Molecular testing of 163 patients with Morquio A (Mucopolysaccharidosis IVA) identifies 39 novel GALNS mutations.

Morquio A (Mucopolysaccharidosis IVA; MPS IVA) is an autosomal recessive lysosomal storage disorder caused by partial or total deficiency of the enzyme galactosamine-6-sulfate sulfatase (GALNS; also known as N-acetylgalactosamine-6-sulfate sulfatase) encoded by the GALNS gene. Patients who inherit two mutated GALNS gene alleles have a decreased ability to degrade the glycosaminoglycans (GAGs) keratan sulfate and chondroitin 6-sulfate, thereby causing GAG accumulation within lysosomes and consequently pleiotropic disease. GALNS mutations occur throughout the gene and many mutations are identified only in single patients or families, causing difficulties both in mutation detection and interpretation. In this study, molecular analysis of 163 patients with Morquio A identified 99 unique mutations in the GALNS gene believed to negatively impact GALNS protein function, of which 39 are previously unpublished, together with 26 single-nucleotide polymorphisms. Recommendations for the molecular testing of patients, clear reporting of sequence findings, and interpretation of sequencing data are provided.

Expert recommendations for the laboratory diagnosis of MPS VI.

Mucopolysaccharidosis VI (MPS VI) is a lysosomal storage disease caused by a deficiency of N-acetylgalactosamine 4-sulfatase (arylsulfatase B, ASB). This enzyme is required for the degradation of dermatan sulfate. In its absence, dermatan sulfate accumulates in cells and is excreted in large quantities in urine. Specific therapeutic intervention is available; however, accurate and timely diagnosis is crucial for maximal benefit. To better understand the current practices for diagnosis and to establish diagnostic guidelines, an international MPS VI laboratory diagnostics scientific summit was held in February of 2011 in Miami, Florida. The various steps in the diagnosis of MPS VI were discussed including urinary glycosaminoglycan (uGAG) analysis, enzyme activity analysis, and molecular analysis. The following conclusions were reached. Dilute urine samples pose a significant problem for uGAG analysis and MPS VI patients can be missed by quantitative uGAG testing alone as dermatan sulfate may not always be excreted in large quantities. Enzyme activity analysis is universally acknowledged as a key component of diagnosis; however, several caveats must be considered and the appropriate use of reference enzymes is essential. Molecular analysis supports enzyme activity test results and is essential for carrier testing, subsequent genetic counseling, and prenatal testing. Overall the expert panel recommends caution in the use of uGAG screening alone to rule out or confirm the diagnosis of MPS VI and acknowledges enzyme activity analysis as a critical component of diagnosis. Measurement of another sulfatase enzyme to exclude multiple sulfatase deficiency was recommended prior to the initiation of therapy. When feasible, the use of molecular testing as part of the diagnosis is encouraged. A diagnostic algorithm for MPS VI is provided.

An integrated strategy for the diagnosis of neuronal ceroid lipofuscinosis types 1 (CLN1) and 2 (CLN2) in eleven Latin American patients.

The neuronal ceroid lipofuscinoses (NCLs) are a family of progressive neurodegenerative diseases that are characterized by the cellular accumulation of ceroid lipofuscin-like bodies. NCL type 1 (CLN1) and type 2 (CLN2) are caused by deficiencies of the lysosomal enzymes palmitoyl-protein thioesterase 1 (PPT-1) and tripeptidyl peptidase 1 (TPP-1), respectively. In this study, 118 Latin American patients were examined for NCL using an integrated multidisciplinary program. This revealed two patients affected by CLN1 and nine by CLN2. Both CLN1 patients had a juvenile-onset phenotype with mutation studies of one patient demonstrating the known mutation p.Arg151X and a novel mutation in intron 3, c.363-3T>G. Six of the CLN2 patients presented with the 'classical' late-infantile phenotype. The remaining three patients, who were siblings, presented with a 'protracted' phenotype and had a higher level of residual TPP-1 activity than the 'classical' CLN2 patients. Genotype analysis of the TPP1 gene in the 'classical' CLN2 patients showed the presence of the known mutation p.Arg208X and the novel mutations p.Leu104X, p.Asp276Val, and p.Ala453Val. The siblings with the 'protracted' phenotype were heterozygous for two known TPP1 mutations, p.Gln66X and c.887-10A>G. This multidisciplinary program is also being used to diagnose other NCL types.

