Molecular and Clinical Analyses of Greig Cephalopolysyndactyly and Pallister-Hall Syndromes: Robust Phenotype Prediction from the Type and Position of GLI3 Mutations

Mutations in the GLI3 zinc-finger transcription factor gene cause Greig cephalopolysyndactyly syndrome (GCPS) and Pallister-Hall syndrome (PHS), which are variable but distinct clinical entities. We hypothesized that GLI3 mutations that predict a truncated functional repressor protein cause PHS and that functional haploinsufficiency of GLI3 causes GCPS. To test these hypotheses, we screened patients with PHS and GCPS for GLI3 mutations. The patient group consisted of 135 individuals: 89 patients with GCPS and 46 patients with PHS. We detected 47 pathological mutations (among 60 probands); when these were combined with previously published mutations, two genotype-phenotype correlations were evident. First, GCPS was caused by many types of alterations, including translocations, large deletions, exonic deletions and duplications, small in-frame deletions, and missense, frameshift/nonsense, and splicing mutations. In contrast, PHS was caused only by frameshift/nonsense and splicing mutations. Second, among the frameshift/nonsense mutations, there was a clear genotype-phenotype correlation. Mutations in the first third of the gene (from open reading frame [ORF] nucleotides [nt] 1-1997) caused GCPS, and mutations in the second third of the gene (from ORF nt 1998-3481) caused primarily PHS. Surprisingly, there were 12 mutations in patients with GCPS in the 3\u27 third of the gene (after ORF nt 3481), and no patients with PHS had mutations in this region. These results demonstrate a robust correlation of genotype and phenotype for GLI3 mutations and strongly support the hypothesis that these two allelic disorders have distinct modes of pathogenesis

Molecular and Clinical Analyses of Greig Cephalopolysyndactyly and Pallister-Hall Syndromes: Robust Phenotype Prediction from the Type and Position of GLI3 Mutations

Mutations in the GLI3 zinc-finger transcription factor gene cause Greig cephalopolysyndactyly syndrome (GCPS) and Pallister-Hall syndrome (PHS), which are variable but distinct clinical entities. We hypothesized that GLI3 mutations that predict a truncated functional repressor protein cause PHS and that functional haploinsufficiency of GLI3 causes GCPS. To test these hypotheses, we screened patients with PHS and GCPS for GLI3 mutations. The patient group consisted of 135 individuals: 89 patients with GCPS and 46 patients with PHS. We detected 47 pathological mutations (among 60 probands); when these were combined with previously published mutations, two genotype-phenotype correlations were evident. First, GCPS was caused by many types of alterations, including translocations, large deletions, exonic deletions and duplications, small in-frame deletions, and missense, frameshift/nonsense, and splicing mutations. In contrast, PHS was caused only by frameshift/nonsense and splicing mutations. Second, among the frameshift/nonsense mutations, there was a clear genotype-phenotype correlation. Mutations in the first third of the gene (from open reading frame [ORF] nucleotides [nt] 1–1997) caused GCPS, and mutations in the second third of the gene (from ORF nt 1998–3481) caused primarily PHS. Surprisingly, there were 12 mutations in patients with GCPS in the 3′ third of the gene (after ORF nt 3481), and no patients with PHS had mutations in this region. These results demonstrate a robust correlation of genotype and phenotype for GLI3 mutations and strongly support the hypothesis that these two allelic disorders have distinct modes of pathogenesis

Immunological identification, localization and characterization of the Lowe syndrome (OCRL) protein

