An Alu-mediated duplication in NMNAT1, involved in NAD biosynthesis, causes a novel syndrome, SHILCA, affecting multiple tissues and organs.

We investigated the genetic origin of the phenotype displayed by three children from two unrelated Italian families, presenting with a previously unrecognized autosomal recessive disorder that included a severe form of spondylo-epiphyseal dysplasia, sensorineural hearing loss, intellectual disability and Leber congenital amaurosis (SHILCA), as well as some brain anomalies that were visible at the MRI. Autozygome-based analysis showed that these children shared a 4.76 Mb region of homozygosity on chromosome 1, with an identical haplotype. Nonetheless, whole-exome sequencing failed to identify any shared rare coding variants, in this region or elsewhere. We then determined the transcriptome of patients' fibroblasts by RNA sequencing, followed by additional whole-genome sequencing experiments. Gene expression analysis revealed a 4-fold downregulation of the gene NMNAT1, residing indeed in the shared autozygous interval. Short- and long-read whole-genome sequencing highlighted a duplication involving 2 out of the 5 exons of NMNAT1 main isoform (NM_022787.3), leading to the production of aberrant mRNAs. Pathogenic variants in NMNAT1 have been previously shown to cause non-syndromic Leber congenital amaurosis (LCA). However, no patient with null biallelic mutations has ever been described, and murine Nmnat1 knockouts show embryonic lethality, indicating that complete absence of NMNAT1 activity is probably not compatible with life. The rearrangement found in our cases, presumably causing a strong but not complete reduction of enzymatic activity, may therefore result in an intermediate syndromic phenotype with respect to LCA and lethality.

A new case report of severe mucopolysaccharidosis type VII: diagnosis, treatment with haematopoietic cell transplantation and prenatal diagnosis in a second pregnancy.

A new patient with severe mucopolysaccharidosis (MPS) type VII is reported. Non-immune hydrops fetalis (NIHF) was diagnosed during pregnancy. At birth, he showed generalized hydrops and dysmorphic features typical of MPS. Many diagnoses were excluded before reaching the diagnosis of MPS VII at 8 months of life. During the first year of life he had frequent respiratory infections associated with restrictive and obstructive bronchopneumopathy and underwent three surgical interventions: decompression of the spinal cord at the craniocervical junction, bilateral inguinal hernia, and bilateral clubfoot. At 14 months of life he underwent successful haematopoietic cell transplantation (HCT). During the following 10 months, his bronchopneumopathy progressively worsened, needing chronic pharmacological treatment and O2 administration. The patient died of respiratory insufficiency during a respiratory syncytial virus infection at 25 months of age. Molecular analysis showed the homozygous variant c.1617C > T, leading to the synonymous mutation p.Ser539=. This caused aberrant splicing with partial skipping of exon 10 (r.1616_1653del38) and complete skipping of exon 9 (r.1392_1476del85; r.1616_1653del38). No transcript of normal size was evident. The parents were both confirmed to be carriers. In a subsequent pregnancy, a prenatal diagnosis showed an affected fetus. Ultrasound examination before abortion showed NIHF. The skin and placenta examination by electron microscopy showed foamy intracytoplasmic vacuoles with a weakly electron-dense substrate. MPS VII is a very rare disease but it is possible that some cases go undiagnosed for several reasons, including that MPS VII, and other lysosomal storage diseases, are not included in the work-up for NIHF in many institutions, and the presence of anasarca at birth may be confounding for the recognition of the typical facial characteristics of the disease. This is the eighth patient affected by MPS VII who has undergone HCT. It is not possible to draw conclusions about the efficacy of HCT in MPS VII. Treatment with enzyme replacement is now available and will probably be beneficial for the patients who have a milder form with no or little cognitive involvement. Increased awareness among clinicians is needed for prompt diagnosis and to offer the correct treatment as early as possible.

Validity of ß-D-glucosidase activity measured in dried blood samples for detection of potential Gaucher disease patients.