The cDNA-deduced amino acid sequence for trichohyalin, a differentiation marker in the hair follicle, contains a 23 amino acid repeat.

Trichohyalin is a highly expressed protein within the inner root sheath of hair follicles and is similar, or identical, to a protein present in the hair medulla. In situ hybridization studies have shown that trichohyalin is a very early differentiation marker in both tissues and that in each case the trichohyalin mRNA is expressed from the same single copy gene. A partial cDNA clone for sheep trichohyalin has been isolated and represents approximately 40% of the full-length trichohyalin mRNA. The carboxy-terminal 458 amino acids of trichohyalin are encoded, and the first 429 amino acids consist of full- or partial-length tandem repeats of a 23 amino acid sequence. These repeats are characterized by a high proportion of charged amino acids. Secondary structure analyses predict that the majority of the encoded protein could form alpha-helical structures that might form filamentous aggregates of intermediate filament dimensions, even though the heptad motif obligatory for the intermediate filament structure itself is absent. The alternative structural role of trichohyalin could be as an intermediate filament-associated protein, as proposed from other evidence.

Analysis of the sheep trichohyalin gene: potential structural and calcium-binding roles of trichohyalin in the hair follicle.

Trichohyalin is a structural protein that is produced and retained in the cells of the inner root sheath and medulla of the hair follicle. The gene for sheep trichohyalin has been purified and the complete amino acid sequence of trichohyalin determined in an attempt to increase the understanding of the structure and function of this protein in the filamentous network of the hardened inner root sheath cells. Sheep trichohyalin has a molecular weight of 201,172 and is characterized by the presence of a high proportion of glutamate, arginine, glutamine, and leucine residues, together totaling more than 75% of the amino acids. Over 65% of trichohyalin consists of two sets of tandem peptide repeats which are based on two different consensus sequences. Trichohyalin is predicted to form an elongated alpha-helical rod structure but does not contain the sequences required for the formation of intermediate filaments. The amino terminus of trichohyalin contains two EF hand calcium-binding domains indicating that trichohyalin plays more than a structural role within the hair follicle. In situ hybridization studies have shown that trichohyalin is expressed in the epithelia of the tongue, hoof, and rumen as well as in the inner root sheath and medulla of the hair follicle.

Congenital disorder of glycosylation type Ia in a Malaysian family: clinical outcome and description of a novel PMM2 mutation.

There are few reports of congenital disorders of glycosylation (CDGs) in the Asian population, although they have been reported worldwide. We identified a Malaysian infant female at 2 days of life with CDG type Ia. The diagnosis was suspected on the basis of inverted nipples and abnormal fat distribution. She had cerebellar hypoplasia and developed coagulopathy, hypothyroidism and severe pericardial effusion and died at 7 months of life. The diagnosis was supported by abnormal serum transferrin isoform pattern that showed elevated levels of the disialotransferrin isoform and trace levels of the asialotransferrin isoform. Enzyme testing of peripheral leukocytes showed decreased level of phosphomannomutase (PMM) activity (0.6 nmol/min per mg protein, normal range 1.6-6.2) and a normal level of phosphomannose isomerase activity (19 nmol/min per mg protein, normal range 12-25), indicating a diagnosis of CDG type Ia. Mutation study of the PMM2 gene showed the patient was heterozygous for both the common p.R141H (c.422T>A) mutation and a novel sequence change in exon 7, c.618C>A. The latter change is predicted to result in the replacement of the highly conserved phenylalanine residue at position 206 with a leucine residue (p.F206L) and occurs in the same codon as the previously reported p.F206S mutation. Analysis of 100 control chromosomes has shown that the p.F206L sequence change is not present, making it highly likely that this change is functionally important. To the best of our knowledge, this is the first report of CDG in the Malay population. Prenatal diagnosis was successfully performed in a subsequent pregnancy for this family.

