Identification of novel mutations in patients with Shwachman-Diamond syndrome

Shwachman-Diamond syndrome (SDS) is a rare autosomal recessive disease, mainly characterized by exocrine pancreatic insufficiency, hematological dysfunction and skeletal abnormalities. The SDS disease locus was mapped to chromosome 7q11 and disease-associated mutations were reported in the Shwachman-Bodian-Diamond syndrome (SBDS) gene. SBDS is a member of a highly conserved protein family with putative orthologs in diverse species including archaea and eukaryotes. It is widely expressed in many tissues and its function is still unknown. In the present study we analyzed the genotype of 15 unrelated Italian SDS patients. After sequencing the whole coding region we were able to complete all genotypes of the SDS patients tested. A total of eleven distinct mutations were identified. The most frequent mutations are due to gene conversion events between SBDS and its unprocessed pseudogene, named SBDSP. We described four new gene conversions involving exon 2 and three novel mutations that are not a result of gene conversion events. In two out of the fifteen cases, the family analysis evidenced an apparently unexpected inheritance of SDS alleles between parents and affected children. In the first case we found a new large gene conversion event, that caused the failure of the amplification of the father's allele and in the second what could be explained as a de novo gene conversion. Both cases have important implications for genetic counseling and molecular genetic analysis. In a disorder caused by gene conversions of variable extension these findings emphasize the necessity of testing patient's parents and the significance of the choice of primers.     

Identification of four novel PMM2 mutations in congenital disorders of glycosylation (CDG) Ia French patients

We screened 11 unrelated French patients with congenital disorders of glycosylation (CDG) Ia for PMM2 mutations. Twenty one missense mutations on the 22 chromosomes (95%) including four novel mutations were identified: C9Y (G26A) in exon 1, L32R (TA95GC) in exon 2, and T226S (C677G) and C241S (G722C) in exon 8. We studied the PMM activity of these four novel mutant proteins and of the R141H mutant protein in an E coli expression system. The T226S, C9Y, L32R, and C241S mutant proteins have decreased specific activity (23 to 41% of normal), are all more or less thermolabile, and R141H has no detectable activity. Our results indicate that the new mutations identified here are less severe than the inactive R141H mutant protein, conferring residual PMM activity compatible with life.


Keywords: CDG; phosphomannomutase; PMM2 mutations     

Identification of novel VMD2 gene mutations in patients with best vitelliform macular dystrophy

ABSTRACT We report five novel VMD2 mutations in Best's macular dystrophy patients (S16F, I73N, R92H, V235L, and N296S). An SSCP analysis of the VMD2 11 exons revealed electrophoretic mobility shifts exclusively in exons 2, 3, 4, 6 and 8. Direct sequencing indicated that these shifts are caused by mono-allelic transition in exons 2, 4, 6, 8 and transversion in exons 3 and 6. Five novel "silent" polymorphisms are also reported: 213T>C, 323C>A, 1514A>G, 1661C>T, and 1712T>C. Hum Mutat 17:235, 2001.     

Identification of 11 novel mutations in USH2A among Japanese patients with Usher syndrome type 2

Usher syndrome (USH) is an autosomal recessive disorder characterized by retinitis pigmentosa and hearing loss. USH type 2 (USH2) is the most common type of USH and is frequently caused by mutations in USH2A , which accounts for 74–90% of USH2 cases. This is the first study reporting the results of scanning for USH2A mutations in Japanese patients with USH2. In 8 of 10 unrelated patients, we identified 14 different mutations. Of these mutations, 11 were novel. Although the mutation spectrum that we identified differed from that for Caucasians, the incidence of mutations in USH2A was 80% for all patients tested, which is consistent with previous findings. Further, c.8559-2A>G was identified in four patients and accounted for 26.7% of mutated alleles; it is thus a frequent mutation in Japanese patients. Hence, mutation screening for c.8559-2A>G in USH2A may prove very effective for the early diagnosis of USH2.     

Identification and characterization of five novel MAN2B1 mutations in Italian patients with alpha-mannosidosis

Mutation analysis performed on six Italian families with alpha-mannosidosis type II allowed the identification of five new mutations in the MAN2B1 gene: c.157G>T, c.562C>T, c.599A>T, c.293dupA, c.2402G>A (p.E53X, p.R188X, p.H200L, p.Y99VfsX61, p.G801D). Protein residues G801 and H200 are conserved among the four mammalian alpha-mannosidases cloned to date: human, cattle, cat and mouse. In vitro expression studies demonstrated that both missense mutations expressed no residual alpha-mannosidase activity indicating that they are disease-causing mutations. Modelling into the three-dimensional structure revealed that the p.H200L could involve the catalytic mechanism, whereas p.G801D would affect the correct folding of the enzyme.     

