Shira G. Ziegler

Multicenter Analysis of Glucocerebrosidase Mutations in Parkinson's Disease

BACKGROUND Recent studies indicate an increased frequency of mutations in the gene encoding glucocerebrosidase (GBA), a deficiency of which causes Gauchers disease, among patients with Parkinsons disease. We aimed to ascertain the frequency of GBA mutations in an ethnically diverse group of patients with Parkinsons disease. METHODS Sixteen centers participated in our international, collaborative study: five from the Americas, six from Europe, two from Israel, and three from Asia. Each center genotyped a standard DNA panel to permit comparison of the genotyping results across centers. Genotypes and phenotypic data from a total of 5691 patients with Parkinsons disease (780 Ashkenazi Jews) and 4898 controls (387 Ashkenazi Jews) were analyzed, with multivariate logistic-regression models and the Mantel-Haenszel procedure used to estimate odds ratios across centers. RESULTS All 16 centers could detect two GBA mutations, L444P and N370S. Among Ashkenazi Jewish subjects, either mutation was found in 15% of patients and 3% of controls, and among non-Ashkenazi Jewish subjects, either mutation was found in 3% of patients and less than 1% of controls. GBA was fully sequenced for 1883 non-Ashkenazi Jewish patients, and mutations were identified in 7%, showing that limited mutation screening can miss half the mutant alleles. The odds ratio for any GBA mutation in patients versus controls was 5.43 across centers. As compared with patients who did not carry a GBA mutation, those with a GBA mutation presented earlier with the disease, were more likely to have affected relatives, and were more likely to have atypical clinical manifestations. CONCLUSIONS Data collected from 16 centers demonstrate that there is a strong association between GBA mutations and Parkinsons disease.

Glucocerebrosidase mutations are also found in subjects with early-onset parkinsonism from Venezuela

