Distribution of Exonic Variants in Glycogen Synthesis and Catabolism Genes in Late Onset Pompe Disease (LOPD).

Pompe disease (PD) is a monogenic autosomal recessive disorder caused by biallelic pathogenic variants of the GAA gene encoding lysosomal alpha-glucosidase; its loss causes glycogen storage in lysosomes, mainly in the muscular tissue. The genotype-phenotype correlation has been extensively discussed, and caution is recommended when interpreting the clinical significance of any mutation in a single patient. As there is no evidence that environmental factors can modulate the phenotype, the observed clinical variability in PD suggests that genetic variants other than pathogenic GAA mutations influence the mechanisms of muscle damage/repair and the overall clinical picture. Genes encoding proteins involved in glycogen synthesis and catabolism may represent excellent candidates as phenotypic modifiers of PD. The genes analyzed for glycogen synthesis included UGP2, glycogenin (GYG1-muscle, GYG2, and other tissues), glycogen synthase (GYS1-muscle and GYS2-liver), GBE1, EPM2A, NHLRC1, GSK3A, and GSK3B. The only enzyme involved in glycogen catabolism in lysosomes is α-glucosidase, which is encoded by GAA, while two cytoplasmic enzymes, phosphorylase (PYGB-brain, PGL-liver, and PYGM-muscle) and glycogen debranching (AGL) are needed to obtain glucose 1-phosphate or free glucose. Here, we report the potentially relevant variants in genes related to glycogen synthesis and catabolism, identified by whole exome sequencing in a group of 30 patients with late-onset Pompe disease (LOPD). In our exploratory analysis, we observed a reduced number of variants in the genes expressed in muscles versus the genes expressed in other tissues, but we did not find a single variant that strongly affected the phenotype. From our work, it also appears that the current clinical scores used in LOPD do not describe muscle impairment with enough qualitative/quantitative details to correlate it with genes that, even with a slightly reduced function due to genetic variants, impact the phenotype.

Morquio B disease: From pathophysiology towards diagnosis.

Morquio B disease is an attenuated phenotype within the spectrum of beta galactosidase (GLB1) deficiencies. It is characterised by dysostosis multiplex, ligament laxity, mildly coarse facies and heart valve defects due to keratan sulphate accumulation, predominantly in the cartilage. Morquio B patients have normal neurological development, setting them apart from those with the more severe GM1 gangliosidosis. Morquio B disease, with an incidence of 1:250.000 to 1:1.000.000 live births, is very rare. Here we report the clinical-biochemical data of nine patients. High amounts of keratan sulfate were detected using LC-MS/MS in the patients' urinary samples, while electrophoresis, the standard procedure of qualitative glycosaminoglycans analysis, failed to identify this metabolite in any of the patients' samples. We performed molecular analyses at gene, gene expression and protein expression levels, for both isoforms of the GLB1 gene, lysosomal GLB1, and the cell-surface expressed Elastin Binding Protein. We characterised three novel GLB1 mutations [c.75 + 2 T > G, c.575A > G (p.Tyr192Cys) and c.2030 T > G (p.Val677Gly)] identified in three heterozygous patients. We also set up a copy number variation assay by quantitative PCR to evaluate the presence of deletions/ insertions in the GLB1 gene. We propose a diagnostic plan, setting out the specific clinical- biochemical and molecular features of Morquio B, in order to avoid misdiagnoses and improve patients' management.

Dietary lipids in glycogen storage disease type III: A systematic literature study, case studies, and future recommendations.

