and two year(s) respectively revealed frontal accentuated brain atrophy and significantly delayed myelination. CONCLUSION: We report siblings with two novel mutations in PGAP1. Other that the common symptoms related to PGAP1 mutations including non-progressive psychomotor retardation, neonatal feeding problems, microcephaly and brain atrophy these patients displayed severely delayed myelination and recurrent apneas thereby widing the clinical spectrum associated with such mutations.

West syndrome, microcephaly, grey matter heterotopia and hypoplasia of corpus callosum due to a novel ARFGEF2 mutation.

West syndrome (WS) is an epileptic encephalopathy of childhood, defined by the presence of clustered spasms usually occurring before the age of 1 year, hypsarrhythmia on EEG that is notoriously difficult to define, and developmental arrest or regression. The incidence of WS is 1:3200 live births with an aetiology-dependent prognosis. Up to 80% of children with symptomatic WS suffer from mental retardation, and approximately 50% develop Lennox-Gastaut syndrome. Using homozygosity mapping followed by exome sequencing, we identified a ADP-ribosylation factor (ARF) guanine nucleotide-exchange factor two (brefeldin A-inhibited) (ARFGEF2) mutation in five related infants with WS. ARFGEF2 is involved in the activation of ARFs by accelerating replacement of bound guanosine diphosphate (GDP) with Guanosine triphosphate (GTP), and is involved in Golgi transport. A mutation in ARFGEF2 has been previously described only once, causing microcephaly and periventricular heterotopia. Here, we describe a novel ARFGEF2 mutation in five related patients presenting with WS, microcephaly, periventricular heterotopia and thin corpus callosum.

rnaset2 mutant zebrafish model familial cystic leukoencephalopathy and reveal a role for RNase T2 in degrading ribosomal RNA.

T2-family acidic endoribonucleases are represented in all genomes. A physiological role for RNase T2 has yet to be defined for metazoa. RNASET2 mutation in humans is linked with a leukoencephalopathy that arises in infancy characterized by cortical cysts and multifocal white matter lesions. We now show localization of RNASET2 within lysosomes. Further, we demonstrate that loss of rnaset2 in mutant zebrafish results in accumulation of undigested rRNA within lysosomes within neurons of the brain. Further, by using high field intensity magnetic resonance microimaging, we reveal white matter lesions in these animals comparable to those observed in RNASET2-deficient infants. This correlates with accumulation of Amyloid precursor protein and astrocytes at sites of neurodegeneration. Thus we conclude that familial cystic leukoencephalopathy is a lysosomal storage disorder in which rRNA is the best candidate for the noxious storage material.

Assessment of myelination in hypomyelinating disorders by quantitative MRI.

PURPOSE: To apply myelin-sensitive quantitative magnetic resonance imaging (MRI) techniques in defined hypomyelinating conditions and to identify spatial patterns of myelination as criteria for characterization of undefined disorders. MATERIALS AND METHODS: Seven patients were included, based on the diagnosis of mitochondrial cytopathy, Pelizaeus-Merzbacher disease, GJA12/GJC2-related Pelizaeus-Merzbacher-like disease, hypomyelination with atrophy of the basal ganglia and cerebellum, and leukoencephalopathy with ataxia, delayed dentition, and hypomyelination. The control group comprised 23 children and adolescents (age range 2.6-22.4 years). The 3T MRI protocol consisted of high-resolution T1- and T2-weighted 3D MRI, diffusion tensor (DTI), and magnetization transfer (MT) imaging. Parameter maps of mean diffusivity, fractional anisotropy, and MT saturation were displayed as pseudocolor overlays and assessed by region-of-interest and histogram analysis. RESULTS: Structural MRI revealed widespread signal alterations in white matter, but were hampered by signal heterogeneity. Quantitative DTI and MT reflected the degree of hypomyelination and discriminative patterns of myelination emerged on MT saturation maps. CONCLUSION: The quantitative parameters in the defined hypomyelination conditions provide additional criteria to further classify undefined white matter disorders.

[Unilateral optic atrophy in a 9-year-old patient]. / Einseitige Optikusatrophie bei einem 9-jährigen Patienten.

