clinical homogeneity and allelic heterogeneity in seven patients

Background Larsen syndrome is an autosomal dominant skeletal dysplasia characterized by large joint dislocations and craniofacial dysmorphism. It is caused by missense or small in-frame deletions in the FLNB gene. To further characterize the phenotype and the mutation spectrum of this condition, we investigated seven probands, five sporadic individuals and a mother-son-duo with Larsen syndrome. Methods The seven patients from six unrelated families were clinically and radiologically evaluated. All patients were screened for mutations in selected exons and exon-intron boundaries of the FLNB gene by Sanger sequencing. FLNB transcript analysis was carried out in one patient to analyse the effect of the sequence variant on pre-mRNA splicing. Results All patients exhibited typical facial features and joint dislocations. Contrary to the widely described advanced carpal ossification, we noted delay in two patients. We identified the five novel mutations c.4927G > A/p.(Gly1643Ser), c.4876G > T / p.(Gly1626Trp), c.4664G > A / p.(Gly1555Asp), c.2055G > C / p.Gln685delins10 and c.5021C > T / p.(Ala1674Val) as well as a frequently observed mutation in Larsen syndrome [c.5164G > A/p.(Gly1722Ser)] in the hotspot regions. FLNB transcript analysis of the c.2055G > C variant revealed insertion of 27 bp intronic sequence between exon 13 and 14 which gives rise to in-frame deletion of glutamine 685 and insertion of ten novel amino acid residues (p.Gln685delins10). Conclusions All seven individuals with Larsen syndrome had a uniform clinical phenotype except for delayed carpal ossification in two of them. Our study reveals five novel FLNB mutations and confirms immunoglobulin-like (Ig) repeats 14 and 15 as major hotspot regions. The p.Gln685delins10 mutation is the first Larsen syndrome-associated alteration located in Ig repeat 5. All mutations reported so far leave the filamin B protein intact in accordance with a gain-of-function effect. Our findings underscore the characteristic clinical picture of FLNB-associated Larsen syndrome and add Ig repeat 5 to the filamin B domains affected by the clustered mutations

Homozygous deletion of exons 2 and 3 of NPC2 associated with Niemann–Pick disease type C

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/134235/1/ajmga37794.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/134235/2/ajmga37794-sup-0001-SuppData-S1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/134235/3/ajmga37794_am.pd

Confirmation of a Rare Genetic Leukoencephalopathy due to a Novel Bi-allelic Variant in RPIA

Ribose 5-phosphate isomerase deficiency is a rare genetic leukoencephalopathy caused by pathogenic sequence variants in RPIA, that encodes ribose 5-phosphate isomerase, an enzyme in the pentose phosphate pathway. Till date, only three individuals with ribose 5-phosphate isomerase deficiency have been described in literature. We report on a subject with RPIA associated progressive leukoencephalopathy with elevated urine arabitol and ribitol levels and a novel missense variant c.770T > C p.(Ile257Thr) in exon 8 of RPIA. We also compare the phenotypes of all the four subjects. Our report confirms the phenotype and the genetic cause of this condition

Bi‐allelic missense variant, p.Ser35Leu in EXOSC1 is associated with pontocerebellar hypoplasia

RNA exosome is a highly conserved ribonuclease complex essential for RNA processing and degradation. Bi‐allelic variants in exosome subunits EXOSC3, EXOSC8 and EXOSC9 have been reported to cause pontocerebellar hypoplasia type 1B, type 1C and type 1D, respectively, while those in EXOSC2 cause short stature, hearing loss, retinitis pigmentosa and distinctive facies. We ascertained an 8‐months‐old male with developmental delay, microcephaly, subtle dysmorphism and hypotonia. Pontocerebellar hypoplasia and delayed myelination were noted on neuroimaging. A similarly affected elder sibling succumbed at the age of 4‐years 6‐months. Chromosomal microarray returned normal results. Exome sequencing revealed a homozygous missense variant, c.104C > T p.(Ser35Leu) in EXOSC1 (NM_016046.5) as the possible candidate. In silico mutagenesis revealed loss of a polar contact with neighboring Leu37 residue. Quantitative real‐time PCR indicated no appreciable differences in EXOSC1 transcript levels. Immunoblotting and blue native PAGE revealed reduction in the EXOSC1 protein levels and EXO9 complex in the proband, respectively. We herein report an individual with the bi‐allelic variant c.104C>T p.(Ser35Leu) in EXOSC1 and clinical features of pontocerebellar hypoplasia type 1. Immunoblotting and blue native PAGE provide evidence for the pathogenicity of the variant. Thus, we propose EXOSC1 as a novel candidate gene for pontocerebellar hypoplasia.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/167044/1/cge13928.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/167044/2/cge13928_am.pd