A potential role of dietary lipids in the management of hepatic glycogen storage diseases (GSDs) has been proposed, but no consensus on management guidelines exists. The aim of this study was to describe current experiences with dietary lipid manipulations in hepatic GSD patients. An international study was set up to identify published and unpublished cases describing hepatic GSD patients with a dietary lipid manipulation. A literature search was performed according to the Cochrane Collaboration methodology through PubMed and EMBASE (up to December 2018). All delegates who attended the dietetics session at the IGSD2017, Groningen were invited to share unpublished cases. Due to multiple biases, only data on GSDIII were presented. A total of 28 cases with GSDIII and a dietary lipid manipulation were identified. Main indications were cardiomyopathy and/or myopathy. A high fat diet was the most common dietary lipid manipulation. A decline in creatine kinase concentrations (n = 19, P < .001) and a decrease in cardiac hypertrophy in paediatric GSDIIIa patients (n = 7, P < .01) were observed after the introduction with a high fat diet. This study presents an international cohort of GSDIII patients with different dietary lipid manipulations. High fat diet may be beneficial in paediatric GSDIIIa patients with cardiac hypertrophy, but careful long-term monitoring for potential complications is warranted, such as growth restriction, liver inflammation, and hepatocellular carcinoma development.

Resting energy expenditure in argininosuccinic aciduria and in other urea cycle disorders.

No data are available on the specific energy needs of patients affected with Urea Cycle disorders (UCD) and especially argininosuccinic aciduria (ASA). In our experience, ASA patients tend to develop central adiposity and hypertriglyceridemia when treated with apparently adequate energy intake, while the other UCD do not. The aim of this study was to evaluate anthropometric parameters, body composition, risk of metabolic syndrome (MS) and resting energy expenditure (REE), both by indirect calorimetry (IC) and predictive equations, in UCD patients. Hypertension (5/13), pathological waist circumference-to-height ratio (WtHr) (6/13), hypertriglyceridemia (12/13), reduced HDL cholesterol (12/13), and MS (5/13) were found in ASA group. In the ASA cohort, the mean and median IC-REE were 88% of what was predicted by Food and Agriculture Organization of the United Nations and Harris-Benedict equations. The "other UCD" cohort did not show hypertension, dyslipidaemia nor MS; IC-REE was similar to the REE predicted by equations. A significant difference was seen for the presence of hypertension, dyslipidaemia, pathological WtHr, MS and IC-REE/predictive equations-REE in the two cohorts. ASA patients have a risk of overfeeding if their energy requirement is not assessed individually with IC. Excessive energy intake might increase the cardiovascular risk of ASA patients. We suggest to test ASA individuals with IC every year if the patient is sufficiently collaborative. We speculate that most of the features seen in ASA patients might depend on an imbalance of Krebs cycle. Further studies are needed to verify this hypothesis.

Chronic liver involvement in urea cycle disorders.

The increased survival of urea cycle disorders (UCDs) patients has led the attention to clinical manifestations that characterize the long-term disease course. Acute and chronic liver disease have been anecdotally reported since the very first description of UCDs. However, a detailed analysis of long-term liver involvement in large patient cohorts is still needed. Chronic liver damage in UCDs has probably a multifactorial origin, but the specific underlying mechanisms of liver disease have not yet been well elucidated. In this study, we report on chronic liver involvement and on associated metabolic abnormalities in a large cohort of 102 UCD patients, followed by two reference centers in Italy. Chronic liver involvement was observed in over 60% of UCDs patients, and comparison between individual diseases showed a significant higher frequency in argininosuccinate lyase deficiency (ASLD) and in hyperornithinemia-hyperammonemia-homocitrullinemia (HHH) syndrome with elevation of transaminases and of gamma-GT in ASLD, and of alpha-fetoprotein in HHH syndrome. Also, consistent with a chronic hepatic dysfunction, ultrasound examination revealed more pronounced abnormalities in ASLD and in HHH syndrome, when compared to other UCDs. Our study highlights in a large UCDs patients' cohort that chronic liver disease is a common finding in UCDs, often with a distinct phenotype between different diseases. Furthers studies are needed to elucidate the specific involvement of different metabolic pathways in the pathogenesis of liver dysfunction in UCDs.

Novel mutations in two unrelated Italian patients with SSADH deficiency.