Identification and in silico analyses of novel TGFBR1 and TGFBR2 mutations in Marfan syndrome-related disorders

Very recently, heterozygous mutations in the genes encoding transforming growth factor beta receptors I (TGFBR1) and II (TGFBR2) have been reported in Loeys-Dietz aortic aneurysm syndrome (LDS). In addition, dominant TGFBR2 mutations have been identified in Marfan syndrome type 2 (MFS2) and familial thoracic aortic aneurysms and dissections (TAAD). In the past, mutations of these genes were associated with atherosclerosis and several human cancers. Here, we report a total of nine novel and one known heterozygous sequence variants in the TGFBR1 and TGFBR2 genes in nine of 70 unrelated individuals with MFS-like phenotypes who previously tested negative for mutations in the gene encoding the extracellular matrix protein fibrillin-1 (FBN1). To assess the pathogenic impact of these sequence variants, in silico analyses were performed by the PolyPhen, SIFT, and Fold-X algorithms and by means of a 3D homology model of the TGFBR2 kinase domain. Our results showed that in all but one of the patients the pathogenic effect of at least one sequence variant is highly probable (c.722C > T, c.799A > C, and c.1460G > A in TGFBR1 and c.773T > G, c.1106G > T, c.1159G > A, c.1181G > A, and c.1561T > C in TGFBR2). These deleterious alleles occurred de novo or segregated with the disease in the families, indicating a causative association between the sequence variants and clinical phenotypes. Since TGFBR2 mutations found in patients with MFS-related disorders cannot be distinguished from heterozygous TGFBR2 mutations reported in tumor samples, we emphasize the importance of segregation analysis in affected families. In order to be able to find the mutation that is indeed responsible for a MFS-related phenotype, we also propose that genetic testing for sequence alterations in TGFBR1 and TGFBR2 should be complemented by mutation screening of the FBN1 gene.     

Identification of novel mutations in FOXL2 associated with premature ovarian failure

Premature ovarian failure (POF) affects approximately 1% of women and is known to be caused by sex chromosome abnormalities, iatrogenic agents and autoimmune diseases, but in the majority of cases the cause is unknown. However, several families have been identified as having an inherited predisposition to POF, suggesting a genetic component to the condition in these cases. The FOXL2 gene of 70 POF patients from New Zealand and Slovenia was screened for mutations. In a Slovenian POF patient, a novel 30 bp deletion was identified that was predicted to remove 10 out of 14 alanines (A221_A230del), from the polyalanine tract downstream of the winged helix/forkhead domain of the FOXL2 protein. A novel single nucleotide substitution, 772(1009)T>A, which is predicted to change amino acid 258 from tyrosine to asparagine (Y258N), was identified in a New Zealand POF patient. Neither mutation was identified in 200 normal control chromosomes from 100 control samples. Three previously unreported single nucleotide substitutions, considered to be non-functional polymorphisms, were also identified.     

Identification of 11 novel mutations in 49 Korean patients with mucopolysaccharidosis type II

Mucopolysaccharidosis type II (MPS II) or Hunter syndrome is a rare lysosomal storage disorder caused by a deficiency of iduronate-2-sulfatase (IDS). As MPS II is X-linked, patients are usually males with heterogeneous mutations ranging from point mutations to gross deletions and recombination. In 2003, we reported a mutation analysis of 25 patients with MPS II. In this study, 31 mutations in another 49 Korean patients (45 families) with MPS II are reported: 12 missense, nine deletions, four splicing, two nonsense, two insertions, one deletion/insertion, and IDS-IDS2 recombination mutations. Among these mutations, 11 were novel ones (4 missense mutations: Ser61Pro, Pro97Arg, Pro228Ala, and Pro261Ala; 5 deletions: c.344delA, c.420delG, c.768delT, c.1112delC and c.1402delC; 1 deletion/insertion: c.1222delinsTA; and 1 insertion mutation: c.359_360insATCC). The IDS-IDS2 recombination mutations were most frequently observed; all patients with this mutation had the severe MPS II phenotype. However, most of the patients (5/7) with the G374G splicing mutation had an attenuated phenotype, except for two sibling cases with the severe phenotype. Except for a few recurrent mutations such as the G374G, R443X, L522P, and recombination mutations, each patient had a unique individual mutation. Therefore, careful interpretation of genotype-phenotype correlations is warranted.     