We read with interest the publications by Clark and colleagues1 and Sato and associates2 regarding their pilot association studies of glucocerebrosidase mutations and Parkinson’s disease. Clearly, several different research findings now support an association between altered glucocerebrosidase and parkinsonism. Initially, parkinsonian manifestations were noted in a rare subset of patients with Gaucher disease, the inherited deficiency of glucocerebrosidase.3,4 Subsequently, neuropathological examination of brains from patients with Gaucher disease and early-onset, treatment-refractory parkinsonism found Lewy bodies in regions specifically affected in Gaucher disease, including layers CA4–CA2 of the hippocampus.5 Family studies complemented these observations, revealing a greater than anticipated incidence of parkinsonism among Gaucher disease carriers.6 Thus, both clinical observations and neuropathological data suggested that alterations in glucocerebrosidase may contribute to a vulnerability to the development of parkinsonian symptoms. To explore this association, the glucocerebrosidase gene (GBA) has been examined by different screening techniques in five different cohorts with parkinsonism. Initially, GBA was sequenced in 57 brain samples from subjects with a primary diagnosis of Parkinson’s disease, obtained from five different North American tissue banks.7 Alterations in GBA were identified in 12 samples (21%); 4 with polymorphisms (T369M or E326K) and 8 with Gaucher mutations (N370S, L444P, K198T, R329C), of which 2 were N370S homozygotes. All eight Gaucher carriers or homozygotes were among the 28 younger Parkinson subjects who died before age 75 (27%). Sequencing of GBA in 44 controls from the same brain banks revealed no mutations, although polymorphisms were identified in 2 subjects (4.5%). Subsequently, another series of 26 brain samples from British subjects diagnosed with Parkinson’s disease between 42 and 78 years were obtained from the Parkinson’s Disease Society Tissue Bank of the Imperial College, United Kingdom (registered charity 948776). Sequencing revealed two (8%) carried GBA mutations (D140H E326K, RecNciI).8 In a third series, investigators from Israel screened 99 Ashkenazi Jewish probands with Parkinson’s disease for six known glucocerebrosidase mutations.9 They identified 31 patients (31.3%) with mutations N370S or c.84insG, including 3 N370S homozygotes. This frequency was considerably higher than the 10.7% incidence of mutation N370S reported in a similar cohort of 160 Jewish subjects with parkinsonism from New York City.1 In a fifth series, a group from Toronto screened for seven glucocerebrosidase mutations in 88 unrelated Caucasian subjects of Canadian origin with clinically diagnosed parkinsonism, selected for an early age of onset or a positive family history.2 They identified mutations in 5.6% of this Canadian cohort, as opposed to 0.8% in their screened normal controls. We have now screened an ethnically different group of 33 subjects diagnosed with early-onset Parkinson’s disease, who were seen at the Movement Disorder Unit at the Hospital Universitario in Caracas, Venezuela. In this cohort, the age at symptom onset ranged from 24 to 50 years, with a mean of 36 years. None of the probands had Ashkenazi Jewish ancestry. All exons and most flanking intronic regions of GBA were sequenced from lymphocyte DNA as previously described.10 Four unrelated probands (12%) carried GBA mutations L444P, N370S, or RecNciI, a recombinant allele that comprises alterations L444P, A456P, and V460V (Table 1). Because three different mutant alleles were identified, this finding was unlikely to be due to a founder effect. All 4 probands were at least initially levodopa responsive, and only 1 had a positive family history of Parkinson’s disease. We subsequently sequenced DNA from a series of 31 adult patients (over age 40) with no clinical signs of parkinsonism, who were seen at the Research Institute Fundación Instituto de Estudios Avanzados in Caracas, Venezuela. This control group included Venezuelan citizens of mixed ethnic backgrounds, including some with Italian, Peruvian, Spanish, or Colombian ancestry. All exons were examined in 29 of the controls, but in 2, sequencing of exons 1 to 4 was incomplete. One control (3.2%) carried an alteration in exon 10, D443N, but no other mutations were identified in the control group. D443N, which has not been described previously, is predicted to be located in Domain II of glucocerebrosidase, on the outer surface of the enzyme.11 These studies demonstrate that GBA mutations are observed with an increased frequency not only among Ashkenazi Jews with Parkinson’s disease but also among subjects with parkinsonism from diverse ethnic backgrounds. This study, in particular, replicates this finding in a cohort with early-onset Parkinson’s disease. It provides further evidence that altered glucocerebrosidase may serve as a risk factor for the development of parkinsonian symptoms. By unraveling the relationship between altered glucocerebrosidase and parkinsonism, we may advance our understanding of the etiology, genetics, and pathogenesis of Parkinson’s disease.

PKHD1 Sequence Variations in 78 Children and Adults with Autosomal Recessive Polycystic Kidney Disease and Congenital Hepatic Fibrosis

PKHD1, the gene mutated in autosomal recessive polycystic kidney disease (ARPKD)/congenital hepatic fibrosis (CHF), is an exceptionally large and complicated gene that consists of 86 exons and has a number of alternatively spliced transcripts. Its longest open reading frame contains 67 exons that encode a 4074 amino acid protein called fibrocystin or polyductin. The phenotypes caused by PKHD1 mutations are similarly complicated, ranging from perinatally-fatal PKD to CHF presenting in adulthood with mild kidney disease. To date, more than 300 mutations have been described throughout PKHD1. Most reported cohorts include a large proportion of perinatal-onset ARPKD patients; mutation detection rates vary between 42% and 87%. Here we report PKHD1 sequencing results on 78 ARPKD/CHF patients from 68 families. Differing from previous investigations, our study required survival beyond 6 months and included many adults with a CHF-predominant phenotype. We identified 77 PKHD1 variants (41 novel) including 19 truncating, 55 missense, 2 splice, and 1 small in-frame deletion. Using computer-based prediction tools (GVGD, PolyPhen, SNAP), we achieved a mutation detection rate of 79%, ranging from 63% in the CHF-predominant group to 82% in the remaining families. Prediction of the pathogenicity of missense variants will remain challenging until a functional assay is available. In the meantime, use of PKHD1 sequencing data for clinical decisions requires caution, especially when only novel or rare missense variants are identified.