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a rare autosomal recessive disorder of γ-aminobutyric acid (GABA) catabolism caused by mutations in the gene coding for succinic semialdehyde dehydrogenase (ALDH5A1). The abnormal levels of GHB detected in the brain and in all physiological fluids of SSADHD patients represent a diagnostic biochemical hallmark of the disease. Here we report on the clinical and molecular characterization of two unrelated Italian patients and the identification of two novel mutations: a 22 bp DNA duplication in exon 1, c.114_135dup, p.(C46AfsX97), and a non-sense mutation in exon 10, c.1429C > T, p.(Q477X). The two patients showed very different clinical phenotypes, coherent with their age. These findings enrich the characterization of SSADHD families and contribute to the knowledge on the progression of the disease.

Refining the Phenotype of Recurrent Rearrangements of Chromosome 16.

Chromosome 16 is one of the most gene-rich chromosomes of our genome, and 10% of its sequence consists of segmental duplications, which give instability and predisposition to rearrangement by the recurrent mechanism of non-allelic homologous recombination. Microarray technologies have allowed for the analysis of copy number variations (CNVs) that can contribute to the risk of developing complex diseases. By array comparative genomic hybridization (CGH) screening of 1476 patients, we detected 27 cases with CNVs on chromosome 16. We identified four smallest regions of overlapping (SROs): one at 16p13.11 was found in seven patients; one at 16p12.2 was found in four patients; two close SROs at 16p11.2 were found in twelve patients; finally, six patients were found with atypical rearrangements. Although phenotypic variability was observed, we identified a male bias for Childhood Apraxia of Speech associated to 16p11.2 microdeletions. We also reported an elevated frequency of second-site genomic alterations, supporting the model of the second hit to explain the clinical variability associated with CNV syndromes. Our goal was to contribute to the building of a chromosome 16 disease-map based on disease susceptibility regions. The role of the CNVs of chromosome 16 was increasingly made clear in the determination of developmental delay. We also found that in some cases a second-site CNV could explain the phenotypic heterogeneity by a simple additive effect or a pejorative synergistic effect.

Mis-splicing of the GALNS gene resulting from deep intronic mutations as a cause of Morquio a disease.

BACKGROUND: Mucopolysaccharidosis-IVA (Morquio A disease) is a lysosomal disorder in which the abnormal accumulation of keratan sulfate and chondroitin-6-sulfate is consequent to mutations in the galactosamine-6-sulfatase (GALNS) gene. Since standard DNA sequencing analysis fails to detect about 16% of GALNS mutant alleles, gross DNA rearrangement screening and uniparental disomy evaluation are required to complete the molecular diagnosis. Despite this, the second pathogenic GALNS allele generally remains unidentified in ~ 5% of Morquio-A disease patients. METHODS: In an attempt to bridge the residual gap between clinical and molecular diagnosis, we performed an mRNA-based evaluation of three Morquio-A disease patients in whom the second mutant GALNS allele had not been identified. We also performed sequence analysis of the entire GALNS gene in two patients. RESULTS: Different aberrant GALNS mRNA transcripts were characterized in each patient. Analysis of these transcripts then allowed the identification, in one patient, of a disease-causing deep intronic GALNS mutation. The aberrant mRNA products identified in the other two individuals resulted in partial exon loss. Despite sequencing the entire GALNS gene region in these patients, the identity of a single underlying pathological lesion could not be unequivocally determined. We postulate that a combination of multiple variants, acting in cis, may synergise in terms of their impact on the splicing machinery. CONCLUSIONS: We have identified GALNS variants located within deep intronic regions that have the potential to impact splicing. These findings have prompted us to incorporate mRNA analysis into our diagnostic flow procedure for the molecular analysis of Morquio A disease.

Hepatocellular carcinoma in Gaucher disease: an international case series.

Gaucher disease (GD) is associated with an increased risk for malignancies. Next to hematological malignancies, the development of solid tumors in several organs has been described. The liver is one of the major storage sites involved in GD pathogenesis, and is also affected by liver-specific complications. In this case series, we describe 16 GD type 1 (GD1) patients from eight different referral centers around the world who developed hepatocellular carcinoma (HCC). Potential factors contributing to the increased HCC risk in GD patients are studied. Eleven patients had undergone a splenectomy in the past. Liver cirrhosis, one of the main risk factors for the development of HCC, was present in nine out of 14 patients for whom data was available. Three out of seven examined patients showed a transferrin saturation > 45%. In these three patients the presence of iron overload after histopathological examination of the liver was shown. Chronic hepatitis C infection was present in three of 14 examined cases. We summarized all findings and made a comparison to the literature. We recommend that GD patients, especially those with prior splenectomy or iron overload, be evaluated for signs of liver fibrosis and if found to be monitored for HCC development.