Gender dimorphism in siblings with schizophrenia-like psychosis due to Niemann-Pick disease type C.

We describe the differential presentation of schizophrenia-like psychosis in two siblings with the 'variant' biochemical presentation of adult Niemann-Pick disease type C. The male sibling presented with psychosis at age 16 years and cognitive and motor disturbance at age 25 years, whereas his elder sister, sharing the same mutation but showing less severe biochemical, neuroimaging and ocular motor parameters, presented with a similar schizophrenia-like illness with associated cognitive and motor disturbance at age 31 years. Their illness onset, course and response to treatment mirrors the sex dimorphism seen in schizophrenia, and is suggestive of an interaction between the neurobiology of their metabolic disorder and sex differences in neurodevelopment.

Congenital disorder of glycosylation type Ia: heterogeneity in the clinical presentation from multivisceral failure to hyperinsulinaemic hypoglycaemia as leading symptoms in three infants with phosphomannomutase deficiency.

We describe three patients with congenital disorder of glycosylation (CDG) type Ia, all of whom had persistent hyperinsulinaemic hypoglycaemia responding to diazoxide therapy as a common feature. The first patient, an infant girl, presented with recurrent vomiting, failure to thrive, liver impairment, hypothyroidism and a pericardial effusion. The second patient, also female, had a milder disease with single organ involvement, presenting as isolated hyperinsulinaemic hypoglycaemia, not associated with any cognitive impairment. The third patient, a boy presented with multi-organ manifestations including congenital hypothyroidism, persistent hyperinsulinaemic hypoglycaemia, coagulopathy, olivopontocerebellar hypoplasia and recurrent pancreatitis. All three patients had a type 1 serum transferrin isoform pattern, and were subsequently found to have low phosphomannomutase activity, confirming the diagnosis of CDG type Ia. Our findings emphasize that CDG should be considered as a differential diagnosis in patients with persistent hyperinsulinaemic hypoglycaemia and that it may even occasionally be the leading symptom in CDG Ia.

Quantitative effects of hedgehog and decapentaplegic activity on the patterning of the Drosophila wing.

BACKGROUND: Members of the hedgehog (hh) gene family encode a novel class of proteins implicated in positional signalling in both invertebrates and vertebrates. In Drosophila, the hh gene has been shown to regulate patterning of the imaginal discs, the precursors of the insect limbs. In a remarkably similar fashion, the function and expression of the sonic hedgehog (shh) gene is closely associated with the 'zone of polarizing activity' (ZPA) that controls antero-posterior patterning of the vertebrate limb. Both of these functions suggest a role for hedgehog family proteins as morphogens. An alternative possibility, however, is that hh and its homologues act to control the expression of other instructive signalling molecules. RESULTS: We have explored this issue by examining the effects on Drosophila wing patterning of ectopically expressing varying levels of hh and shh, as well as of the putative hh target gene, decapentaplegic (dpp), a member of the transforming growth factor-beta family of signalling molecules. We find that different levels of hh activity can induce graded changes in the patterning of the wing, and that zebrafish shh acts in a similar though attenuated fashion. Varying levels of ectopic hh and shh activity can differentially activate transcription of the patched and dpp genes. Furthermore, ectopic expression of dpp alone is sufficient to induce the pattern alterations caused by ectopic hh or shh activity. CONCLUSION: Thus, hh family proteins can elicit different responses in a dose-dependent manner in the imaginal disc. The principal function of hh, however, is to activate transcription of dpp at the compartment boundary, thereby establishing a source of dpp activity that is the primary determinant of antero-posterior patterning.

Secretion of the amino-terminal fragment of the hedgehog protein is necessary and sufficient for hedgehog signalling in Drosophila.