Cognitive Outcome in Treated Patients with Chronic Neuronopathic Gaucher Disease

OBJECTIVE To investigate the spectrum and prevalence of cognitive deficits among children with type 3 (chronic neuronopathic) Gaucher disease (GD). STUDY DESIGN A case review study identified 32 children (male/female; 17:15) with type 3 GD who had received enzyme replacement therapy (ERT) or a bone marrow transplant. The diagnosis of GD was established by enzymatic assay and DNA testing. Subjects were assessed with standard neuropsychological testing, and data from the most recent evaluation were included. RESULTS Neuropsychometric assessments demonstrated a wide spectrum of full-scale IQ scores ranging from 39 to 124 (mean 75). About 60% of subjects had intellectual skills below average. There were significant discrepancies between verbal and performance IQ, with a range between -6 and 38 points (P = .02). This gap was more prominent in older subjects, with better performance in the verbal areas. No correlation was observed between intelligence measures and genotype or the extent of systemic involvement. The dosage, age at initiation, and the length of ERT had no significant effect on IQ scores. CONCLUSIONS In type 3 GD, cognitive deficits, characterized by visual-spatial dysfunction, are common but underappreciated and appear resistant to ERT.

Natural History of Pulmonary Fibrosis in Two Subjects With the Same Telomerase Mutation

UNLABELLED Previous studies have identified subclinical lung disease in family members of probands with familial pulmonary fibrosis, but the natural history of preclinical pulmonary fibrosis is uncertain. The purpose of this study was to determine whether individuals with preclinical lung disease will develop pulmonary fibrosis. After a 27-year interval, two subjects with manifestations of preclinical familial pulmonary fibrosis, including asymptomatic alveolar inflammation and alveolar macrophage activation, were reevaluated for lung disease. CT scans of the chest, pulmonary function tests, and BAL were performed, and genomic DNA was analyzed for mutations in candidate genes associated with familial pulmonary fibrosis. One subject developed symptomatic familial pulmonary fibrosis and was treated with oxygen; her sister remained asymptomatic but had findings of pulmonary fibrosis on high-resolution CT scan of the chest. High concentrations of lymphocytes were found in BAL fluid from both subjects. Genetic sequencing and analyses identified a novel heterozygous mutation in telomerase reverse transcriptase (TERT, R1084P), resulting in telomerase dysfunction and short telomeres in both subjects. In familial pulmonary fibrosis, asymptomatic preclinical alveolar inflammation associated with mutation in TERT and telomerase insufficiency can progress to fibrotic lung disease over 2 to 3 decades. TRIAL REGISTRY ClinicalTrials.gov; No.: NCT00071045; URL: www.clinicaltrials.gov.

The phenotype of the musculocontractural type of Ehlers‐Danlos syndrome due to CHST14 mutations

The musculocontractural type of Ehlers‐Danlos syndrome (MC‐EDS) has been recently recognized as a clinical entity. MC‐EDS represents a differential diagnosis within the congenital neuromuscular and connective tissue disorders spectrum. Thirty‐one and three patients have been reported with MC‐EDS so far with bi‐allelic mutations identified in CHST14 and DSE, respectively, encoding two enzymes necessary for dermatan sulfate (DS) biosynthesis. We report seven additional patients with MC‐EDS from four unrelated families, including the follow‐up of a sib‐pair originally reported with the kyphoscoliotic type of EDS in 1975. Brachycephaly, a characteristic facial appearance, an asthenic build, hyperextensible and bruisable skin, tapering fingers, instability of large joints, and recurrent formation of large subcutaneous hematomas are always present. Three of seven patients had mildly elevated serum creatine kinase. The oldest patient was blind due to retinal detachment at 45 years and died at 59 years from intracranial bleeding; her affected brother died at 28 years from fulminant endocarditis. All patients in this series harbored homozygous, predicted loss‐of‐function CHST14 mutations. Indeed, DS was not detectable in fibroblasts from two unrelated patients with homozygous mutations. Patient fibroblasts produced higher amounts of chondroitin sulfate, showed intracellular retention of collagen types I and III, and lacked decorin and thrombospondin fibrils compared with control. A great proportion of collagen fibrils were not integrated into fibers, and fiber bundles were dispersed into the ground substance in one patient, all of which is likely to contribute to the clinical phenotype. This report should increase awareness for MC‐EDS.

