Marsha Zeigler

Molecular basis of variant pseudo-Hurler polydystrophy (mucolipidosis IIIC)

Mucolipidosis IIIC, or variant pseudo-Hurler polydystrophy, is an autosomal recessive disease of lysosomal hydrolase trafficking. Unlike the related diseases, mucolipidosis II and IIIA, the enzyme affected in mucolipidosis IIIC (N-Acetylglucosamine-1-phosphotransferase [GlcNAc-phosphotransferase]) retains full transferase activity on synthetic substrates but lacks activity on lysosomal hydrolases. Bovine GlcNAc-phosphotransferase has recently been isolated as a multisubunit enzyme with the subunit structure alpha(2)beta(2)gamma(2). We cloned the cDNA for the human gamma-subunit and localized its gene to chromosome 16p. We also showed, in a large multiplex Druze family that exhibits this disorder, that MLIIIC also maps to this chromosomal region. Sequence analysis of the gamma-subunit cDNA in patients from 3 families identified a frameshift mutation, in codon 167 of the gamma subunit, that segregated with the disease, indicating MLIIIC results from mutations in the phosphotransferase gamma-subunit gene. This is to our knowledge the first description of the molecular basis for a human mucolipidosis and suggests that the gamma subunit functions in lysosomal hydrolase recognition.

The clinical spectrum of fetal Niemann-Pick type C.

Niemann–Pick type C (NPC) disease is a lysosomal neurovisceral storage disease. The spectrum of the clinical presentation as well as the severity of the disease and the age of presentation may be highly variable. Fetal presentation is rarely described in the literature. Here, we report on seven new cases of fetal onset NPC of whom two were diagnosed in utero and five postnatally. The fetal clinical presentation, included, in utero splenomegaly (6/7), in utero hepatomegaly (5/7), in utero ascites (4/7), intra uterine growth retardation (IUGR) (2/7), and oligohydramnios (2/7). Placentomegaly was present in two of the three pregnancies examined. Congenital thrombocytopenia (4/4), congenital anemia (2/4), and petechial rash (2/5) were diagnosed immediately after birth. Three patients were born preterm. Pregnancy and postnatal outcome were remarkably poor with one case of intrauterine fetal death, one elective termination of pregnancy, and four patients who died within the first months of life from a rapidly fatal neonatal cholestatic disease. NPC1 gene mutation analysis identified all of the mutant alleles including three novel mutations. Splenomegaly, hepatomegaly, and ascites were the most consistent prenatal ultrasonographic findings of the NPC fetuses. We suggest that once identified these findings, should raise the suspicion of fetal NPC. Our study further expands the antenatal clinical spectrum of NPC and provides clues to its prenatal diagnosis.

Clinical-biochemical correlation in molecularly characterized patients with Niemann-Pick type C

Purpose: Niemann-Pick disease type C (NP-C) is an autosomal recessive lipid storage disease manifested by an impairment in cellular cholesterol homeostasis. The clinical phenotype of NP-C is extremely variable, ranging from an acute neonatal form to an adult late-onset presentation. To facilitate phenotype-genotype studies, we have analyzed multiple Israeli NP-C families.Methods: The severity of the disease was assessed by the age at onset, hepatic involvement, neurological deterioration, and cholesterol esterification studies. Screening of the entire NPC1 coding sequence allowed for molecular characterization and identification of disease causing mutations.Results: A total of nine NP-C index cases with mainly neurovisceral involvement were characterized. We demonstrated a possible link between the severity of the clinical phenotype and the cholesterol esterification levels in fibroblast cultures following 24 hours of in vitro cholesterol loading. In addition, we identified eight novel mutations in the NPC1 gene.Conclusions: Our results further support the clinical and allelic heterogeneity of NP-C and point to possible association between the clinical and the biochemical phenotype in distinct affected Israeli families.

