Late-onset sensory-motor axonal neuropathy, a novel SLC12A6-related phenotype.

We describe five families from different regions in Norway with a late-onset autosomal-dominant hereditary polyneuropathy sharing a heterozygous variant in the SLC12A6 gene. Mutations in the same gene have previously been described in infants with autosomal-recessive hereditary motor and sensory neuropathy with corpus callosum agenesis and mental retardation (Andermann syndrome), and in a few case reports describing dominantly acting de novo mutations, most of them with onset in childhood. The phenotypes in our families demonstrated heterogeneity. Some of our patients only had subtle to moderate symptoms and some individuals even no complaints. None had CNS manifestations. Clinical and neurophysiological evaluations revealed a predominant sensory axonal polyneuropathy with slight to moderate motor components. In all 10 patients the identical SLC12A6 missense variant, NM_001365088.1 c.1655G>A p.(Gly552Asp), was identified. For functional characterization, the mutant potassium chloride cotransporter 3 was modelled in Xenopus oocytes. This revealed a significant reduction in potassium influx for the p.(Gly552Asp) substitution. Our findings further expand the spectrum of SLC12A6 disease, from biallelic hereditary motor and sensory neuropathy with corpus callosum agenesis and mental retardation and monoallelic early-onset hereditary motor and sensory neuropathy caused by de novo mutations, to late-onset autosomal-dominant axonal neuropathy with predominant sensory deficits.

Epidemiology and natural history in 101 subjects with FKRP-related limb-girdle muscular dystrophy R9. The Norwegian LGMDR9 cohort study (2020).

We aimed to investigate the epidemiology and natural history of FKRP-related limb-girdle muscular dystrophy R9 (LGMDR9) in Norway. We identified 153 genetically confirmed subjects making the overall prevalence 2.84/100,000, the highest reported figure worldwide. Of the 153 subjects, 134 (88 %) were homozygous for FKRP c.826C>A giving a carrier frequency for this variant of 1/101 in Norway. Clinical questionnaires and patient notes from 101 subjects, including 88 c.826C>A homozygotes, were reviewed, and 43/101 subjects examined clinically. Age of onset in c.826C>A homozygotes demonstrated a bimodal distribution. Female subjects showed an increased cumulative probability of wheelchair dependency and need for ventilatory support. Across the cohort, the need for ventilatory support preceded wheelchair dependency in one third of the cases, usually due to sleep apnea. In c.826C>A homozygotes, occurrence of cardiomyopathy correlated positively with male gender but not with age or disease stage. This study highlights novel gender differences in both loss of ambulation, need for ventilatory support and the development of cardiomyopathy. Our results confirm the need for vigilance in order to detect respiratory insufficiency and cardiac involvement, but indicate that these events affect males and females differently.

Kennedy disease in two sisters with biallelic CAG expansions of the androgen receptor gene.

We present a retrospective 21-year follow-up of two sisters with X-linked biallelic CAG expansions in the androgen receptor (AR) gene causing Kennedy disease. Two sisters inherited CAG expansions from their mother who was a carrier and their father who had Kennedy disease. Genetic testing revealed alleles comprising 43/45, and 43/43 CAG repeats in the younger and older sister, respectively. They were referred to a neurologist for further evaluation. Both reported similar symptoms with chronic backache, pain and cramps in upper- and lower extremities, and fasciculations in their faces and extremities. Neurological examination demonstrated postural hand tremor in both and EMG revealed chronic neurogenic changes. Reevaluation of the patients at ages 74 and 83 showed slight progression of clinical manifestations. As opposed to male patients, these two females showed minimal disease progression and have maintained normal level of function into old age.

Clinical manifestations and novel pathogenic variants in SOX10 in eight Danish probands with Waardenburg syndrome.