Skin abnormalities as an early predictor of neurologic outcome in Gaucher disease.

To the Editor: Gaucher disease (GD) (MIM 230800, 230900, and 231000), the inherited deficiency of glucocerebrosidase, is classically divided into three types, where type 2, the acute neuronopathic form, is universally progressive and fatal in the first years of life (1–4). Because of the wide range of prognoses in GD, early diagnosis is critical for appropriate treatment decisions and genetic counseling. Currently, substantial neurological deficits may already exist when the diagnosis of type 2 GD is confirmed. Although there are some relevant genotype/phenotype correlations, neither the genotype nor the residual enzyme activity have been consistently reliable in predicting a patient’s clinical course (2, 5). The involvementof the epidermis is reported solely in patients with type 2 GD, where some infants have ichthyotic skin and have been considered ‘collodion babies’ (4, 6–9). In several previous cases, electronmicroscopy revealedabnormal arrays of loosely packed lamellar body-derived sheets in the stratum corneum in infants with type 2 GD (6–9), as well as in a null allele mouse model of GD (7, 8). Moreover, the ratio of glucosylceramide to ceramide is reversed in the skin exclusively from patients with type 2 GD (6). In normal epidermis, glucosylceramide hydrolysis yields ceramide, a critical component of the intercellular lamellae mediating the epidermal permeability barrier (7). We report an infant with type 2 GD, born at 38 weeks gestation to non-consanguineous parents of Western European ancestry, in whom ultrastructural abnormalities were identified in skin prior to the development of more typical neurological manifestations of type 2 GD. The pregnancy was complicated by gestational diabetes and parvovirus exposure, and a cesarean section was performed at 38 weeks. The infant weighed 2.41 kg and had moderately decreased tone, jaundice, generalized petechiae, thrombocytopenia requiring multiple platelet transfusions, and a distended abdomen from hepatosplenomegaly. Parvoviral and TORCH titers were negative. A bone marrow biopsy at 5 weeks revealed characteristic Gaucher cells, confirmed by 7% of normal glucocerebrosidase activity in fibroblasts. Direct sequencing demonstrated genotype L444P/Q414R. Initially, his neurologic examination results and skin appearance were described as normal, and enzyme replacement therapy was begun at age 7 weeks at a dose of 60 U/kg/week. At 9 weeks, he weighed 3.86 kg with all parameters below the 3rd percentile. He had an enlarged liver (4 cm) and spleen (10 cm), scrotal hernias, decreased truncal tone, a strong suck reflex, normal spontaneous and reflex-induced saccadic eye movements, and intermittent strabismus (Fig.1, panel a). He could momentarily lift his head and had a social smile. Although the skin appeared normal, a full-thickness skin biopsy of the forearm was performed for ultrastructural analysis and preserved in half-strength Karnovsky fixative. At 6 months, a gastrostomy tube was placed for poor suck, failure to thrive and reflux. He had minimal weight gain and persistent organomegaly. Severe growth retardation, convergent strabismus, abdominal distention with prominent vasculature and significant axial hypertonia with scissoring and opistotonic posturing were evident. Absent horizontal saccadic eye movements and severe hepatosplenomegaly were documented. Ultrastructural analysis of the outer epidermis revealed disorganized lamellar membranes within the stratum corneum (panels b–d; arrows), interspersed with amorphous, electron-dense non-lamellar microclefts that may represent non-lamellar phase separations due to pockets of accumulated hydrophilic glucosylceramide (panel c; asterisk). These findings were consistent with a significant epidermal lipid processing abnormality, previously reported in type 2 GD (6, 9). The abnormal epidermal ultrastructural findings preceded and predicted the severe neurologic outcome. These unique abnormalities continue to provide a means to discriminate type 2 from Clin Genet 2006: 69: 355–357 Copyright # 2006 Blackwell Munksgaard Printed in Singapore. All rights reserved No claim to original US government works

Cellular and clinical report of new Griscelli syndrome type III cases.