OBJECTIVES: Gaucher disease (GD) diagnosis relies on the demonstration of deficient ß-D-glucosidase (GBA) activity in cellular homogenates. Diagnosis process, however, can be delayed as (i) some GD symptoms are non-specific; and (ii) diagnostic tests are performed in specialized laboratories. These difficulties negatively impact on timely access of patients to therapy. GBA assay in dried blood spots (DBS) represents a method facilitating early identification of patients who will be finally diagnosed with gold standard assay of nucleated cells. Aim of this study is to investigate the DBS analytical performance compared with gold standard method. DESIGN & METHODS: A cross-sectional study started by comparing data of 50 DBS and 50 homogenate samples from the same subjects (25 known-GD and 25 controls). The subsequent phase examined 443 DBS samples. Along with these, 73 blood samples were sent for leukocyte separation and/or EBV-lymphoblast cell lines, and 1 skin biopsy for fibroblast cell lines. Overall the study included a total of 493 subjects. RESULTS: While the results from this first validation group did not yield false positive/negative values, when the analysis was extended to 443 DBS, 14.4% (64 samples) of positive results was yielded. Among these, only 15 were confirmed as GD values with gold standard test. In addition, a thorough examination of some clinical data also revealed 2 false negative results which were confirmed by both enzymatic and molecular analyses. CONCLUSIONS: DBS test could be useful as screening method although with cautions, whereas the standardized GBA assay should remain the gold standard for laboratory diagnosis of Gaucher disease.

A novel homozygous splicing mutation in PSAP gene causes metachromatic leukodystrophy in two Moroccan brothers.

Prosaposin (PSAP) gene mutations, affecting saposin B (Sap-B) domain, cause a rare metachromatic leukodystrophy (MLD) variant in which arylsulfatase A (ARSA) activity is normal. To date, only 10 different PSAP mutations have been associated with a total of 18 unrelated MLD patients worldwide. In this study, we report for the first time a family with Moroccan origins in which the proband, presenting with a late-infantile onset of neurological involvement and a brain MRI with the typical tigroid MLD pattern, showed normal values of ARSA activity in the presence of an abnormal pattern of urinary sulfatides. In view of these findings, PSAP gene was analyzed, identifying the newly genomic homozygous c.909 + 1G > A mutation occurring within the invariant GT dinucleotide of the intron 8 donor splice site. Reverse transcriptase-polymerase chain reaction (RT-PCR), showing the direct junction of exon 7 to exon 9, confirmed the skipping of the entire exon 8 (p.Gln260_Lys303) which normally contains two cysteine residues (Cys271 and Cys265) involved in disulfide bridges. Our report provides further evidence that phenotypes of patients with Sap-B deficiency vary widely depending on age of onset, type, and severity of symptoms. Awareness of this rare MLD variant is crucial to prevent delayed diagnosis or misdiagnosis and to promptly provide an accurate genetic counseling, including prenatal diagnosis, to families.

99mTc-sestamibi scintigraphy to monitor the long-term efficacy of enzyme replacement therapy on bone marrow infiltration in patients with Gaucher disease.

UNLABELLED: Assessing the skeletal response to enzyme replacement therapy (ERT) in Gaucher disease (GD) is problematic. We investigated the reliability of (99m)Tc-sestamibi scintigraphy in monitoring changes in bone marrow involvement induced by ERT. METHODS: In 52 GD patients, the efficacy of ERT on bone marrow disease was monitored using at least 2 sequential (99m)Tc-sestamibi scans; 17 patients were receiving ERT at enrollment, and 35 were ERT-naïve. We elaborated a dose-response model by statistical analysis based on linear mixed models. RESULTS: Patients whose marrow disease improved had received a significantly higher ERT dose per month than patients who did not improve. Significantly more patients reached near-disappearance of marrow disease if their disease burden at enrollment had been lower and the duration of clinical signs shorter. The response of the marrow scintigraphic score was more pronounced in ERT-naïve patients. No relevant effect of ERT on marrow disease was observed until platelet count and splenomegaly had improved. CONCLUSION: Although based on localized evaluation, changes in the (99m)Tc-sestamibi score closely correlated with the main determinants of ERT, with a definite dose-response relationship. The threshold at which ERT induced any improvement in bone marrow disease was 35-36 U/kg/mo; in ERT-naïve patients, the scintigraphic score declined by 1 unit after ERT at 28 U/kg/mo.

Lysosomal lipase deficiency: molecular characterization of eleven patients with Wolman or cholesteryl ester storage disease.