Optimizing the molecular diagnosis of GALNS: novel methods to define and characterize Morquio-A syndrome-associated mutations.

Morquio A syndrome (MPS IVA) is a systemic lysosomal storage disorder caused by the deficiency of N-acetylgalactosamine-6-sulfatase (GALNS), encoded by the GALNS gene. We studied 37 MPS IV A patients and defined genotype-phenotype correlations based on clinical data, biochemical assays, molecular analyses, and in silico structural analyses of associated mutations. We found that standard sequencing procedures, albeit identifying 14 novel small GALNS genetic lesions, failed to characterize the second disease-causing mutation in the 16% of the patients' cohort. To address this drawback and uncover potential gross GALNS rearrangements, we developed molecular procedures (CNV [copy-number variation] assays, QF-PCRs [quantitative fluorescent-PCRs]), endorsed by CGH-arrays. Using this approach, we characterized two new large deletions and their corresponding breakpoints. Both deletions were heterozygous and included the first exon of the PIEZO1 gene, which is associated with dehydrated hereditary stomatocitosis, an autosomal-dominant syndrome. In addition, we characterized the new GALNS intronic lesion c.245-11C>G causing m-RNA defects, although identified outside the GT/AG splice pair. We estimated the occurrence of the disease in the Italian population to be approximately 1:300,000 live births and defined a molecular testing algorithm designed to help diagnosing MPS IVA and foreseeing disease progression.

Progression of renal damage in glycogen storage disease type I is associated to hyperlipidemia: a multicenter prospective Italian study.

Angiotensin converting enzyme (ACE)-inhibitors decrease glomerular hyperfiltration but not microalbuminuria and proteinuria in glycogen storage disease type I. In the current study, we demonstrated that severe hyperlipidemia is associated with ACE-inhibitor ineffectiveness. We underline the importance of adequate metabolic control in glycogen storage disease type I. A combination therapy with ACE-inhibitors and lipid lowering drugs might be considered.

Homozygous MTTP and APOB mutations may lead to hepatic steatosis and fibrosis despite metabolic differences in congenital hypocholesterolemia.

BACKGROUND & AIMS: Non-alcoholic steatohepatitis leading to fibrosis occurs in patients with abetalipoproteinemia (ABL) and homozygous or compound heterozygous familial hypobetalipoproteinemia (Ho-FHBL). We wanted to establish if liver alterations were more frequent in one of both diseases and were influenced by comorbidities. METHODS: We report genetic, clinical, histological and biological characteristics of new cases of ABL (n =7) and Ho-FHBL (n = 7), and compare them with all published ABL (51) and Ho-FHBL (22) probands. RESULTS: ABL patients, diagnosed during infancy, presented mainly with diarrhea, neurological and ophthalmological impairments and remained lean, whereas Ho-FHBL were diagnosed later, with milder symptoms often becoming overweight in adulthood. Despite subtle differences in lipid phenotype, liver steatosis was observed in both groups with a high prevalence of severe fibrosis (5/27 for Ho-FHBL vs. 4/58 for ABL (n.s.)). Serum triglycerides concentration was higher in Ho-FHBL whereas total and HDL-cholesterol were similar in both groups. In Ho-FHBL liver alterations were found to be independent from the apoB truncation size and apoB concentrations. CONCLUSIONS: Our findings provide evidence for major liver abnormalities in both diseases. While ABL and Ho-FHBL patients have subtle differences in lipid phenotype, carriers of APOB mutations are more frequently obese. These results raise the question of a complex causal link between apoB metabolism and obesity. They suggest that the genetic defect in VLDL assembly is critical for the occurrence of liver steatosis leading to fibrosis and shows that obesity and insulin resistance might contribute by increasing lipogenesis.

