Characterization of a partial pseudogene homologous to the Hermansky-Pudlak syndrome gene HPS-1; relevance for mutation detection

The HPS-1 gene is the first gene found to be responsible for the autosomal recessive disorder Hermansky-Pudlak syndrome (HPS). HPS is characterized by oculocutaneous albinism, a platelet storage pool deficiency, and ceroid lipofuscinosis. The HPS-1 gene has been mapped to chromosome 10q23.1-23.3 and encodes a 79-kDa protein of unknown function with no homology to any known protein. A sequence database search has revealed that a portion of clone HS 1119A7 shows high sequence similarity to HPS-1 cDNA. By performing sequence alignments and PCR amplification of cDNA from several human tissues, we have shown that part of this clone consists of an unprocessed partial HPS-1 pseudogene located on chromosome 22q12.2-12.3. The pseudogene contains several intact HPS-1 exons and shows 95% sequence homology to the HPS-1 cDNA. Exon 6 of the pseudogene has 100% sequence homology to exon 6 of HPS-1 itself. In the pseudogene, this exon is surrounded by portions of both its normal flanking introns. These data provide the first characterization of an HPS-1 pseudogene, called HPS1-psi1. During amplification of exon 6 of the HPS-1 gDNA for mutation identification, the pseudogene might also be amplified, leading to a false positive for mutation. In addition, amplification of specific parts of the HPS-1 cDNA (e.g., exons 2-5) for mutation detection might lead to false positives for mutations, if the cDNA is contaminated with gDNA. This calls for caution when employing these screening approaches.     

Familial posterior fossa brain tumors of infancy secondary to germline mutation of the hSNF5 gene

We have identified a family afflicted over multiple generations with posterior fossa tumors of infancy, including central nervous system (CNS) malignant rhabdoid tumor (a subset of primitive neuroectodermal tumors, or PNET) and choroid plexus carcinoma. Various hereditary tumor syndromes, including Li-Fraumeni syndrome, Gorlin syndrome, and Turcot syndrome, have been linked to increased risk of developing CNS PNETs and choroid plexus tumors. Malignant rhabdoid tumors of the CNS and kidney show loss of heterozygosity at chromosome 22q11. The hSNF5 gene on chromosome 22q11 has recently been identified as a candidate tumor-suppressor gene in sporadic CNS and renal malignant rhabdoid tumors. We describe a family in which both affected and some unaffected family members were found to have a germline splice-site mutation of the hSNF5 gene, leading to exclusion of exon 7 from the mature cDNA and a subsequent frameshift. Tumor tissue shows loss of the wild-type hSNF5 allele, in keeping with a tumor-suppressor gene. These findings suggest that germline mutations in hSNF5 are associated with a novel autosomal dominant syndrome with incomplete penetrance that predisposes to malignant posterior fossa brain tumors in infancy.     

Clinical and molecular studies in two families with Fraser syndrome: a new FRAS1 gene mutation, prenatal ultrasound findings and implications for genetic counselling

Fraser syndrome is a rare autosomal recessive genetic disorder characterized by cryptophthalmus, variable expression of cutaneous syndactyly of fingers and toes, genital ambiguity and renal agenesis/dysgenesis. We present here molecular and clinical findings of four fetuses with FS from two families. Molecular genetic studies in the two families revealed mutations in FRAS1 gene allowing better genetic counselling and subsequent prenatal diagnosis in one of the two families. In family one, a nonsense mutation (c.3730C>T, p.R1244X) previously described in a Polish patient was found. In family two a novel nonsense mutation previously not known was detected (c.370C>T, p.R124X). PGD is planned for family 1.     

Disease-causing Mutations in Exon 11 of the Medium-Chain Acyl-CoA Dehydrogenase Gene