BACKGROUND: The Drosophila segment polarity gene hedgehog encodes a member of a family of secreted proteins that are involved in a variety of patterning processes, in both vertebrates and invertebrates. Some of these processes depend upon short-range or contact-dependent interactions, whereas others seem to involve long-range signalling. Two different models have been proposed to account for the execution of these contrasting processes by the same proteins: one postulates that Hedgehog acts exclusively over short distances, its long-range influences being effected through regulation of other signalling factors; the second postulates that different aspects of Hedgehog activity are mediated by distinct forms of the protein that are generated by autoproteolysis. RESULTS: We have investigated these models by mutating the hedgehog coding region such that only the amino-terminal or carboxy-terminal half of the protein is secreted. Deletion of the carboxy-terminal portion has little effect on the signalling activity of the protein, whereas abolishing the secretion of the amino-terminal half leads to a complete loss of signalling. In addition, we find that increases in the level of expression within the normal hedgehog transcriptional domain of either the wild-type protein or the carboxy-terminal-deleted form expand the range of activity to a limited extent, but have only minor effects on cell identity. CONCLUSIONS: In Drosophila, all of the signalling activity of Hedgehog resides in the amino-terminal portion of the protein, the secretion of which is essential for its function. The range of Hedgehog is limited by the close association of the amino-terminal peptide with the cell surface but can be extended by elevating the level of its expression.

A novel mutation af Cln3 associated with delayed-classic juvenile ceroid lipofuscinois and autophagic vacuolar myopathy.

Juvenile neuronal-ceroid-lipofuscinosis (JNCL) is a lysosomal storage disease caused by mutations in CLN3. The most frequent mutation is a 1.02-kb deletion that, when homozygous, causes the classical clinical presentation. Patients harboring mutations different than the major deletion show a marked clinical heterogeneity, including protracted disease course with possible involvement of extraneuronal tissues. Cardiac involvement is relatively rare in JNCL and it is usually due to myocardial storage of ceroid-lipofuscinin. Only recently, histopathological findings of autophagic vacuolar myopathy (AVM) were detected in JNCL patients with severe cardiomyopathy. We describe a 35-year-old male showing a delayed-classic JNCL with visual loss in childhood and neurological manifestations only appearing in adult life. He had an unusual CLN3 genotype with an unreported deletion (p.Ala349_Leu350del) and the known p.His315Glnfs*67 mutation. Autophagic vacuolar myopathy was shown by muscle biopsy. At clinical follow-up, moderately increased CPK levels were detected whereas periodic cardiac assessments have been normal to date. Adult neurologists should be aware of protracted JNCL as cause of progressive neurological decline in adults. The occurrence of autophagic vacuolar myopathy necessitates periodic cardiac surveillance, which is not usually an issue in classic JNCL due to early neurological death.

Mapping of the trichohyalin gene: co-localization with the profilaggrin, involucrin, and loricrin genes.

The chromosomal location of the gene encoding the human hair follicle protein trichohyalin has been determined by in situ hybridization. The human gene has been localized to the region 1q21.1-1q23 (probably 1q21.3) using a sheep trichohyalin cDNA probe. The genes encoding three other epithelial proteins, namely, profilaggrin, involucrin, and loricrin, are also located in the same region of chromosome 1, which, together with their similar gene and protein structures, suggests that the four proteins form a novel superfamily of epithelial structural proteins.

An Australasian diagnostic service for the neuronal ceroid lipofuscinoses.

The neuronal ceroid lipofuscinoses (NCLs) are a family of related genetic disorders that together are believed to affect one child in every 12,500 births in the USA. Our laboratory has developed a diagnostic service for classical late infantile neuronal ceroid lipofuscinosis (LINCL) by assay of tripeptidyl-peptidase I (TPP-I) activity using the fluorogenic peptide substrate Ala-Ala-Phe aminomethylcoumarin, followed by a screen for three mutations in the CLN2 gene. In addition, we have also begun to offer a limited diagnostic service for the juvenile (JNCL) and infantile (INCL) forms of the disease on the basis of mutation analysis of the CLN3 and CLN1 genes, respectively. Retrospective analysis of Australasian patients with a clinical suspicion of NCL has revealed that six are affected by LINCL, six by JNCL and, to date, two by INCL. Mutation analysis of our LINCL patients has shown that the three screened mutations, namely, the nonsense mutation R208X and the splice mutations IVS5-1 G > C and IVS5-1 G > A, constitute 83% of alleles.