Lysinuric Protein Intolerance: not only a disorder for pediatric nephrologists. Case report.

INTRODUCTION: Lysinuric Protein Intolerance (LPI) is a multisystemic inborn error of metabolism with a variable clinical expressivity, that usually begins in childhood with growth failure and gastroenterological/neurological problems related to the altered urea cycle and later complications involving the renal, pulmonary and immuno-hematological systems. CASE REPORT: We present the case of a 40-year-old woman suffering from chronic kidney disease in the context of a LPI, whose diagnosis was challenging because the signs of the disease were always blurred and the patient never manifested critical episodes typical of this multisystemic disease. In addition to renal disease, splenomegaly, thrombocytopenia, elevated lactate dehydrogenase (LDH), hyperferritinemia and hypertriglyceridemia were also present. A thorough investigation of the patient's food preferences revealed her spontaneous aversion to protein-containing foods and excessive drowsiness during the occurrence of infectious episodes or on the rare occasions of excessive protein intake, although without ever coming to medical attention. These nuanced signs led us to suspect an impairment of the urea cycle and ultimately allowed us to narrow down the diagnosis to LPI through biochemical and genetic investigations. CONCLUSION: Nephrologists should consider LPI in the differential diagnosis, whenever a patient presents with mixed proteinuria, tubular dysfunction and/or chronic kidney failure of unknown origin. In these circumstances, we suggest looking for other signs such as growth failure, signs and symptoms ascribed to urea-cycle impairment, pulmonary involvement, hepatosplenomegaly and laboratory alterations such as pancytopenia, hyperferritinemia, lipid abnormalities, elevated LDH.

Long-term outcome of a cohort of Italian patients affected with alpha-Mannosidosis.

Alpha-mannosidosis (MIM #248500) is an ultra-rare autosomal recessive lysosomal storage disease with multi-system involvement and a wide phenotypic spectrum. Information on long-term outcomes remains poor. We present the long-term outcomes (median, 19 years) of nine patients with alpha-mannosidosis, three females and six males, followed at a single center. The findings of the nine patients were collected from medical records and reported as mean ± SD or median, and range. The age of onset of the first symptoms ranged from 0-1 to 10 years. The diagnostic delay ranged from 2 to 22 years (median= 11 years). Coarse face, hearing, heart valves, joints, gait, language, dysarthria, psychiatric symptoms, I.Q., MRI, walking disabilities, orthopedic disturbances and surgeries showed a slow worsening over the decades. Our patients showed a slowly worsening progressive outcome over the decades. Psychiatric symptoms were present in 100% of our population and improved with the appropriate pharmacological intervention. This aspect requires attention when following up on these patients. Our description of the long-term evolution of alpha-mannosidosis patients may provide basic knowledge for understanding the effects of specific treatments.

Fertility and pregnancy in women affected by glycogen storage disease type I, results of a multicenter Italian study.

BACKGROUND: Life expectancy of patients with glycogen storage disease (GSD) type I has improved considerably, opening new problems correlated with adult age. In females polycystic ovaries (PCOs) has been described as frequently associated with the disease, however successful pregnancies have been reported. Whether or not GSD I is associated with impaired reproductive function is still unclear. PATIENTS AND METHODS: Data about female patients with GSD Ia and Ib, who were 16 years or older, were obtained from clinical records and interviews. RESULTS: A total of 32 women with GSD I (25 GSD Ia, 7 GSD Ib), median age 26 years (range 16-55), were included. 9/32 patients had delayed menarche, 17/32 had irregular cycles, 8/22 had documented polycystic ovaries. Five successful spontaneous pregnancies in four patients with GSD Ia and two in a woman with GSD Ib were reported. The latter had development and enlargement of hepatic adenomas during pregnancies. CONCLUSION: Despite the high prevalence of irregular menstruation cycles and polycystic ovaries, fertility seems not to be impaired in women with GSD I. During pregnancy monitoring for adenoma development is mandatory.

