Assessing the effects of PMM2 variants on protein stability.

Phosphomannomutase 2 deficiency, PMM2-CDG, is the most frequent disorder of protein N-glycosylation. It is an autosomal recessive disease with a broad clinical and biochemical phenotype. Trying to predict the impact of novel variants is often a challenge due to the high number of variants and the difficulty to establish solid genotype-phenotype correlations. A potential useful strategy is to use computational chemistry calculations as a tool from which relevant information on the structural impact of novel variants may be deduced. Here we present our analyses based on four well-known PMM2 deleterious variants (p.(Leu32Arg), p.(Asp65Tyr), p.(Phe119Leu), p.(Arg141His)) and the polymorphic p.(Glu197Ala) for which we have predicted the effect on protein stability. Our work predicts the effect of different amino acid residues on the conformation and stability of PMM2. These computational simulations are, therefore, an extremely useful methodology which, in combination with routinely used in silico methods of pathogenicity prediction, may help to reveal the structural impact of novel variants at the protein level, potentially leading to a better understanding of target biological molecules.

RFT1-CDG: Absence of Epilepsy and Deafness in Two Patients with Novel Pathogenic Variants.

This report is on two novel patients with RFT1-CDG. Their phenotype is characterized by mild psychomotor disability, behavioral problems, ataxia, and mild dysmorphism. Neither of them shows signs of epilepsy, which was observed in all RFT1-CDG patients reported to date (n = 14). Also, deafness, which is often associated with this condition, was not observed in our patients. Molecular analysis of RFT1 showed biallelic missense variants including three novel ones: c.827G > A (p.G276D), c.73C > T (p.R25W), and c.208T > C (p.C70R).

Clinical, biochemical and molecular characterization of cystinuria in a cohort of 12 patients.

Cystinuria is a rare autosomal inherited disorder characterized by impaired transport of cystine and dibasic aminoacids in the proximal renal tubule. Classically, cystinuria is classified as type I (silent heterozygotes) and non-type I (heterozygotes with urinary hyperexcretion of cystine). Molecularly, cystinuria is classified as type A (mutations on SLC3A1 gene) and type B (mutations on SLC7A9 gene). The goal of this study is to provide a comprehensive clinical, biochemical and molecular characterization of a cohort of 12 Portuguese patients affected with cystinuria in order to provide insight into genotype-phenotype correlations. We describe seven type I and five non-type I patients. Regarding the molecular classification, seven patients were type A and five were type B. In SLC3A1 gene, two large genomic rearrangements and 13 sequence variants, including four new variants c.611-2A>C; c.1136+44G>A; c.1597T (p.Y533N); c.*70A>G, were found. One large genomic rearrangement was found in SLC7A9 gene as well as 24 sequence variants including 3 novel variants: c.216C>T (p.C72C), c.1119G>A (p.S373S) and c.*82C>T. In our cohort the most frequent pathogenic mutations were: large rearrangements (33.3% of mutant alleles) and a missense mutation c.1400T>C (p.M467T) (11.1%). This report expands the spectrum of SLC3A1 and SLC7A9 mutations and provides guidance in the clinical implementation of molecular assays in routine genetic counseling of Portuguese patients affected with cystinuria.

The molecular landscape of phosphomannose mutase deficiency in iberian peninsula: identification of 15 population-specific mutations.

PMM2-CDG is an autosomal recessive disorder and the most frequent form of congenital disorder of N-glycosylation, with more than 100 mutations identified to date. Sixty-six patients from 58 unrelated families were diagnosed as PMM2-CDG (CDG-Ia) based on clinical signs or because of a previous affected sibling. They all presented a type 1 serum transferrin isoform pattern, and, in most cases, the disease was confirmed by determining PMM2 activity in fibroblasts and/or lymphocytes. Residual PMM2 activity in fibroblasts ranged from not detectable to 60% of the mean controls. DNA and RNA were isolated from fresh blood or fibroblasts from patients to perform molecular studies of the PMM2 gene, resulting in the identification of 30 different mutations, four of them newly reported here (p.Y102C, p.T118S, p.P184T, and p.D209G). From these 30 mutations, 15 have only been identified among Iberian PMM2-CDG patients. As in other Caucasian populations, p.R141H was the most frequent mutation (24 alleles, prevalence 20.6%), but less than in other European series in which this mutation represents 35-43% of the disease alleles. The next frequent mutations were p.D65Y (12 alleles, prevalence 10.3%) and p.T237M (9 alleles, prevalence 7.6%), while p.F119L and p.E139K, the most frequent changes in Scandinavian and French populations, respectively, were not found in our patients. The most common genotype was [p.R141H] + [p.T237M], and four homozygous patients for p.Y64C, p.D65Y, p.P113L, and p.T237M were detected. The broad mutational spectrum and the diversity of phenotypes found in the Iberian populations hamper genotype-phenotype correlation.