Mucolipidosis Type IV: The Effect of Increased Lysosomal pH on the Abnormal Lysosomal Storage

Mucolipidosis type IV (MLIV) is a neurodegenerative channelopathy that is caused by the deficiency of TRPML1 activity, a nonselective cation channel. TRPML1 is a lysosomal membrane protein, and thus, MLIV is a lysosomal storage disorder. The basic, specific function of TRPML1 has not been yet clarified. A recent report (Soyombo AA, Tjon-Kon-Sang S, Rbaibi Y, Bashllari E, Bisceglia J, Muallem S, Kiselyov K: J Biol Chem 281:7294–7301, 2006) indicated that TRPML1 functions as an outwardly proton channel whose function is the prevention of overacidification of these organelles. Thus, in MLIV the lysosomal pH is lower than normal. Furthermore, attempts by these investigators to increase slightly the lysososmal pH with either Nigericin or Chloroquine suggested corrective effect of the abnormal storage in MLIV cells. We investigated this approach using these agents with cultured fibroblasts from severely affected and milder patients. Our data indicated that there was no reduction in the total number of storage vesicles by either agent, although Nigericin resulted in a change in the nature of the storage materials, reducing the presence of lamellated substances (lipids) so that the storage vesicles contained predominantly granulated substances. On the other hand, transfection with the normal MCOLN1 cDNA (the gene coding for TRPML1) resulted in the removal of almost all the storage materials.

TAT gene mutation analysis in three Palestinian kindreds with oculocutaneous tyrosinaemia type II; characterization of a silent exonic transversion that causes complete missplicing by exon 11 skipping

SummaryDeficiency of the hepatic cytosolic enzyme tyrosine aminotransferase (TAT) causes marked hypertyrosinaemia leading to painful palmoplantar hyperkeratoses, pseudodendritic keratitis and variable mental retardation (oculocutaneous tyrosinaemia type II or Richner–Hanhart syndrome). Parents may therefore seek prenatal diagnosis, but this is not possible by biochemical assays as tyrosine does not accumulate in amniotic fluid and TAT is not expressed in chorionic villi or amniocytes. Molecular analysis is therefore the only possible approach for prenatal diagnosis and carrier detection. To this end, we sought TAT gene mutations in 9 tyrosinaemia II patients from three consanguineous Palestinian kindreds. In two kindreds (7 patients), the only potential abnormality identified after sequencing all 12 exons and exon–intron boundaries was homozygosity for a silent, single-nucleotide transversion c.1224G > T (p.T408T) at the last base of exon 11. This was predicted to disrupt the 5′ donor splice site of exon 11 and result in missplicing. However, as TAT is expressed exclusively in liver, patient mRNA could not be obtained for splicing analysis. A minigene approach was therefore used to assess the effect of c.1224G > T on exon 11 splicing. Transfection experiments with wild-type and c.1224G > T mutant minigene constructs demonstrated that c.1224G > T results in complete exon 11 skipping, illustrating the utility of this approach for confirming a putative splicing defect when cDNA is unavailable. Homozygosity for a c.1249C > T (R417X) exon 12 nonsense mutation (previously reported in a French patient) was identified in both patients from the third kindred, enabling successful prenatal diagnosis of an unaffected fetus using chorionic villous tissue.

Niemann Pick disease type A in Israeli Arabs: 677delT, a common novel single mutation

A novel single base pair deletion in the acid sphingomyelinase (ASM) gene ( 677delT in the cDNA) was identified in 12 Israeli Arab families with Niemann‐Pick disease (NPD) type A. This deletion creates a premature stop codon which explains the complete deficiency of ASM activity in these patients and the severe clinical manifestation. A single mutation in 12 families living in a relatively small geographical region suggests a founder effect and explains the high frequency of this disease in this population. This is in contrast to multiple mutations found in two other lysosomal storage disorders prevalent in this population, namely, Hurler disease (MPSI) and metachromatic leukodystrophy. Mutation analysis is therefore an important tool in characterizing the grounds for the high frequency of inherited diseases as well as a basis for prevention programs for prevalent diseases through carrier identification and the ascertainment of high risk families. Hum Mutat 12:136, 1998.