Mutation Analysis of PKD1 and PKD2 Genes in a Large Italian Cohort Reveals Novel Pathogenic Variants Including A Novel Complex Rearrangement.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited disease of the kidney. It occurs in adulthood but is also rarely diagnosed in early childhood. The majority of the disease-causing variants observed in ADPKD patients are in two genes: PKD1 and PKD2. METHODS: 237 patients from 198 families with a clinical diagnosis of ADPKD were screened for PKD1 and PKD2 genetic variants using Sanger sequencing and Multiple Ligation-dependent Probe Amplification (MLPA) analysis. RESULTS: Disease-causing (diagnostic) variants were identified in 173 families (211 patients), 156 on PKD1 and 17 on PKD2. Variants of unknown significance (VUS) were detected in 6 additional families, while no mutations were found in the remaining 19 families. Among the diagnostic variants detected, 51 were novel. In ten families, seven large rearrangements were found and the molecular breakpoints of 3 rearrangements were identified. Renal survival was significantly worse for PKD1 mutated patients, particularly those carrying truncating mutations. In patients with PKD1 truncating ( PKD1-T) mutations, disease onset was significantly earlier than in patients with PKD1 non-truncating (PKD1-NT) variants or PKD2 mutated patients. CONCLUSIONS: Comprehensive genetic testing confirms its utility in diagnosing patients with ADPKD and contributes to explaining the clinical heterogeneity observed in this disease. Moreover, the genotype-phenotype correlation can allow a more accurate disease prognosis.

A GWAS in the pandemic epicenter highlights the severe COVID-19 risk locus introgressed by Neanderthals.

Large GWAS indicated that genetic factors influence the response to SARS-CoV-2. However, sex, age, concomitant diseases, differences in ancestry, and uneven exposure to the virus impacted the interpretation of data. We aimed to perform a GWAS of COVID-19 outcome in a homogeneous population who experienced a high exposure to the virus and with a known infection status. We recruited inhabitants of Bergamo province-that in spring 2020 was the epicenter of the SARS-Cov-2 pandemic in Europe-via an online questionnaire followed by personal interviews. Cases and controls were matched by age, sex and risk factors. We genotyped 1195 individuals and replicated the association at the 3p21.31 locus with severity, but with a stronger effect size that further increased in gravely ill patients. Transcriptome-wide association study highlighted eQTLs for LZTFL1 and CCR9. We also identified 17 loci not previously reported, suggestive for an association with either COVID-19 severity or susceptibility.

CFH and CFHR structural variants in atypical Hemolytic Uremic Syndrome: Prevalence, genomic characterization and impact on outcome.

Introduction: Atypical hemolytic uremic syndrome (aHUS) is a rare disease that manifests with microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure, and is associated with dysregulation of the alternative complement pathway. The chromosomal region including CFH and CFHR1-5 is rich in repeated sequences, favoring genomic rearrangements that have been reported in several patients with aHUS. However, there are limited data on the prevalence of uncommon CFH-CFHR genomic rearrangements in aHUS and their impact on disease onset and outcomes. Methods: In this study, we report the results of CFH-CFHR Copy Number Variation (CNV) analysis and the characterization of resulting structural variants (SVs) in a large cohort of patients, including 258 patients with primary aHUS and 92 with secondary forms. Results: We found uncommon SVs in 8% of patients with primary aHUS: 70% carried rearrangements involving CFH alone or CFH and CFHR (group A; n=14), while 30% exhibited rearrangements including only CFHRs (group B; n=6). In group A, 6 patients presented CFH::CFHR1 hybrid genes, 7 patients carried duplications in the CFH-CFHR region that resulted either in the substitution of the last CFHR1 exon(s) with those of CFH (CFHR1::CFH reverse hybrid gene) or in an internal CFH duplication. In group A, the large majority of aHUS acute episodes not treated with eculizumab (12/13) resulted in chronic ESRD; in contrast, anti-complement therapy induced remission in 4/4 acute episodes. aHUS relapse occurred in 6/7 grafts without eculizumab prophylaxis and in 0/3 grafts with eculizumab prophylaxis. In group B, 5 subjects had the CFHR31-5::CFHR410 hybrid gene and one had 4 copies of CFHR1 and CFHR4. Compared with group A, patients in group B exhibited a higher prevalence of additional complement abnormalities and earlier disease onset. However, 4/6 patients in this group underwent complete remission without eculizumab treatment. In secondary forms we identified uncommon SVs in 2 out of 92 patients: the CFHR31-5::CFHR410 hybrid and a new internal duplication of CFH. Discussion: In conclusion, these data highlight that uncommon CFH-CFHR SVs are frequent in primary aHUS and quite rare in secondary forms. Notably, genomic rearrangements involving the CFH are associated with a poor prognosis but carriers respond to anti-complement therapy.