The SRY-related HMG box gene 10 (SOX10), located on 22q13.1, encodes a member of the SOX family of transcription factors involved in the regulation of embryonic development and in the determination of cell fate and differentiation. SOX10 is one of the six causal genes for Waardenburg syndrome, which is a dominantly inherited auditory-pigmentary disorder characterized by sensorineural hearing impairment and abnormal pigmentation of the hair, skin and iris. Waardenburg syndrome is categorized into four subtypes based on clinical features (WS1-WS4). Here we present eight families (eleven patients) harboring pathogenic variants in SOX10. The patients displayed both allelic and clinical variability: bilateral profound hearing impairment (11/11), malformations of the semicircular canals (5/11), motor skill developmental delay (5/11), pigmentary defects (3/11) and Hirschsprung's disease (3/11) were some of the clinical manifestations observed. The patients demonstrate a spectrum of pathogenic SOX10 variants, of which six were novel (c.267del, c.299_300insA, c.335T >C, c.366_376del, c.1160_1179dup, and exon 3-4 deletion), and two were previously reported (c.336G>A and c.422T>C). Six of the variants occurred de novo whereas two were dominantly inherited. The pathogenic SOX10 variants presented here add novel information to the allelic variability of Waardenburg syndrome and illustrate the considerable clinical heterogeneity.

Protein homeostasis in LGMDR9 (LGMD2I) - The role of ubiquitin-proteasome and autophagy-lysosomal system.

AIMS: Limb-girdle muscular dystrophy R9 (LGMDR9) is an autosomal recessive disorder caused by mutations in the fukutin-related protein gene (FKRP), encoding a glycosyltransferase involved in α-dystroglycan modification. Muscle atrophy, a significant feature of LGMDR9, occurs by a change in the normal balance between protein synthesis and protein degradation. The ubiquitin-proteasome system (UPS) and autophagy-lysosomal system play a key role in protein degradation in skeletal muscle cells, but their involvement in the pathology of LGMDR9 is still largely unknown. We have aimed at clarifying whether proteolysis through the UPS and the autophagy-lysosomal pathway is dysregulated in LGMDR9 patients. METHODS: Vastus lateralis biopsies from 8 normal controls and 12 LGMDR9 patients harbouring the c.826C>A/c.826C>A FKRP genotype were assessed for protein markers related to UPS, the autophagy-lysosomal system and endoplasmic reticulum (ER) stress/unfolded protein response (UPR), followed by ultrastructural analysis by transmission electron microscopy (TEM). RESULTS: Protein levels of E3 ubiquitin ligases Atrogin-1 and MuRF1 showed a pattern similar to normal controls. Elevation of the autophagy markers Atg7, LC3B-II, decreased level of p62 as well as downregulation of the negative autophagy regulator mTORC1, indicated an activation of autophagy in LGMDR9. Mitophagy markers Bnip3 and Parkin were decreased. TEM analysis demonstrated accumulation of autophagosome-like structures in LGMDR9 muscle. There was also an increase in the expression of ER stress/UPR markers PDI, peIF2α and CHOP and a decrease in IRE1α. However, GRP94, Bip and Calnexin remained unchanged. CONCLUSION: Our findings indicate that autophagy and ER stress are induced in LGMDR9 muscle.

Limb girdle muscular dystrophy type 2I: No correlation between clinical severity, histopathology and glycosylated α-dystroglycan levels in patients homozygous for common FKRP mutation.

Limb girdle muscular dystrophy type 2I (LGMD2I) is a progressive disorder caused by mutations in the FuKutin-Related Protein gene (FKRP). LGMD2I displays clinical heterogeneity with onset of severe symptoms in early childhood to mild calf and thigh hypertrophy in the second or third decade. Patients homozygous for the common FKRP mutation c.826C>A (p.Leu276Ile) show phenotypes within the milder end of the clinical spectrum. However, this group also manifests substantial clinical variability. FKRP deficiency causes hypoglycosylation of α-dystroglycan; a component of the dystrophin associated glycoprotein complex. α-Dystroglycan hypoglycosylation is associated with loss of interaction with laminin α2, which in turn results in laminin α2 depletion. Here, we have attempted to clarify if the clinical variability seen in patients homozygous for c.826C>A is related to alterations in muscle fibre pathology, α-DG glycosylation levels, levels of laminin α2 as well as the capacity of α-DG to bind to laminin. We have assessed vastus lateralis muscle biopsies from 25 LGMD2I patients harbouring the c.826C>A/c.826C>A genotype by histological examination, immunohistochemistry and immunoblotting. No clear correlation was found between clinical severity, as determined by self-reported walking function, and the above features, suggesting that more complex molecular processes are contributing to the progression of disease.

Usher syndrome in Denmark: mutation spectrum and some clinical observations.