Relevance of expanded neonatal screening of medium-chain acyl co-a dehydrogenase deficiency: outcome of a decade in galicia (Spain).

Neonatal screening of medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is of major importance due to the significant morbidity and mortality in undiagnosed patients. MCADD screening has been performed routinely in Galicia since July 2000, and until now 199,943 newborns have been screened. We identified 11 cases of MCADD, which gives an incidence of 1/18,134. During this period, no false negative screens have been detected. At diagnosis, all identified newborns were asymptomatic. Our data showed that octanoylcarnitine (C8) and C8/C10 ratio are the best markers for screening of MCADD. C8 was increased in all patients and C8/C10 was increased in all but one patient.The common mutation, c.985A > G, was found in homozygosity in seven newborns and in compound heterozygosity in three, while one patient did not carry the common mutation at all. In addition, two novel mutations c.245G > C (p.W82S) and c.542A > G (p.D181G) were identified. Ten of the 11 identified newborns did not experience any episodes of decompensation. The patient with the highest level of medium chain acylcarnitines at diagnosis, who was homozygous for the c.985A > G mutation, died at the age of 2 years due to a severe infection.This is the first report of the results from neonatal screening for MCADD in Spain. Our data provide further evidence of the benefits of MCADD screening and contribute to better understanding of this disease.

Quantitative analysis of five sterols in amniotic fluid by GC-MS: application to the diagnosis of cholesterol biosynthesis defects.

Cholesterol and its precursors, namely 7-dehydrocholesterol, desmosterol and lathosterol are important biochemical markers of cholesterol biosynthesis, and their quantification in body fluids is useful for the diagnosis of cholesterol biosynthesis pathway disorders. A rapid and sensitive gas chromatographic-mass spectrometric method was developed and validated for quantitative analysis of five sterols (cholesterol, 7-dehydrocholesterol, desmosterol, lathosterol and sitosterol) in amniotic fluid. The method was linear for all compounds (r(2)>0.99), and intra and inter-assay coefficients of variation were typically below 5%, and inaccuracy was within a +/-12% interval. The method was applied to 330 amniotic fluid samples, grouped by gestational age between 13 and 22 weeks of pregnancy, in order to establish reference intervals for sterols in this specimen. The obtained concentrations (mumol/L) for each sterol was as follows: 22.1758+/-4.2716 at 13 weeks and 78.5082+/-12.9041 at 22 weeks for cholesterol; 0.0039+/-0.0007 at 13 weeks and 0.1150+/-0.0212 at 22 weeks for 7-dehydrocholesterol; 0.1562+/-0.0406 at 13 weeks and 0.7691+/-0.0821 at 22 weeks for desmosterol; 0.0272+/-0.0035 at 13 weeks and 0.8551+/-0.1791 at 22 weeks for lathosterol; and 0.0404+/-0.0039 at 13 weeks and 0.2326+/-0.0386 at 22 weeks for sitosterol. The method was also applied to one pathological sample that showed decreased levels of cholesterol, and higher concentration of 7-dehydrocholesterol, which is consistent with a 7-dehydrocholesterol-reductase deficiency. Our results showed that as long as pregnancy goes on, the concentrations of cholesterol and precursors increase in amniotic fluid, which is related to the increased need for cholesterol by the fetus. The reference range of each sterol in amniotic fluid was calculated at different gestational ages and will be useful for the interpretation and validation of biochemical prenatal diagnosis of inborn errors of sterol biosynthesis.

Congenital disorder of glycosylation type Ia: searching for the origin of common mutations in PMM2.