Hunter syndrome in Jews in Israel: further evidence for prenatal selection favoring the Hunter allele

SummaryAmong all the Jewish families with Hunter patients in Israel, 10 were Ashkenazi or Moroccan in origin. In those families, there was a paucity of new mutations. In addition, a significant deviation of the segregation ratio between the Hunter gene and the normal allele was demonstrated among the offspring of heterozygous mothers or siblings of affected children in these families. These results confirm and extend our previous observations suggesting selection in favor of the X chromosome carrying the Hunter allele among Ashkenazi and Moroccan Jews.

Genetic screening for Krabbe disease: learning from the past and looking to the future.

In Israel, Krabbe disease is frequent in two Moslem Arab villages in the Jerusalem area. In this paper we present our experience of almost four decades with diagnosis of Krabbe disease, carrier screening and prenatal diagnosis. The screening program is well accepted by the community, and there is a clear trend towards premarital testing. The screening program and prenatal diagnosis have led to a decrease in the incidence of Krabbe disease from 1.6 per 1,000 live births to 0.82 per 1,000.

Aspartylglucosaminuria among Palestinian Arabs

Aspartylglucosaminuria (AGU) is a rare disorder of glycoprotein metabolism caused by the deficiency of the lysosomal enzyme aspartylglucosaminidase (AGA). AGU is inherited as an autosomal recessive trait and occurs with a high frequency in Finland because of a founder effect. While very few patients with AGU have been reported from non-Finnish origin, we diagnosed the disorder in 8 patients originating from 3 unrelated families, all Palestinian Arabs from the region of Jerusalem. The clinical diagnosis of AGU is often difficult, in particular early in the course of the disease, and most of the patients are diagnosed after the age of 5 years. However, since these patients excrete early large amounts of aspartylglucosamine in urine, biochemical screening is easy by urine chromatography.

Niemann Pick Disease type A in Israeli Arabs: 677delT, a common novel single mutation. Mutations in brief no. 161. Online.

The maturity-onset diabetes of the young (MODY), an autosomal dominant form of non-insulin dependent diabetes mellitus (NIDDM), is caused by mutations in the glucokinase (GK, MODY 2) and in the hepatocyte nuclear factor 1a (MODY 3) and 4a (MODY 1) genes. We have screened the glucokinase gene by the polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) in fifteen subjects with clinical characteristics of MODY and one parent with NIDDM, impaired glucose tolerance or gestational diabetes. PCR products with abnormal mobility in DGGE were directly sequenced. We have identified four mutant alleles, three of them (G80S, E221K, G227C) are new missense mutations located in or near the region of the active site cleft of the enzyme. The mutations co-segregate with hyperglycemia in the families of the three probands, whose biochemical and clinical phenotype is similar to other individuals with MODY 2 mutations.A novel single base pair deletion in the acid sphingomyelinase (ASM) gene (677delT in the cDNA) was identified in 12 Israeli Arab families with Niemann-Pick disease (NPD) type A. This deletion creates a premature stop codon which explains the complete deficiency of ASM activity in these patients and the severe clinical manifestation. A single mutation in 12 families living in a relatively small geographical region suggests a founder effect and explains the high frequency of this disease in this population. This is in contrast to multiple mutations found in two other lysosomal storage disorders prevalent in this population, namely, Hurler disease (MPSI) and metachromatic leukodystrophy. Mutations analysis is therefore an important tool in characterizing the grounds for the high frequency of inherited diseases as well as a basis for prevention programs for prevalent diseases through carrier identification and the ascertainment of high risk families.Mutation analysis of DNA from cultured amniocytes with absent branched‐chain α‐ketoacid dehydrogenase activity revealed a C to T transition producing a nonsense mutation (R242X) in exon 7 of the gene encoding the E1a subunit of this multienzyme complex (BCKDHA). This pregnancy occured in a large consanguinous pedigree with multiple individuals with maple syrup urine disease (MSUD). PCR amplification of the region surrounding exon 7 allowed the identification of this mutation as well as two other previously identified mutations which cause MSUD. hum Mutat 12:136, 1998.