Identification of fifteen novel PHEX gene mutations in Finnish patients with hypophosphatemic rickets

We have carried out a mutation screening of the PHEX gene in Finnish patients with hypophosphatemia. A total of 100% (5/5) of the familial HYP patients (X-linked hypophosphatemia) and 93% (14/15) of the sporadic cases were found to carry a mutation in the PHEX gene. We identified 18 mutations, of which 15 were novel. We report also a new polymorphism 46bp upstream of exon 16. Two families were segregating the same nonsense mutation in exon 1 (R20X), but since this mutation has been previously reported in three independent studies, we consider it to be a mutational hotspot rather than a Finnish founder mutation. We did not find PHEX gene mutations in two additional hypophosphatemia families in which the mode of inheritance was other than X-linked dominant. Also, no mutation could be detected in a patient with suspected oncogenic osteomalacia (OHO).     

Identification of 9 novel FBN1 mutations in German patients with Marfan syndrome

We report 9 new mutations in German patients presenting with classical Marfan syndrome. All mutations occur in exons with calcium‐binding (cb) epidermal growth factor‐like (EGF) domains. Five mutations are missense involving exons 12, 27, 30, 44, and 52 with the resultant substitution of cysteine by phenylalanine (C504F), cysteine by tyrosine (C1129Y), tyrosine by cysteine (Y1261C), cysteine by serine (C1833S), and cysteine by tyrosine (C2142Y), respectively. The other four mutations are single base deletions in exons 39, 43, 48, and 58, at nucleotide A4826, C5311, T6018, and A7291, respectively, each resulting in frameshift with premature termination. Four mutations were detected in sporadic cases and are likely to be de novo. Hum Mutat 14:181, 1999. © 1999 Wiley‐Liss, Inc.     

Mutation analysis in patients with Wilson disease: Identification of 4 novel mutations

In order to obtain novel mutations in the recently discovered Wilson disease gene, we screened 5 unrelated German individuals for mutations in the 21 exons and their flanking intronic sequences. We detected 9 mutations affecting the Wilson disease gene. Four of those, designated 802‐808delTGTAAGT, 2008‐2013delTATATG, Cys985Thr, and Ile1148Thr have not yet been reported. One patient had a homozygous mutation whereas the remaining four subjects were compound heterozygous. Therefore these data confirm, that mutations causing Wilson disease are frequently found in affected subjects and they are very heterogenous. Hum Mutat 14:88, 1999. © 1999 Wiley‐Liss, Inc.     

Identification of novel mutations in Spanish patients with muscle carnitine palmitoyltransferase II deficiency

Carnitine palmitoyltransferase II (CPT II) deficiency is the most common recessively inherited disorder of lipid metabolism affecting skeletal muscle and the most frequent cause of hereditary myoglobinuria. We studied 5 Spanish patients with CPT II deficiency from four unrelated families. Four patients had the typical clinical phenotype of muscle CPT II deficiency with recurrent episodes of myoglobinuria, triggered by prolonged exercise, fasting, or fever, and marked elevation of creatine kinase values during metabolic crisis. One patient had exercise-related myalgia, cramps and moderate elevation of serum CK values, but had never had myoglobinuria. Molecular analysis showed that three patients were heterozygous for the S113L mutation and one patient heterozygous for the P50H substitution. To identify the mutations in the other alleles of our patients we amplified and sequenced genomic DNA fragments encompassing the entire coding region and intron/exon boundaries of the CPT2 gene. We found the recently reported 178 insT/del 25 bp in one patient. Three novel mutations were identified: a Y120C substitution that leads to a nonconservative amino acid replacement; a 36-38 insGC mutation that results in premature termination of the translation; and an I502T substitution that affects a conserved amino acid residue in the CPT II protein. Our data confirm the molecular heterogeneity of patients with CPT II deficiency, and suggest that the ethnic origin has to be taken into account before performing mutation analysis in these patients.     

Identification of novel SDHD mutations in patients with phaeochromocytoma and/or paraganglioma

Familial paraganglioma is a dominantly inherited disorder characterised by the development of highly vascular tumours in the head and neck. Recently, a relationship between hereditary tumours derived from the autonomic nervous system and germline mutations in the gene encoding succinate dehydrogenase complex subunit D (SDHD) is increasingly a subject of study. Familial paraganglioma syndrome is embryologically related to phaeochromocytoma, another neuroendocrine tumour that shows great aetiological and genetic heterogeneity. Some hereditary phaeochromocytomas may be associated with germline mutations in VHL, RET and NF1 genes in genetic disorders such as von Hippel-Lindau disease (VHL), multiple endocrine neoplasia type 2 (MEN 2) and neurofibromatosis type 1 (NF 1), respectively. However, there are many cases that cannot be explained by mutations in these genes. In this report, we describe two previously unreported mutations in two patients from 25 unrelated kindreds with phaeochromocytoma and/or paraganglioma disorders and with or without familial antecedents: a mutation featuring the change of tryptophan to a termination codon in exon 2, and a 4-bp deletion in exon 4 that results in a truncated protein. We also describe one missense substitution of uncertain significance. The patients had previously tested negative for germline mutations in VHL and RET genes and had not been previously selected. The involvement of SDHD mutations in familial phaeochromocytoma and/or paraganglioma predisposition is of considerable interest since other studies have shown these alterations to be associated with highly expressed angiogenic factors.     