BACKGROUND: Usher syndrome (USH) is a genetically heterogeneous deafness-blindness syndrome, divided into three clinical subtypes: USH1, USH2 and USH3. METHODS: Mutations in 21 out of 26 investigated Danish unrelated individuals with USH were identified, using a combination of molecular diagnostic methods. RESULTS: Before Next Generation Sequencing (NGS) became available mutations in nine individuals (1 USH1, 7 USH2, 1 USH3) were identified by Sanger sequencing of USH1C,USH2A or CLRN1 or by Arrayed Primer EXtension (APEX) method. Mutations in 12 individuals (7 USH1, 5 USH2) were found by targeted NGS of ten known USH genes. Five novel pathogenic variants were identified. We combined our data with previously published, and obtained an overview of the USH mutation spectrum in Denmark, including 100 unrelated individuals; 32 with USH1, 67 with USH2, and 1 with USH3. Macular edema was observed in 44 of 117 individuals. Olfactory function was tested in 12 individuals and found to be within normal range in all. CONCLUSION: Mutations that lead to USH1 were predominantly identified in MYO7A (75%), whereas all mutations in USH2 cases were identified in USH2A. The MYO7A mutation c.93C>A, p.(Cys31*) accounted for 33% of all USH1 mutations and the USH2A c.2299delG, p.(Glu767Serfs*21) variant accounted for 45% of all USH2 mutations in the Danish cohort.

Alpha-mannosidosis: correlation between phenotype, genotype and mutant MAN2B1 subcellular localisation.

BACKGROUND: Alpha-mannosidosis is caused by mutations in MAN2B1, leading to loss of lysosomal alpha-mannosidase activity. Symptoms include intellectual disabilities, hearing impairment, motor function disturbances, facial coarsening and musculoskeletal abnormalities. METHODS: To study the genotype-phenotype relationship for alpha-mannosidosis 66 patients were included. Based on the predicted effect of the mutations and the subcellular localisation of mutant MAN2B1 in cultured cells, the patients were divided into three subgroups. Clinical and biochemical data were collected. Correlation analyses between each of the three subgroups of genotype/subcellular localisation and the clinical and biochemical data were done to investigate the potential relationship between genotype and phenotype in alpha-mannosidosis. Statistical analyses were performed using the SPSS software. Analyses of covariance were performed to describe the genotype-phenotype correlations. The phenotype parameters were modelled by the mutation group and age as a covariate. P values of <0.05 were considered as statistically significant. RESULTS: Complete MAN2B1 genotypes were established for all patients. We found significantly higher scores in the Leiter-R test, lower concentrations of CSF-oligosaccharides, higher point scores in the Bruininks-Oseretsky Test of Motor Proficiency subtests (BOT-2); Upper limb coordination and Balance, and a higher FVC% in patients in subgroup 3, harbouring at least one variant that allows localisation of the mutant MAN2B1 protein to the lysosomes compared to subgrou 2 and/or subgroup 1 with no lysosomal localization of the mutant MAN2B1 protein. CONCLUSION: Our results indicate a correlation between the MAN2B1 genotypes and the cognitive function, upper limb coordination, balance, FVC% and the storage of oligosaccharides in CSF. This correlation depends on the subcellular localisation of the mutant MAN2B1 protein.

amamutdb.no: A relational database for MAN2B1 allelic variants that compiles genotypes, clinical phenotypes, and biochemical and structural data of mutant MAN2B1 in α-mannosidosis.

α-Mannosidosis is an autosomal recessive lysosomal storage disorder caused by mutations in the MAN2B1 gene, encoding lysosomal α-mannosidase. The disorder is characterized by a range of clinical phenotypes of which the major manifestations are mental impairment, hearing impairment, skeletal changes, and immunodeficiency. Here, we report an α-mannosidosis mutation database, amamutdb.no, which has been constructed as a publicly accessible online resource for recording and analyzing MAN2B1 variants (http://amamutdb.no). Our aim has been to offer structured and relational information on MAN2B1 mutations and genotypes along with associated clinical phenotypes. Classifying missense mutations, as pathogenic or benign, is a challenge. Therefore, they have been given special attention as we have compiled all available data that relate to their biochemical, functional, and structural properties. The α-mannosidosis mutation database is comprehensive and relational in the sense that information can be retrieved and compiled across datasets; hence, it will facilitate diagnostics and increase our understanding of the clinical and molecular aspects of α-mannosidosis. We believe that the amamutdb.no structure and architecture will be applicable for the development of databases for any monogenic disorder.