Biochemical analysis of GNPTAB missense mutations associated with ML II

Mucolipidosis type II and type III (ML II and III) are rare autosomal recessive disorders of lysosomal hydrolase trafficking respectively caused by completely absent or reduced activity of the enzyme GlcNAc-phosphotransferase, which catalyzes the initial step in the synthesis of mannose-6-phosphate recognition marke. This heterohexameric enzyme composed of three subunits (alpha2 beta2 gamma2), is a product of two distinct genes GNPTAB and GNPTG. Mutations in GNPTAB result ML II and ML III, while mutations in GNPTG are only associated with ML III. To date more than 100 different GNPTAB mutations have been described, causing either ML II alpha\beta or ML III alpha\beta. Although splicing and frameshift mutations are usually associated with more severe phenotypes and missense mutations with milder ones, this typical pattern is not observed for all ML II patients. Here we report the impact of two GNPTAB missense mutations upon the protein: while W81L occurred in the portion of the gene that encodes the apha-subunit, R986C affected a genomic region encoding the beta subunit. To address this issue, the entire coding region of the wild-type GNPTAB was cloned into the pcDNAHisMax TOPO vector and the c.440delC (A147AfsX5), c.2956T>C (R986C) and c.242G>T (W81L) were introduced on this vector using the QuikChange Site-directed Mutagenesis kit. The presence of additional mutations, resulting from possible enzymatic misincorporation, was excluded by sequencing all constructs. COS7 cells were transfected with control and mutant plasmids using Lipofectamine 2000 reagent. Protein expression levels and subcellular location were determined through Western Blot and Immunofluorescence, respectively. Results and Conclusions: We analyzed the protein expression levels of three GNPTAB mutations: c.242G>T (W81L), c.2956T>C (R986C) andc.440delC (A147AfsX5). The frameshift c.440delC (A147AfsX5), predicting to introduce a premature stop codon, was used as negative control. and, as expected, no GNPTAB protein product was detectable. Instead, the analysis of both missense mutations, c.2956T>C (R986C) and c.242G>T (W81L), revealed a decrease in GNPTAB protein expression, compared to the control wild type. This concurs with a previous computational assessment by the Polyphen and SIFT algorithms, predicting that the 2 mutations were likely to be potentially damaging. In addition, computational analysis (http://www.ensembl.org/) revealed that both missense mutations occurred at evolutionarily conserved amino acid residues. The results of all these approaches correlate with the severe ML II phenotype of the patients

Phenotype and genotype in patients with Larsen syndrome: clinical homogeneity and allelic heterogeneity in seven patients

Background: Larsen syndrome is an autosomal dominant skeletal dysplasia characterized by large joint dislocations and craniofacial dysmorphism. It is caused by missense or small in-frame deletions in the FLNB gene. To further characterize the phenotype and the mutation spectrum of this condition, we investigated seven probands, five sporadic individuals and a mother-son-duo with Larsen syndrome. Methods: The seven patients from six unrelated families were clinically and radiologically evaluated. All patients were screened for mutations in selected exons and exon-intron boundaries of the FLNB gene by Sanger sequencing. FLNB transcript analysis was carried out in one patient to analyse the effect of the sequence variant on pre-mRNA splicing. Results: All patients exhibited typical facial features and joint dislocations. Contrary to the widely described advanced carpal ossification, we noted delay in two patients. We identified the five novel mutations c.4927G > A/p.(Gly1643Ser), c. 4876G > T / p.(Gly1626Trp), c.4664G > A / p.(Gly1555Asp), c.2055G > C / p.Gln685delins10 and c.5021C > T / p.(Ala1674Val) as well as a frequently observed mutation in Larsen syndrome [c.5164G > A/ p.(Gly1722Ser)] in the hotspot regions. FLNB transcript analysis of the c. 2055G > C variant revealed insertion of 27 bp intronic sequence between exon 13 and 14 which gives rise to in-frame deletion of glutamine 685 and insertion of ten novel amino acid residues (p.Gln685delins10). Conclusions: All seven individuals with Larsen syndrome had a uniform clinical phenotype except for delayed carpal ossification in two of them. Our study reveals five novel FLNB mutations and confirms immunoglobulin-like (Ig) repeats 14 and 15 as major hotspot regions. The p.Gln685delins10 mutation is the first Larsen syndrome-associated alteration located in Ig repeat 5. All mutations reported so far leave the filamin B protein intact in accordance with a gain-of-function effect. Our findings underscore the characteristic clinical picture of FLNB-associated Larsen syndrome and add Ig repeat 5 to the filamin B domains affected by the clustered mutations