IDUA mutational profiling of a cohort of 102 European patients with mucopolysaccharidosis type I: identification and characterization of 35 novel α-L-iduronidase (IDUA) alleles.

Mutational analysis of the IDUA gene was performed in a cohort of 102 European patients with mucopolysaccharidosis type I. A total of 54 distinct mutant IDUA alleles were identified, 34 of which were novel including 12 missense mutations, 2 nonsense mutations, 12 splicing mutations, 5 micro-deletions, 1 micro-duplication 1 translational initiation site mutation, and 1 'no-stop' change (p.X654RextX62). Evidence for the pathological significance of all novel mutations identified was sought by means of a range of methodological approaches, including the assessment of evolutionary conservation, RT-PCR/in vitro splicing analysis, MutPred analysis and visual inspection of the 3D-model of the IDUA protein. Taken together, these data not only demonstrate the remarkable mutational heterogeneity characterizing type 1 mucopolysaccharidosis but also illustrate our increasing ability to make deductions pertaining to the genotype-phenotype relationship in disorders manifesting a high degree of allelic heterogeneity.

PRDX1 gene-related epi-cblC disease is a common type of inborn error of cobalamin metabolism with mono- or bi-allelic MMACHC epimutations.

BACKGROUND: The role of epigenetics in inborn errors of metabolism (IEMs) is poorly investigated. Epigenetic changes can contribute to clinical heterogeneity of affected patients but could also be underestimated determining factors in the occurrence of IEMs. An epigenetic cause of IEMs has been recently described for the autosomal recessive methylmalonic aciduria and homocystinuria, cblC type (cblC disease), and it has been named epi-cblC. Epi-cblC has been reported in association with compound heterozygosity for a genetic variant and an epimutation at the MMACHC locus, which is secondary to a splicing variant (c.515-1G > T or c.515-2A > T) at the adjacent PRDX1 gene. Both these variants cause aberrant antisense transcription and cis-hypermethylation of the MMACHC gene promotor with subsequent silencing. Until now, only nine epi-cblC patients have been reported. METHODS: We report clinical/biochemical assessment, MMACHC/PRDX1 gene sequencing and genome-wide DNA methylation profiling in 11 cblC patients who had an inconclusive MMACHC gene testing. We also compare clinical phenotype of epi-cblC patients with that of canonical cblC patients. RESULTS: All patients turned out to have the epi-cblC disease. One patient had a bi-allelic MMACHC epimutation due to the homozygous PRDX1:c.515-1G > T variant transmitted by both parents. We found that the bi-allelic epimutation produces the complete silencing of MMACHC in the patient's fibroblasts. The remaining ten patients had a mono-allelic MMACHC epimutation, due to the heterozygous PRDX1:c.515-1G > T, in association with a mono-allelic MMACHC genetic variant. Epi-cblC disease has accounted for about 13% of cblC cases diagnosed by newborn screening in the Tuscany and Umbria regions since November 2001. Comparative analysis showed that clinical phenotype of epi-cblC patients is similar to that of canonical cblC patients. CONCLUSIONS: We provide evidence that epi-cblC is an underestimated cause of inborn errors of cobalamin metabolism and describe the first instance of epi-cblC due to a bi-allelic MMACHC epimutation. MMACHC epimutation/PRDX1 mutation analyses should be part of routine genetic testing for all patients presenting with a metabolic phenotype that combines methylmalonic aciduria and homocystinuria.