The RAB27A/Melanophilin/Myosin‐5a tripartite protein complex is required for capturing mature melanosomes in the peripheral actin network of melanocytes for subsequent transfer to keratinocytes. Mutations in any one member of this tripartite complex cause three forms of Griscelli syndrome (GS), each with distinct clinical features but with a similar cellular phenotype. To date, only one case of GS type III (GSIII), caused by mutations in the Melanophilin (MLPH) gene, has been reported. Here, we report seven new cases of GSIII in three distinct Arab pedigrees. All affected individuals carried a homozygous missense mutation (c.102C>T; p.R35W), located in the conserved Slp homology domain of MLPH, and had hypomelanosis of the skin and hair. We report the first cellular studies on GSIII melanocytes, which demonstrated that MLPH(R35W) causes perinuclear aggregation of melanosomes in melanocytes, typical for GS. Additionally, co‐immunoprecipitation assays showed that MLPH(R35W) lost its interaction with RAB27A, indicating pathogenicity of the R35W mutation.

Hermansky-Pudlak syndrome type 1 in patients of Indian descent.

Hermansky-Pudlak syndrome (HPS) develops from defects in the biogenesis and/or function of lysosome-related organelles essential to membrane and protein trafficking. Of the eight known human subtypes, only HPS-1 and HPS-4 develop pulmonary fibrosis in addition to the general clinical manifestations of oculocutaneous albinism and bleeding diathesis. We identified HPS-1 in three unrelated patients from different regions of India, who presented with iris transillumination, pale fundi, hypopigmentation, nystagmus, decreased visual acuity, and a bleeding diathesis. Two of these patients carried the homozygous mutation c.398+5G>A (IVS5+5G>A) in HPS1, resulting in skipping of exon 5 in HPS1 mRNA. The third patient carried a novel homozygous c.988-1G>T mutation that resulted in in-frame skipping of HPS1 exon 12 and removes 56 amino acids from the HPS1 protein. Given the discovery of HPS-1 in an ethnic group where oculocutaneous albinism (OCA) is highly prevalent, it is possible that HPS in India is under-diagnosed. We recommend that unconfirmed OCA patients in this ethic group be considered for mutational screening of known HPS genes, in particular c.398+5G>A and c.980-1G>T, to ensure that patients can be monitored and treated for clinical complications unique to HPS.

In silico and functional studies of the regulation of the glucocerebrosidase gene

In Gaucher disease (GD), the inherited deficiency of glucocerebrosidase results in the accumulation of glucocerebroside within lysosomes. Although almost 300 mutations in the glucocerebrosidase gene (GBA) have been identified, the ability to predict phenotype from genotype is quite limited. In this study, we sought to examine potential GBA transcriptional regulatory elements for variants that contribute to phenotypic diversity. Specifically, we generated the genomic sequence for the orthologous genomic region ( approximately 39.4kb) encompassing GBA in eight non-human mammals. Computational comparisons of the resulting sequences, using human sequence as the reference, allowed the identification of multi-species conserved sequences (MCSs). Further analyses predicted the presence of two putative clusters of transcriptional regulatory elements upstream and downstream of GBA, containing five and three transcription factor-binding sites (TFBSs), respectively. A firefly luciferase (Fluc) reporter construct containing sequence flanking the GBA gene was used to test the functional consequences of altering these conserved sequences. The predicted TFBSs were individually altered by targeted mutagenesis, resulting in enhanced Fluc expression for one site and decreased expression for seven others sites. Gel-shift assays confirmed the loss of nuclear-protein binding for several of the mutated constructs. These identified conserved non-coding sequences flanking GBA could play a role in the transcriptional regulation of the gene contributing to the complexity underlying the phenotypic diversity seen in GD.