A 9­year-old patient presented with a reduction of visual acuity in the left eye, which was incidentally noticed 2 weeks previously. Funduscopy revealed a mild vitritis, a pale optic disk, narrowed blood vessels and an increased reflex of the retinal surface. The values in the blood examination were normal apart from eosinophilia and an elevated immunoglobulin E (IgE) antibody titer.

A novel remitting leukodystrophy associated with a variant in FBP2.

Leukodystrophies are genetic disorders of cerebral white matter that almost exclusively have a progressive disease course. We became aware of three members of a family with a disorder characterized by a sudden loss of all previously acquired abilities around 1 year of age followed by almost complete recovery within 2 years. Cerebral MRI and myelin sensitive imaging showed a pronounced demyelination that progressed for several months despite signs of clinical improvement and was followed by remyelination. Exome sequencing did not-identify any mutations in known leukodystrophy genes but revealed a heterozygous variant in the FBP2 gene, c.343G>A, p. Val115Met, shared by the affected family members. Cerebral MRI of other family members demonstrated similar white matter abnormalities in all carriers of the variant in FBP2. The FBP2 gene codes for muscle fructose 1,6-bisphosphatase, an enzyme involved in gluconeogenesis that is highly expressed in brain tissue. Biochemical analysis showed that the variant has a dominant negative effect on enzymatic activity, substrate affinity, cooperativity and thermal stability. Moreover, it also affects the non-canonical functions of muscle fructose 1,6-bisphosphatase involved in mitochondrial protection and regulation of several nuclear processes. In patients' fibroblasts, muscle fructose 1,6-bisphosphatase shows no colocalization with mitochondria and nuclei leading to increased reactive oxygen species production and a disturbed mitochondrial network. In conclusion, the results of this study indicate that the variant in FBP2 disturbs cerebral energy metabolism and is associated with a novel remitting leukodystrophy.

Interferon-driven brain phenotype in a mouse model of RNaseT2 deficient leukoencephalopathy.

Infantile-onset RNaseT2 deficient leukoencephalopathy is characterised by cystic brain lesions, multifocal white matter alterations, cerebral atrophy, and severe psychomotor impairment. The phenotype is similar to congenital cytomegalovirus brain infection and overlaps with type I interferonopathies, suggesting a role for innate immunity in its pathophysiology. To date, pathophysiological studies have been hindered by the lack of mouse models recapitulating the neuroinflammatory encephalopathy found in patients. In this study, we generated Rnaset2-/- mice using CRISPR/Cas9-mediated genome editing. Rnaset2-/- mice demonstrate upregulation of interferon-stimulated genes and concurrent IFNAR1-dependent neuroinflammation, with infiltration of CD8+ effector memory T cells and inflammatory monocytes into the grey and white matter. Single nuclei RNA sequencing reveals homeostatic dysfunctions in glial cells and neurons and provide important insights into the mechanisms of hippocampal-accentuated brain atrophy and cognitive impairment. The Rnaset2-/- mice may allow the study of CNS damage associated with RNaseT2 deficiency and may be used for the investigation of potential therapies.

Zebrafish disease model of human RNASET2-deficient cystic leukoencephalopathy displays abnormalities in early microglia.

Human infantile-onset RNASET2-deficient cystic leukoencephalopathy is a Mendelian mimic of in utero cytomegalovirus brain infection with prenatally developing inflammatory brain lesions. We used an RNASET2-deficient zebrafish model to elucidate the underlying disease mechanisms. Mutant and wild-type zebrafish larvae brain development between 2 and 5 days post fertilization (dpf) was examined by confocal live imaging in fluorescent reporter lines of the major types of brain cells. In contrast to wild-type brains, RNASET2-deficient larvae displayed increased numbers of microglia with altered morphology, often containing inclusions of neurons. Furthermore, lysosomes within distinct populations of the myeloid cell lineage including microglia showed increased lysosomal staining. Neurons and oligodendrocyte precursor cells remained unaffected. This study provides a first look into the prenatal onset pathomechanisms of human RNASET2-deficient leukoencephalopathy, linking this inborn lysosomal disease to the innate immune system and other immune-related childhood encephalopathies like Aicardi-Goutières syndrome (AGS).

The Phenotypic Spectrum of PRRT2-Associated Paroxysmal Neurologic Disorders in Childhood.