Wolman Disease (WD) and cholesteryl ester storage disease (CESD) represent two distinct phenotypes of the same recessive disorder caused by the complete or partial deficiency of lysosomal acidic lipase (LAL), respectively. LAL, encoded by the LIPA gene, hydrolyzes cholesteryl esters derived from cell internalization of plasma lipoproteins. WD is a rapidly progressive and lethal disease characterized by intestinal malabsorption, hepatic and adrenal failure. CESD is characterized by hepatic fibrosis, hyperlipidemia and accelerated atherosclerosis. Aim of the study was the identification of LIPA mutations in three WD and eight CESD patients. The WD patients, all deceased before the first year of age, were homozygous for two novel mutations (c.299+1G>A and c.419G>A) or a mutation (c.796G>T) previously reported as compound heterozygosity in a CESD patient. The two mutations (c.419G>A and c.796G>T) resulting in truncated proteins (p.W140* and p.G266*) and the splicing mutation (c.229+1G>A) were associated with undetectable levels of LIPA mRNA in fibroblasts. All eight CESD patients carried the common mutation c.894G>A known to result not only in a major non-functional transcript with the skipping of exon 8 (p.S275_Q298del), but also in a minor normally spliced transcript producing 5-10% residual LAL activity. The c.894G>A mutation was found in homozygosity in four patients and, as compound heterozygosity, in association with a known (p.H295Y and p.G342R) or a novel (p.W140*) mutation in four other CESD patients. Segregation analysis performed in all patients harboring c.895G>A showed its occurrence on the same haplotype suggesting a common founder ancestor. The other WD and CESD mutations were associated with different haplotypes.

Lysosomal storage disorders: molecular basis and laboratory testing.

Lysosomal storage disorders (LSDs) are a large group of more than 50 different inherited metabolic diseases which, in the great majority of cases, result from the defective function of specific lysosomal enzymes and, in few cases, of non-enzymatic lysosomal proteins or non-lysosomal proteins involved in lysosomal biogenesis. The progressive lysosomal accumulation of undegraded metabolites results in generalised cell and tissue dysfunction, and, therefore, multi-systemic pathology. Storage may begin during early embryonic development, and the clinical presentation for LSDs can vary from an early and severe phenotype to late-onset mild disease. The diagnosis of most LSDs--after accurate clinical/paraclinical evaluation, including the analysis of some urinary metabolites--is based mainly on the detection of a specific enzymatic deficiency. In these cases, molecular genetic testing (MGT) can refine the enzymatic diagnosis. Once the genotype of an individual LSD patient has been ascertained, genetic counselling should include prediction of the possible phenotype and the identification of carriers in the family at risk. MGT is essential for the identification of genetic disorders resulting from non-enzymatic lysosomal protein defects and is complementary to biochemical genetic testing (BGT) in complex situations, such as in cases of enzymatic pseudodeficiencies. Prenatal diagnosis is performed on the most appropriate samples, which include fresh or cultured chorionic villus sampling or cultured amniotic fluid. The choice of the test--enzymatic and/or molecular--is based on the characteristics of the defect to be investigated. For prenatal MGT, the genotype of the family index case must be known. The availability of both tests, enzymatic and molecular, enormously increases the reliability of the entire prenatal diagnostic procedure. To conclude, BGT and MGT are mostly complementary for post- and prenatal diagnosis of LSDs. Whenever genotype/phenotype correlations are available, they can be helpful in prognosis and in making decisions about therapy.

Unbalanced GLA mRNAs ratio quantified by real-time PCR in Fabry patients' fibroblasts results in Fabry disease.

Total or partial deficiency of the human lysosomal hydrolase alpha-galactosidase A is responsible for Fabry disease, the X-linked inborn error of glycosphingolipid metabolism. Together with the predominant alpha-galactosidase A gene mRNA product encoding the lysosomal enzyme, a weakly regulated alternatively spliced alpha-galactosidase A transcript is expressed in normal tissues, but its overexpression, due to the intronic g.9331G>A mutation, leads to the cardiac variant. We report the molecular characterization of five Fabry patients including two siblings. Sequencing analysis of the alpha-galactosidase A gene coding region and intron/exon boundaries identified the new c.124A>G (p.M42V) genetic lesion as well as a known deletion in three patients, whereas in the two remaining patients, no mutations were identified. To evaluate possible alpha-galactosidase A gene transcription alterations, both predominant and alternatively spliced mRNAs were quantified by absolute real-time PCR on total RNA preparations from the patients' fibroblasts. An impressive reduction in the predominant alpha-galactosidase A transcript was detected in the last patients (Pt 4 and Pt 5). However, the alternatively spliced mRNA was dramatically overexpressed in one of them, carrying a new intronic lesion (g.9273C>T). These findings strongly suggest a correlation between this new intronic mutation and the unbalanced alpha-galactosidase A mRNAs ratio, which could therefore be responsible for the reduced enzyme activity causing Fabry disease. The real-time assay developed here to investigate the two alpha-galactosidase A mRNAs might play a crucial role in revealing possible genetic lesions and in confirming the pathogenetic mechanisms underlying Fabry disease.