Molecular characterization and phylogenetics of Fennoscandian cowpox virus isolates based on the p4c and atip genes.

BACKGROUND: Cowpox virus (CPXV), a rodent-borne Orthopoxvirus (OPV) that is indigenous to Eurasia can infect humans, cattle, felidae and other animals. Molecular characterization of CPXVs isolated from different geographic locations is important for the understanding of their biology, geographic distribution, classification and evolution. Our aim was to characterize CPXVs isolated from Fennoscandia on the basis of A-type inclusion (ATI) phenotype, restriction fragment length polymorphism (RFLP) profiles of atip gene fragment amplicon, and phylogenetic tree topology in conjunction with the patristic and genetic distances based on full length DNA sequence of the atip and p4c genes. METHODS: ATI phenotypes were determined by transmission electron microcopy and RFLP profiles were obtained by restriction enzyme digestion of the atip gene fragment PCR product. A 6.2 kbp region spanning the entire atip and p4c genes of Fennoscandian CPXV isolates was amplified and sequenced. The phylogenetic affinity of Fennoscandian CPXV isolates to OPVs isolated from other geographic regions was determined on the basis of the atip and p4c genes. RESULTS: Fennoscandian CPXV isolates encoded full length atip and p4c genes. They produce wild type V+ ATI except for CPXV-No-H2. CPXVs were resolved into six and seven species clusters based on the phylogeny of the atip and p4c genes respectively. The CPXVs isolated from Fennoscandia were grouped into three distinct clusters that corresponded to isolates from Norway, Sweden and Finland. CONCLUSION: CPXV is a polyphyletic assemblage of six or seven distinct clusters and the current classification in which CPXVs are united as one single species should be re-considered. Our results are of significance to the classification and evolution of OPVs.

Segregation analysis in families with Charcot-Marie-Tooth disease allows reclassification of putative disease causing mutations.

BACKGROUND: The identification of disease causing, or putative disease causing, mutations in index patients with Charcot-Marie-Tooth disease (CMT) allows for genetic testing of family members. Relevant variants identified in index patients are of either definite, likely or uncertain pathogenicity. The main objective of this study was to make an evaluation of the family investigations performed as part of the assessment of genetic variants of unknown clinical significance (VUS). METHODS: Between 2004 and 2010 molecular genetic family investigations were requested for 87 family members from 41 families harbouring PMP22dup or genetic variants in GJB1, MPZ, MFN2 and NEFL. Relatives were tested for the family mutation and data from the requisitions were evaluated by means of statistical tools. RESULTS: The results within each indication category are presented and discussed in detail. Twenty-two relatives (9 affected) from eight families were included in the segregation analyses, which invoked reclassification of three MFN2 mutations, two of which were de novo substitutions (c.2146_2148dup, c.692C > T). One MFN2 substitution was downgraded due to non-segregation (c.1709 A > G), and a MPZ substitution (c.103 G > A) upgraded due to segregation with the phenotype in the family. CONCLUSIONS: The results allow for the evaluation of the patient phenotypes ascertained in families, as opposed to the phenotypic descriptions of index patients. They indicate that de novo MFN2 mutations are regularly found in patients with a classical CMT2 phenotype. They also demonstrate the importance of a precise clinical and neurophysiologic diagnosis of affected family members. This particularly applies for the examination of variants of uncertain clinical significance. Finally, the fact that 14,6% of affected relatives tested for (probable or certain) pathogenic mutations were mutation negative, demonstrates that clinical evaluation alone is not always sufficient in order to determine their diagnosis. We believe that the results will aid in the estimation and planning of resources required for the various aspects of family evaluations in CMT.

The natural course and complications of alpha-mannosidosis--a retrospective and descriptive study.

Most alpha-mannosidosis patients described have been children and information on the natural course of the disorder has been based on a very limited number of observations. In order to assess the disease presentation in detail and to study disease characteristics, a study was started in 1991 and has been ongoing for over 20 years. Patients with confirmed alpha-mannosidosis were recruited through The International Society for Mannosidosis and Related Diseases (ISMRD) where families affected with alpha-mannosidosis received a questionnaire on general clinical information to be filled out by the responsible physician. The questionnaire was returned by 125 patients (64%). Of these, 45 patients were 15 years old or older at the time of evaluation. The questionnaire allowed us to assess the following features: Facial dysmorphism, columnar disease, arthritis, myopathy, hearing impairment, mental impairment, psychosis, bone disease and motor function as well as general health. This study describes the progression of alpha-mannosidosis and may be helpful in determining the clinical characteristics for assessments of prognosis.