Congenital Disorders of Glycosylation (CDG) are a group of recessive genetic disorders characterized by hypoglycosylation of glycoproteins. CDG-Ia, the most common type, is caused by mutations in the PMM2 gene, coding for a phosphomannomutase (PMM2; EC 5.4.2.8). The mutational spectrum of PMM2 comprises more than 80 different mutations but one of them, R141H, is particularly interesting due to its high frequency among CDG-Ia patients worldwide. In contrast, other mutations are ethnically or geographically restricted, such as D65Y which is only found in patients of Iberian ancestry. In the present study a population genetic approach was used in an attempt to clarify the origins of two important disease causing mutations: R141H and D65Y. Based on SNP and STR genotypic analysis, we ascertained an association between the R141H substitution and a particular haplotype, suggesting a common origin for all the mutated chromosomes. Similar results were found for D65Y, although the associated haplotype was different from that of R141H, suggesting independent origins for these two mutations. Our results enable us to infer an Iberian origin for the D65Y mutation.

Detailed glycan analysis of serum glycoproteins of patients with congenital disorders of glycosylation indicates the specific defective glycan processing step and provides an insight into pathogenesis.

The fundamental importance of correct protein glycosylation is abundantly clear in a group of diseases known as congenital disorders of glycosylation (CDGs). In these diseases, many biological functions are compromised, giving rise to a wide range of severe clinical conditions. By performing detailed analyses of the total serum glycoproteins as well as isolated transferrin and IgG, we have directly correlated aberrant glycosylation with a faulty glycosylation processing step. In one patient the complete absence of complex type sugars was consistent with ablation of GlcNAcTase II activity. In another CDG type II patient, the identification of specific hybrid sugars suggested that the defective processing step was cell type-specific and involved the mannosidase III pathway. In each case, complementary serum proteome analyses revealed significant changes in some 31 glycoproteins, including components of the complement system. This biochemical approach to charting diseases that involve alterations in glycan processing provides a rapid indicator of the nature, severity, and cell type specificity of the suboptimal glycan processing steps; allows links to genetic mutations; indicates the expression levels of proteins; and gives insight into the pathways affected in the disease process.

X-linked adrenoleukodystrophy in patients with idiopathic Addison disease.

UNLABELLED: The two main causes of primary adrenal disease are tuberculosis and auto-immune adrenal destruction. The latter is responsible for about 70% of the cases of primary adrenal insufficiency (Addison disease). Commonly referred to as a rare cause of adrenal failure is X-linked adrenoleukodystrophy (ALD), a demyelinating peroxisomal disorder affecting 1: 20,000 Caucasian males. Albeit primary adrenal insufficiency is a rare entity per se, we decided to study patients with idiopathic Addison disease and establish the frequency of ALD as a cause of adrenal insufficiency. The biochemical defect of ALD was found in 5 out of 24 patients. The small number of cases in our series led us to include in our analysis the published results of two other groups of investigators. This analysis indicates that the proportion of cases in which Addison disease is attributable to ALD is age dependent. It is highest when the adrenal insufficiency manifests before 15 years. This study clearly demonstrates that the proportion of ALD in patients presenting primary adrenal insufficiency has been under-estimated. CONCLUSION: Addison disease manifesting during the first decade of life has a high likelihood of being the first sign of X-linked adrenoleukodystrophy.

Characterization of X-linked adrenoleukodystrophy in different biological specimens from ten Portuguese families.

X-linked adrenoleukodystrophy (ALD) is a severe neurodegenerative disease characterized by progressive demyelination, adrenocortical insufficiency and accumulation in tissues and body fluids of unbranched, saturated very long-chain fatty acids (VLCFA). The diagnosis of ALD is usually based on clinical history, neurological examination and the determination of levels of VLCFA in plasma and cultured skin fibroblasts. In the present paper we report the biochemical findings in plasma, cultured skin fibroblasts and lymphoblastoid cell lines from ALD patients. The results obtained indicate that the increment of the ratios C24:0 to C22:0 and C26:0 to C22:0 and of the concentration C26:0 (micrograms/ml) in plasma was parallel with that of fibroblasts, but not with that of Epstein-Barr virus (EBV)-transformed lymphocytes, suggesting that this cell line is not reliable for diagnosis of ALD by VLCFA analysis. Subsequent studies carried out on family members revealed heterozygotes other than obligate carriers and hemizygotes who were pre-symptomatic or had a misdiagnosis of multiple sclerosis or psychosis.