The oculocerebrorenal syndrome of Lowe is a rare X-linked disorder characterized by congenital cataracts, proximal renal tubular dysfunction, and mental retardation. The OCRL gene, OCRL-1, was identified by positional cloning and found to be highly homologous to a 75 Kd inositol polyphosphate 5-phosphatase, INPP5Bp. This protein is responsible for the majority of phosphatidyl (4,5) bisphosphate (PIP2) 5-phosphatase activity in platelets. Enzymatic assays demonstrate a deficiency of this same activity in Lowe patient\u27s fibroblast when compared to control fibroblasts. This activity suggests a role for the OCRL-1 protein (ocrl-1) in a signaling pathway, in particular, the inositol pathway. To characterize the OCRL-1 protein by immunological methods, antibodies were made to identify the protein. A single protein of 105 Kd was identified using affinity purified sera in western blot analysis of control fibroblast lysates and absent in the fibroblasts of a Lowe\u27s patient who lacked the OCRL-1 transcript. This antisera also recognizes a protein of similar mass in other human cell lines and in many mouse tissue homogenates. Immunofluorescence assays in fibroblast cells using the same purified sera, localize ocrl-1 to the Golgi complex. Brefeldin A (BFA) is a fungal product which reversibly disrupts the Golgi apparatus and recycles resident proteins back to the endoplasmic reticulum (ER). Studies demonstrate that ocrl-1 does not localize to the ER during BFA treatment but remains in a juxtanuclear position similar to proteins specifically localized to the trans-Golgi network (TGN) compartment of the Golgi complex. The recovery from BFA treatment comparing control and Lowe patient fibroblast cells demonstrates a marked difference in the time of Golgi reformation. These preliminary studies demonstrate an important difference which suggests that ocrl-1 may play an important role in vesicle formation. These immunological studies have identified and localized the protein defective in OCRL. Together with recent biochemical data, this information defines this protein as an inositol phospholipid enzyme localized to the Golgi complex with possible roles in vesicle formation. These studies can eventually aid in the elucidation of the precise role of ocrl-1, the pathways it affects and how the absence of ocrl-1 produces Lowe syndrome

Immunological identification, localization and characterization of the Lowe syndrome (OCRL) protein

The oculocerebrorenal syndrome of Lowe is a rare X-linked disorder characterized by congenital cataracts, proximal renal tubular dysfunction, and mental retardation. The OCRL gene, OCRL-1, was identified by positional cloning and found to be highly homologous to a 75 Kd inositol polyphosphate 5-phosphatase, INPP5Bp. This protein is responsible for the majority of phosphatidyl (4,5) bisphosphate (PIP2) 5-phosphatase activity in platelets. Enzymatic assays demonstrate a deficiency of this same activity in Lowe patient\u27s fibroblast when compared to control fibroblasts. This activity suggests a role for the OCRL-1 protein (ocrl-1) in a signaling pathway, in particular, the inositol pathway. To characterize the OCRL-1 protein by immunological methods, antibodies were made to identify the protein. A single protein of 105 Kd was identified using affinity purified sera in western blot analysis of control fibroblast lysates and absent in the fibroblasts of a Lowe\u27s patient who lacked the OCRL-1 transcript. This antisera also recognizes a protein of similar mass in other human cell lines and in many mouse tissue homogenates. Immunofluorescence assays in fibroblast cells using the same purified sera, localize ocrl-1 to the Golgi complex. Brefeldin A (BFA) is a fungal product which reversibly disrupts the Golgi apparatus and recycles resident proteins back to the endoplasmic reticulum (ER). Studies demonstrate that ocrl-1 does not localize to the ER during BFA treatment but remains in a juxtanuclear position similar to proteins specifically localized to the trans-Golgi network (TGN) compartment of the Golgi complex. The recovery from BFA treatment comparing control and Lowe patient fibroblast cells demonstrates a marked difference in the time of Golgi reformation. These preliminary studies demonstrate an important difference which suggests that ocrl-1 may play an important role in vesicle formation. These immunological studies have identified and localized the protein defective in OCRL. Together with recent biochemical data, this information defines this protein as an inositol phospholipid enzyme localized to the Golgi complex with possible roles in vesicle formation. These studies can eventually aid in the elucidation of the precise role of ocrl-1, the pathways it affects and how the absence of ocrl-1 produces Lowe syndrome