Glandular excision in total glandular mastectomy and modified radical mastectomy: a comparison.

Total glandular mastectomy and modified radical mastectomy were compared for the amount of breast tissue remaining after surgery. Multiple biopsies were taken from the anterior chest walls of women following total glandular mastectomy (N = 27) and modified radical mastectomy (N = 28) to try to detect any residual glandular tissue. Regardless of procedure performed, breast tissue was identified histologically in 5 percent of all biopsy specimens (159 and 161, respectively). One of every five operative fields was shown to have glandular elements in at least one of the biopsy sites; the positive biopsies did not form a discernible pattern. The residual breast tissue in each of these patients averaged less than 1 gm. On the basis of this study, modified radical mastectomy and total glandular mastectomy appear to be equally effective in removing most of the breast.

Size and shape of the corpus callosum in adult Niemann-Pick type C reflects state and trait illness variables.

BACKGROUND AND PURPOSE: Variable alterations to the structure of the corpus callosum have been described in adults with NPC, a neurometabolic disorder known to result in both white and gray matter pathology. This study sought to examine the structure of the callosum in a group of adult patients with NPC and compared callosal structure with a group of matched controls, and to relate callosal structure with state and trait illness variables. MATERIALS AND METHODS: Nine adult patients with NPC were matched to control subjects (n = 26) on age and sex. The corpus callosum was segmented from the midsagittal section of T1-weighted images on all subjects, and total area, length, bending angle, and mean thickness were calculated. In addition, 39 regional thickness measures were derived by using a previously published method. All measures were compared between groups, and analyzed alongside symptom measures, biochemical parameters, and ocular-motor measures. RESULTS: The callosal area and mean thickness were significantly reduced in the patient group, and regional thickness differences were greatest in the genu, posterior body, isthmus, and anterior splenium. Global callosal measures correlated significantly with duration of illness and symptom score, and at trend level with degree of filipin staining. Measures of reflexive saccadic peak velocity and gain, and self-paced saccades, correlated strongly with total callosal area. CONCLUSIONS: Callosal structure and size reflect both state and trait markers in adult NPC, and they may be useful biomarkers to index both white and gray matter changes that reflect illness severity and progression.

White and gray matter alterations in adults with Niemann-Pick disease type C: a cross-sectional study.

OBJECTIVE: Niemann-Pick disease type C (NPC) is a progressive neurovisceral disorder with disrupted intracellular cholesterol metabolism that results in significant alterations to neuronal and axonal structure. Adult patients present with ataxia, gaze palsy, impaired cognition, and neuropsychiatric illness, but the neural substrate has not been well-characterized in vivo. Our aim was to investigate a well-characterized sample of adults with confirmed NPC for gray and white matter abnormalities. METHODS: We utilized a combination of optimized voxel-based morphometry of T1-weighted images and tract-based spatial statistics of diffusion tensor images to examine gray matter volume and white matter structural differences in 6 adult patients with NPC and 18 gender- and age-matched controls. RESULTS: Patients with NPC demonstrated bilateral gray matter reductions in large clusters in bilateral hippocampus, thalamus, superior cerebellum, and insula, in addition to smaller regions of inferoposterior cortex. Patients demonstrated widespread reductions in fractional anisotropy in major white matter tracts. Subsequent analysis of measures of axial and radial diffusivity suggest that these changes are contributed to by both impaired myelination and altered axonal structure. CONCLUSIONS: Findings in gray matter areas are broadly consistent with human and animal studies of selective vulnerability of neuronal populations to the neuropathology of NPC, whereas more widespread white matter changes are consistent with the hypothesis that disrupted myelination and axonal structure predate changes to the neuronal cell body. These findings suggest that volumetric analysis of gray matter and diffusion tensor imaging may be useful modalities for indexing illness stage and monitoring response to emerging treatment.