Mild functional effects of a novel GFAP mutant allele identified in a familial case of adult-onset Alexander disease.

Alexander disease is a neurological genetic disorder characterized by progressive white-matter degeneration, with astrocytes containing cytoplasmic aggregates, called Rosenthal fibers, including the intermediate filament glial fibrillary acidic protein (GFAP). The age of onset of the disease defines three different forms, infantile, juvenile and adult, all due to heterozygous GFAP mutations and characterized by a progressive less severe phenotype from infantile to adult forms. In an Italian family with a recurrent mild adult onset of Alexander disease, we have identified two GFAP mutations, coupled on a same allele, leading to p.[R330G; E332K]. Functional studies on this complex allele revealed less severe aggregation patterns compared to those observed with p.R239C GFAP mutant, associated with a severe Alexander disease phenotype. Moreover, in addition to confirming the involvement of the ubiquitin-proteasome system in cleaning cells from aggregates and a dominant effect of the novel mutant protein, in cells expressing the mild p.[R330G; E332K] mutant we have observed that indirect alphaB-crystallin overexpression, induced by high extracellular potassium concentration, could completely rescue the correct filament organization while, under the same experimental conditions, in cells expressing the severe p.R239C mutant only a partial rescue effect could be achieved.

Molecular analysis and characterization of nine novel CTSK mutations in twelve patients affected by pycnodysostosis. Mutation in brief #961. Online.

Molecular characterization of twelve unrelated patients affected by the autosomal recessive osteosclerotic skeletal dysplasia, Pycnodysostosis (cathepsin k deficiency), revealed 11 different genotypes. The mutational profile consisted of 12 different mutations, including nine previously unreported ones, spread throughout the whole gene. One mutation occurred in regions coding predomain, two affected the prodomain and nine others occurred in the mature domain. The novel lesions consisted in six missense mutations c.20T>C (p.L7P), c.494A>G (p.Q165R), c.580G>A (p.G194S), c.746T>C (p.I249T), c.749A>G (p.D250G), c.955G>T (p.G319C), two frameshifts c.60_61dupGA (p.I21RfsX29), c.282dupA (p.S95VfsX9) and a splicing mutation c.890G>A (r.785_890del). The six new missense mutations were examined by western blots of COS-7 cells transfected with mutant CTSK genes. The L7P, occurring within the predicted hydrophobic domain of signal peptide, showed a significantly reduced expression level compared to the wild type control. These findings suggested that the mutation affected targeting and translocation of the nascent lysosomal protein across the endoplasmatic reticulum membrane. The novel amino acid changes were also modeled into the three-dimensional structure that predicted incorrect protein folding for all of them. Molecular characterization of the patients is of particular value for genetic counseling of patients and their families as diagnosis of Pycnodysostosis based on enzyme assay is unpractical and thus not offered routinely.

Germline mutations in HRAS proto-oncogene cause Costello syndrome.

Costello syndrome is a multiple congenital anomaly and mental retardation syndrome characterized by coarse face, loose skin, cardiomyopathy and predisposition to tumors. We identified four heterozygous de novo mutations of HRAS in 12 of 13 affected individuals, all of which were previously reported as somatic and oncogenic mutations in various tumors. Our observations suggest that germline mutations in HRAS perturb human development and increase susceptibility to tumors.

Genetic disorders affecting white matter in the pediatric age.

Pediatric white matter disorders can be distinguished into well-defined leukoencephalopathies, and undefined leukoencephalopathies. The first category may be subdivided into: (a) hypomyelinating disorders; (b) dysmyelinating disorders; (c) leukodystrophies; (d) disorders related to cystic degeneration of myelin; and (e) disorders secondary to axonal damage. The second category, representing up to 50% of leukoencephalopathies in childhood, requires a multidisciplinar approach in order to define novel homogeneous subgroups of patients, possibly representing "new genetic disorders" (such as megalencephalic leukoencepahlopathy with subcortical cysts and vanishing white matter disease that have recently been identified). In the majority of cases, pediatric white matter disorders are inherited diseases. An integrated description of the clinical, neuroimaging and pathophysiological features is crucial for categorizing myelin disorders and better understanding their genetic basis. A review of the genetic disorders affecting white matter in the pediatric age, including some novel entities, is provided.