Identification of novel mutations in the RB1 gene in Mexican patients with retinoblastoma

Retinoblastoma (RB) is a childhood tumor of the eye with an average incidence of one case in every 15,000-20,000 live births and occurs in sporadic or hereditary form. This cancer results from loss or inactivation of the RB1 gene located at 13q14.1. This gene encodes for a 110 Kd nuclear phosphoprotein (pRB) that plays a major role in cell proliferation control. Different types of mutations in the RB1 gene have been reported, but point mutations are the most common. There are no molecular studies on RB1 gene mutation in Mexican patients. In this study, 19 patients with bilateral or unilateral RB were analyzed. Genetic and cytogenetic studies were carried out. Detection of RB1 gene mutations was done using single-strand conformational polymorphism (SSCP). Five conformational polymorphisms were identified in different exons. In all cases, SSCP sequence showed new non-described mutations that produced a frameshift on the open reading frame. The identification of mutations in the RB1 gene contributes to basic knowledge of this neoplasia and permits the possibility to offer adequate genetic counseling to relatives at risk.     

Identification of 2 novel ANTXR2 mutations in patients with hyaline fibromatosis syndrome and proposal of a modified grading system

Juvenile hyaline fibromatosis (JHF) and infantile systemic hyalinosis (ISH) are rare, autosomal recessive disorders of the connective tissue caused by mutations in the gene encoding the anthrax toxin receptor 2 protein (ANTXR2) located on chromosome 4q21. Characteristically, these conditions present with overlapping clinical features, such as nodules and/or pearly papules, gingival hyperplasia, flexion contractures of the joints, and osteolytic bone defects. The present report describes a pair of sibs and three other JHF/ISH patients whose diagnoses were based on typical clinical manifestations and confirmed by histopathologic analyses and/or molecular analysis. A comparison of ISH and JHF, additional thoughts about new terminology (hyaline fibromatosis syndrome) and a modified grading system are also included.     

Identification of 3 novel VHL germ-line mutations in Danish VHL patients

ABSTRACT: BACKGROUND: von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome in which the patients develop retinal and central nervous system hemangioblastomas, pheochromocytomas and clear-cell renal tumors. The autosomal dominant disease is caused by mutations in the VHL gene. METHODS: VHL mutational analysis was carried out by sequencing of the coding sequence and by multiplex ligation-dependent probe amplification analysis. The functional consequence of the variants was investigated using in silico prediction tools. RESULTS: A total of 289 probands suspected of having VHL syndrome have been screened for mutations in the VHL gene. Twenty-six different VHL mutations were identified in 36 families including one in-frame duplication, two frame-shift mutations, four nonsense mutations, twelve missense mutations, three intronic mutations and four large genomic rearrangements. Three of these mutations (c.319 C > T, c.342_343dupGGT and c.520_521dupAA) were novel. CONCLUSIONS: In this study we report the VHL germ-line mutations found in Danish families. We found three novel VHL mutations where two were classified as pathogenic and the latter was classified as a variant of unknown significance. Together, our findings contribute to the interpretation of the potential pathogenicity of VHL germ-line mutations.     

Identification of three novel mutations of the noggin gene in patients with fibrodysplasia ossificans progressiva

We report noggin mutations in three Spanish families with fibrodysplasia ossificans progressiva (FOP). The three propositi have typical FOP findings; in the first and third families the parents are unaffected, while in the second family the father is partially affected. DNA of the three propositi and their parents was screened by sequencing for mutations in the noggin gene (NOG). Sequencing indicated a G to C mutation at nucleotide 274 of the NOG gene in the first propositus, encoding for the G92R substitution at the peptide level; this first mutation is de novo, the corresponding change not being observed in parents. In the second propositus, a G to T mutation at nucleotide 271 encodes for the G91C substitution, transmitted in the corresponding family by the partially affected father. In the third propositus, sequencing indicated a G to A mutation at nucleotide 275, encoding for the G92E substitution; this third mutation is de novo. All three mutations, as well as the Delta42 deletion already reported, resulted in the alteration of the portion of the NOG gene at positions 265-282, encoding for the potential N-myristoylation site at residues 89-GGGGGA-94.