Diagnostic laboratory testing for Charcot Marie Tooth disease (CMT): the spectrum of gene defects in Norwegian patients with CMT and its implications for future genetic test strategies.

BACKGROUND: Current genetic test algorithms for Charcot Marie Tooth (CMT) disease are based on family details and comprehensive clinical and neurophysiological data gathered under ideal conditions for clinical assessment. However, in a diagnostic laboratory setting relying on external test requisitions and patient samples, such conditions are not always met. Our objective was therefore to perform a retrospective evaluation of the data given in laboratory request forms and to assess their quality and applicability with regard to the recommended algorithms for CMT diagnostics. As we are the main test centre for CMT in Norway our results also provide an overview of the spectrum of gene defects in the Norwegian CMT population. METHODS: Genetic testing was performed according to polyneuropathy type; demyelinating/mixed: PMP22 duplication, MPZ, EGR2, LITAF, NEFL, PMP22, GJB1, axonal: MFN2, MPZ, NEFL, and GJB1. RESULTS: Diagnostic testing of index patients was requested in 435 of the 549 cases. Seventy-two (16.6%) positive molecular genetic findings were made. The majority (94.6%) of mutation positive cases showed disease onset before 50 years of age. PMP22 (duplication), MPZ, GJB1 and MFN2 mutations constituted 95.8% of the positive findings. Within the nerve conduction study groups, mutation detection rates were; demyelinating 33.8%; mixed 29.0%; axonal 8.8%; unspecified 16.5%. CONCLUSION: We suggest a simplified algorithm intended for referral centres, dealing with DNA/blood samples, which involves the assessment of age at onset and neurophysiological data followed by testing of four genes; PMP22 (duplication), MPZ, GJB1 and MFN2. Patients negative for mutations in those four genes should be subjected to evaluation at an interdisciplinary inherited neuropathy clinic with the capacity for extended molecular genetic analysis by next generation sequencing.

Hereditary motor neuron disease in a large Norwegian family with a "H46R" substitution in the superoxide dismutase 1 gene.

Mutant genes associated with Charcot Marie Tooth type 2, distal hereditary motor neuropathy and familial amyotrophic lateral sclerosis may cause overlapping clinical phenotypes. We performed whole genome linkage analysis, haplotype analysis, sequencing and detailed clinical and neurophysiological investigations in a large Norwegian kindred with a condition that clinically had been classified as Charcot Marie Tooth type 2. The mutation c.140A>G, p.His47Arg (alias p.His46Arg or H46R) in the superoxide dismutase 1 gene (SOD1) segregated with the disease. The patients present a hereditary motor neuropathy-like clinical picture and long survival (mean 29years). To our knowledge, this is the first extensive report describing a large non-Japanese kindred. The prognostic implications of the condition seen in this family have little in common with what is normally associated with sporadic amyotrophic lateral sclerosis and illustrates the complexity of the genetic etiology of lower motor neuron disease.

Identification of 83 novel alpha-mannosidosis-associated sequence variants: functional analysis of MAN2B1 missense mutations.

The lysosomal storage disorder alpha-mannosidosis is caused by deficiency of the enzyme lysosomal alpha-mannosidase (MAN2B1). In this study, 96 disease-associated sequence variants were identified in 130 unrelated alpha-mannosidosis patients from 30 countries. Eighty-three novel variants were detected, extending the mutation spectrum from 42 to 125. In total, 256 of the 260 mutant alleles (98.5%) were identified. Most of the variants were unique to each family, however, c.2248C>T (p.Arg750Trp) was detected in 50 patients from 16 countries, and accounted for 27.3% of the disease alleles. Haplotype analysis revealed that the c.2248T variant was present on four MAN2B1 haplotype backgrounds, where one major haplotype accounted for 95% of the alleles. The distribution of the c.2248T-associated haplotypes differed remarkably from those of the control populations, suggesting that c.2248C>T has occurred on a few ancestral haplotypes where the major haplotype subsequently has spread by founder effects. The disease-associated missense mutations were introduced into the human MAN2B1 cDNA, expressed in cell culture and assayed for MAN2B1 activity. The majority of the variants were inactive, however, ten showed MAN2B1 activity above background, and more detailed studies are necessary to further evaluate the pathogenicity of these variants.