Wolman disease and cholesteryl ester storage disease diagnosed by histological and ultrastructural examination of intestinal and liver biopsy.

Deficient activity of lysosomal acid lipase (LAL) results in massive accumulation of cholesteryl esters and triglycerides in most tissues of the body. The deficiency state is expressed in two major phenotypes: Wolman disease (WD) and cholesteryl ester storage disease (CESD). WD occurs in infancy and is nearly always fatal before the age of 1 year, whereas CESD can be more benign and may not be detected until adulthood. Since there are no specific routine laboratory observations that suggest these metabolic diseases, diagnosis is based on the clinical picture combined with LAL deficiency in cultured skin fibroblasts or peripheral lymphocytes. Both disorders are rather rare, considering that about a hundred of cases have been described up to now. This study describes the histological and ultrastructural aspects disclosed by intestinal or liver biopsy in three cases of WD and in two cases of CESD. Furthermore, it emphasizes the role of morphological findings in pointing the diagnosis towards a metabolic storage disease.

Severity of bone marrow involvement in patients with Gaucher's disease evaluated by scintigraphy with 99mTc-sestamibi.

UNLABELLED: Gaucher's disease is a lysosomal storage disorder due to a genetically transmitted deficiency of the enzyme glucocerebrosidase. In the most common form of the disease (type 1), accumulation of glucosylceramide in the reticuloendothelial cells of liver, spleen, and bone marrow leads to visceromegaly, anemia, thrombocytopenia, and osteopenia. Skeletal manifestations secondary to infiltration of the bone marrow by Gaucher's cells are detectable by radiography only in advanced stages. Imaging of bone marrow involvement can be performed indirectly by magnetic resonance techniques or by bone marrow scintigraphy with radiocolloids. However, both procedures lack specificity because the normal bone marrow, rather than the pathologic process, is imaged. The aim of this study was to assess the reliability of (99m)Tc-sestamibi scintigraphy for direct evaluation of bone marrow involvement. METHODS: Seventy-two patients with type 1 and 2 patients with type 3 Gaucher's disease (35 males, 39 females) were enrolled in the study. The mean age +/- SD was 31.9 +/- 16.5 y (range, 3-76 y), and the average duration of the disease manifestations when performing scintigraphy was 12.95 y (median, 10.5 y; range, 0-44 y). Forty-three of 74 patients had never received enzyme replacement therapy (ERT), whereas 31 patients were already being treated with ERT. (99m)Tc-Sestamibi was injected intravenously (6-8 MBq/kg of body weight) and imaging was recorded at the lower limbs 30 min after injection, at the plateau of tracer accumulation in the involved bone marrow. The scans were evaluated visually, assigning a semiquantitative score based on the extension and intensity of uptake in the bone marrow of the lower limbs (0 = no uptake; 8 = maximum uptake). The scintigraphic score was entered into complex statistical analysis, which included a series of clinical and blood chemistry parameters defining overall severity of the disease. RESULTS: (99m)Tc-Sestamibi scintigraphy showed that 71 of 74 patients had some degree of bone marrow involvement. The scintigraphic score was highly correlated with an overall clinical severity score index (SSI) and with various parameters contributing to the SSI, either positively or negatively. The highest correlation of the scintigraphic score was found with an overall biochemical marker of disease severity (serum chitotriosidase). ERT-naive patients showed high correlation of the scintigraphic score with the clinical SSI, with a radiographically based score, and with serum chitotriosidase. In the ERT-treated patients, the scintigraphic score was correlated with the clinical SSI, with hepatomegaly, and with hemoglobin. CONCLUSION: (99m)Tc-Sestamibi uptake reliably identifies bone marrow infiltration by Gaucher's cells. The scintigraphic score is helpful for defining the severity of bone marrow involvement and for comparing patients. (99m)Tc-Sestamibi scintigraphy, which provides topographic information about the sites involved by the disease, is highly correlated with other parameters of disease severity and appears to correlate with response to ERT.