Pathogenic variants in PRRT2, encoding the proline-rich transmembrane protein 2, have been associated with an evolving spectrum of paroxysmal neurologic disorders. Based on a cohort of children with PRRT2-related infantile epilepsy, this study aimed at delineating the broad clinical spectrum of PRRT2-associated phenotypes in these children and their relatives. Only a few recent larger cohort studies are on record and findings from single reports were not confirmed so far. We collected detailed genetic and phenotypic data of 40 previously unreported patients from 36 families. All patients had benign infantile epilepsy and harbored pathogenic variants in PRRT2 (core cohort). Clinical data of 62 family members were included, comprising a cohort of 102 individuals (extended cohort) with PRRT2-associated neurological disease. Additional phenotypes in the cohort of patients with benign sporadic and familial infantile epilepsy consist of movement disorders with paroxysmal kinesigenic dyskinesia in six patients, infantile-onset movement disorders in 2 of 40 individuals, and episodic ataxia after mild head trauma in one girl with bi-allelic variants in PRRT2. The same girl displayed a focal cortical dysplasia upon brain imaging. Familial hemiplegic migraine and migraine with aura were reported in nine families. A single individual developed epilepsy with continuous spikes and waves during sleep. In addition to known variants, we report the novel variant c.843G>T, p.(Trp281Cys) that co-segregated with benign infantile epilepsy and migraine in one family. Our study highlights the variability of clinical presentations of patients harboring pathogenic PRRT2 variants and expands the associated phenotypic spectrum.

Clinical, radiological and possible pathological overlap of cystic leukoencephalopathy without megalencephaly and Aicardi-Goutières syndrome.

BACKGROUND: Cystic leukoencephalopathy without megalencephaly is a disorder related in some cases to RNASET2 mutations and characterized by bilateral anterior temporal subcortical cysts and multifocal lobar white matter lesions with sparing of central white matter structures. This phenotype significantly overlaps with the sequelae of in utero cytomegalovirus (CMV) infection, including the presence of intracranial calcification in some cases. Aicardi-Goutières syndrome (AGS) is another inherited leukodystrophy with cerebral calcification mimicking congenital infection. Clinical, radiological and biochemical criteria for the diagnosis of AGS have been established, although the breadth of phenotype associated with mutations in the AGS-related genes is much greater than previously envisaged. PATIENTS AND METHODS: We describe the clinical, biochemical and radiological findings of five patients demonstrating a phenotype reminiscent of AGS. RESULTS: All patients were found to carry biallelic mutations of RNASET2. CONCLUSIONS: Our patients illustrate the clinical and radiological overlap that can be seen between RNASET2-related leukodystrophy and AGS in some cases. Our data highlight the need to include both disorders in the same differential diagnosis, and hint at possible shared pathomechanisms related to auto-inflammation which are worthy of further investigation.

Mutations in SNORD118 cause the cerebral microangiopathy leukoencephalopathy with calcifications and cysts.

Although ribosomes are ubiquitous and essential for life, recent data indicate that monogenic causes of ribosomal dysfunction can confer a remarkable degree of specificity in terms of human disease phenotype. Box C/D small nucleolar RNAs (snoRNAs) are evolutionarily conserved non-protein-coding RNAs involved in ribosome biogenesis. Here we show that biallelic mutations in the gene SNORD118, encoding the box C/D snoRNA U8, cause the cerebral microangiopathy leukoencephalopathy with calcifications and cysts (LCC), presenting at any age from early childhood to late adulthood. These mutations affect U8 expression, processing and protein binding and thus implicate U8 as essential in cerebral vascular homeostasis.

Identification of ten novel mutations in patients with eIF2B-related disorders.