Fukutin-related protein resides in the Golgi cisternae of skeletal muscle fibres and forms disulfide-linked homodimers via an N-terminal interaction.

Limb-Girdle Muscular Dystrophy type 2I (LGMD2I) is an inheritable autosomal, recessive disorder caused by mutations in the FuKutin-Related Protein (FKRP) gene (FKRP) located on chromosome 19 (19q13.3). Mutations in FKRP are also associated with Congenital Muscular Dystrophy (MDC1C), Walker-Warburg Syndrome (WWS) and Muscle Eye Brain disease (MEB). These four disorders share in common an incomplete/aberrant O-glycosylation of the membrane/extracellular matrix (ECM) protein α-dystroglycan. However, further knowledge on the FKRP structure and biological function is lacking, and its intracellular location is controversial. Based on immunogold electron microscopy of human skeletal muscle sections we demonstrate that FKRP co-localises with the middle-to-trans-Golgi marker MG160, between the myofibrils in human rectus femoris muscle fibres. Chemical cross-linking experiments followed by pairwise yeast 2-hybrid experiments, and co-immune precipitation, demonstrate that FKRP can exist as homodimers as well as in large multimeric protein complexes when expressed in cell culture. The FKRP homodimer is kept together by a disulfide bridge provided by the most N-terminal cysteine, Cys6. FKRP contains N-glycan of high mannose and/or hybrid type; however, FKRP N-glycosylation is not required for FKRP homodimer or multimer formation. We propose a model for FKRP which is consistent with that of a Golgi resident type II transmembrane protein.

Molecular and cellular characterization of novel {alpha}-mannosidosis mutations.

α-Mannosidosis is a lysosomal storage disorder caused by mutations in the MAN2B1 gene. The clinical presentation of α-mannosidosis is variable, but typically includes mental retardation, skeletal abnormalities and immune deficiency. In order to understand the molecular aetiology of α-mannosidosis, we describe here the subcellular localization and intracellular processing of 35 MAN2B1 variants, including 29 novel missense mutations. In addition, we have analysed the impact of the individual mutations on the three-dimensional structure of the human MAN2B1. We categorize the MAN2B1 missense mutations into four different groups based on their intracellular processing, transport and secretion in cell culture. Impaired transport to the lysosomes is a frequent cause of pathogenicity and correlates with a lack of protein processing (groups 1 and 3). Mutant MAN2B1 proteins that find their way to the lysosomes are processed, but less efficiently than the wild-types (groups 2 and 4). The described four categories of missense mutations likely represent different pathogenic mechanisms. We demonstrate that the severity of individual mutations cannot be determined based only on their position in the sequence. Pathogenic mutations cluster into amino acids which have an important role on the domain interface (arginines) or on the folding of the enzyme (prolines, glycines, cysteines). Tolerated mutations generally include surface mutations and changes without drastic alteration of residue volume. The expression system and structural details presented here provide opportunities for the development of pharmacological therapy by screening or design of small molecules that might assist MAN2B1 folding and hence, transport and activity.

Prevalence, mutation spectrum and phenotypic variability in Norwegian patients with Limb Girdle Muscular Dystrophy 2I.

Mutations in the FKRP (Fukutin Related Protein) gene produce a range of phenotypes including Limb Girdle Muscular Dystrophy Type 2I (LGMD2I). In order to investigate the prevalence, the mutation spectrum and possible genotype-phenotype correlation, we studied a cohort of Norwegian patients with LGMD2I, ascertained in a 4-year period. In this retrospective study of genetically tested patients, we identified 88 patients from 69 families, who were either homozygous or compound heterozygous for FKRP mutations. This gives a minimum prevalence of 1/54,000 and a corresponding carrier frequency of 1/116 in the Norwegian population. Seven different FKRP mutations, including three novel changes, were detected. Seventy-six patients were homozygous for the common c.826C>A mutation. These patients had later disease onset than patients who were compound heterozygous - 14.0 vs. 6.1 years. We detected substantial variability in disease severity among homozygous patients.