Autosomal recessive inherited mutations in each of the five eukaryotic initiation factor 2B (eIF2B) subunits are known to cause white matter abnormalities with a wide continuum of clinical signs and severity leading to the concept of eIF2B-related disorders. The clinical spectrum extends from fatal infantile forms to adult forms with slow or absent neurological deterioration. In this study 15 well-characterised patients with the classical form of leukoencephalopathy with vanishing white matter (VWM) or with phenotypic variants like ovarioleukodystrophy were investigated for mutations in the genes EIF2B1, EIF2B2, EIF2B3, EIF2B4, and EIF2B5 encoding eIF2B. We identified one novel nonsense mutation (EIF2B4, c.625C>T, p.Arg209X), one novel frameshift mutation (EIF2B5, c.453_454del, p.Tyr152fsX12), eight novel missense muations (EIF2B1, c.547G>T, p.Val183Phe; EIF2B2, c. 586C>T, p.Pro196Ser; EIF2B4, c.806T>G, p.Leu269Arg; EIF2B5, c.203T>C, p.Leu68Ser; EIF2B5, c.220G>A, p.Ala74Thr; EIF2B5, c.805C>G, p.Arg269Gly; EIF2B5, c.929G>T, p.Cys310Phe; EIF2B5, c.1003T>C, p.Cys335Arg), and eight previously described alterations.

Phenotypic and molecular insights into CASK-related disorders in males.

BACKGROUND: Heterozygous loss-of-function mutations in the X-linked CASK gene cause progressive microcephaly with pontine and cerebellar hypoplasia (MICPCH) and severe intellectual disability (ID) in females. Different CASK mutations have also been reported in males. The associated phenotypes range from nonsyndromic ID to Ohtahara syndrome with cerebellar hypoplasia. However, the phenotypic spectrum in males has not been systematically evaluated to date. METHODS: We identified a CASK alteration in 8 novel unrelated male patients by targeted Sanger sequencing, copy number analysis (MLPA and/or FISH) and array CGH. CASK transcripts were investigated by RT-PCR followed by sequencing. Immunoblotting was used to detect CASK protein in patient-derived cells. The clinical phenotype and natural history of the 8 patients and 28 CASK-mutation positive males reported previously were reviewed and correlated with available molecular data. RESULTS: CASK alterations include one nonsense mutation, one 5-bp deletion, one mutation of the start codon, and five partial gene deletions and duplications; seven were de novo, including three somatic mosaicisms, and one was familial. In three subjects, specific mRNA junction fragments indicated in tandem duplication of CASK exons disrupting the integrity of the gene. The 5-bp deletion resulted in multiple aberrant CASK mRNAs. In fibroblasts from patients with a CASK loss-of-function mutation, no CASK protein could be detected. Individuals who are mosaic for a severe CASK mutation or carry a hypomorphic mutation still showed detectable amount of protein. CONCLUSIONS: Based on eight novel patients and all CASK-mutation positive males reported previously three phenotypic groups can be distinguished that represent a clinical continuum: (i) MICPCH with severe epileptic encephalopathy caused by hemizygous loss-of-function mutations, (ii) MICPCH associated with inactivating alterations in the mosaic state or a partly penetrant mutation, and (iii) syndromic/nonsyndromic mild to severe ID with or without nystagmus caused by CASK missense and splice mutations that leave the CASK protein intact but likely alter its function or reduce the amount of normal protein. Our findings facilitate focused testing of the CASK gene and interpreting sequence variants identified by next-generation sequencing in cases with a phenotype resembling either of the three groups.

Identification of twelve novel mutations in patients with classic and variant forms of maple syrup urine disease.

Maple syrup urine disease (MSUD) is an autosomal recessive metabolic disorder of panethnic distribution caused by a deficiency of the activity of branched-chain alpha-ketoacid dehydrogenase (BCKD) complex. Mutations in the human BCKD genes E1alpha (BCKDHA), E1beta (BCKDHB) and E2 (DBT) are known to result in MSUD, referred to as type Ia, Ib and II mutations respectively. In this study 16 patients with the classic severe form of MSUD and three patients with milder variant forms of the disease were investigated for mutations in the E1alpha-, E1beta- and E2-gene by single-strand conformation polymorphism (SSCP) analysis and DNA sequencing. The patients' clinical and biochemical phenotypes were well characterized. One novel type Ia missense mutation, eight novel type Ib (three missense, two nonsense, two small deletions, one small duplication) and three novel type II (two missense, one splice site) mutations were identified in patients. Moreover, eleven previously described mutations were detected: five type Ia (four missense, one nonsense), three type Ib mutations (two missense, one nonsense) and three type II mutations (two missense, one small deletion). Fourteen patients are homozygous for one single mutation, five patients are compound-heterozygous for two different mutations affecting one of the three genes. Thus, in all 19 patients the identified mutations can most probably be considered the molecular basis of the disease.