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most commonly recognized defect of the mitochondrial β-oxidation in humans. It is a potentially fatal, autosomal recessive inherited defect. Most patients with MCAD deficiency are homozygous for a single disease-causing mutation (G985), causing a change from lysine to glutamate at position 304 (K304E) in the mature MCAD. Only seven non-G985 mutations, all of which are rare, have been reported. Because the G985 mutation and three of the non-G985 mutations are located in exon 11, it has been suggested that this exon may be a mutational hot spot. Here we describe the results from sequence analysis of exon 11 and part of the flanking introns from 36 compound heterozygous patients with MCAD deficiency. We have identified four previously unknown disease-causing mutations (M301T, S311R, R324X, and E359X) and two silent mutations in exon 11. Our results show that exon 11 is not especially mutation prone. We demonstrate that two of the identified disease-causing mutations can be detected by restriction enzyme digestion of the PCR product from the assay for the G985 mutation, a discovery that makes this assay even more useful than before. On the basis of expression of wild-type and mutant MCAD protein in COS-7 cells, we show that the identified mutations abolish MCAD enzyme activity and that they therefore must be disease causing. The M301T, S311R, and K304E mutations are located in helix H, which makes up part of the dimer-dimer interface of the MCAD tetramer. On the basis of the three-dimensional structure of MCAD and the results from the COS-7 expression experiments, we speculate that the primary effect of the M301T and S311R mutations is on correct folding/tetramer assembly, as it has previously been observed for the K304E mutation.     

A new autosomal recessive form of Stickler syndrome is caused by a mutation in the COL9A1 gene

Stickler syndrome is characterized by ophthalmic, articular, orofacial, and auditory manifestations. It has an autosomal dominant inheritance pattern and is caused by mutations in COL2A1, COL11A1, and COL11A2. We describe a family of Moroccan origin that consists of four children with Stickler syndrome, six unaffected children, and two unaffected parents who are distant relatives (fifth degree). All family members were clinically investigated for ear, nose, and throat; ophthalmologic; and radiological abnormalities. Four children showed symptoms characteristic of Stickler syndrome, including moderate-to-severe sensorineural hearing loss, moderate-to-high myopia with vitreoretinopathy, and epiphyseal dysplasia. We considered the COL9A1 gene, located on chromosome 6q13, to be a candidate gene on the basis of the structural association with collagen types II and XI and because of the high expression in the human inner ear indicated by cDNA microarray. Mutation analysis of the coding region of the COL9A1 gene showed a homozygous R295X mutation in the four affected children. The parents and four unaffected children were heterozygous carriers of the R295X mutation. Two unaffected children were homozygous for the wild-type allele. None of the family members except the homozygous R295X carriers had any signs of Stickler syndrome. Therefore, COL9A1 is the fourth identified gene that can cause Stickler syndrome. In contrast to the three previously reported Stickler syndrome-causing genes, this gene causes a form of Stickler syndrome with an autosomal recessive inheritance pattern. This finding will have a major impact on the genetic counseling of patients with Stickler syndrome and on the understanding of the pathophysiology of collagens. Mutation analysis of this gene is recommended in patients with Stickler syndrome with possible autosomal recessive inheritance.     

Identification of one novel mutation in the EVC2 gene in a Chinese family with Ellis–van Creveld syndrome

Ellis–van Creveld syndrome (EvC) is a rare autosomal recessive skeletal dysplasia characterized by short limbs, short ribs, postaxial polydactyly, and dysplastic nails and teeth. It is caused by biallelic mutations in the EVC or EVC2 gene. Here, we identified a novel nonsense mutation p.W828X (c.2484G>A) in exon 14 and a recurrent nonsense mutation p. R399X (c.1195C>T) in exon 10 of EVC2 gene in a Chinese boy with EvC. Identification of a novel genotype in EvC will provide clues to the phenotype–genotype relations and may assist not only in the clinical diagnosis of EvC but also in the interpretation of genetic information used for prenatal diagnosis and genetic counseling.     

Analysis of two Arab families reveals additional support for a DFNB2 nonsyndromic phenotype of MYO7A

Variants in the head and tail domains of the MYO7A gene, encoding myosin VIIA, cause Usher syndrome type 1B (USH1B) and nonsyndromic deafness (DFNB2, DFNA11). In order to identify the genetic defect(s) underling profound deafness in two consanguineous Arab families living in UAE, we have sequenced a panel of 19 genes involved in Usher syndrome and nonsyndromic deafness in the index cases of the two families. This analysis revealed a novel homozygous insertion of AG (c.1952_1953insAG/p.C652fsX11) in exon 17 of the MYO7A gene in an Iraqi family, and a homozygous point mutation (c.5660C>T/p.P1887L) in exon 41 affecting the same gene in a large Palestinian family. Moreover, some individuals from the Palestinian family also harbored a novel heterozygous truncating variant (c.1267C>T/p.R423X) in the DFNB31 gene, which is involved in autosomal recessive nonsyndromic deafness type DFNB31 and Usher syndrome type II. Assuming an autosomal recessive mode of inheritance in the two inbred families, we conclude that the homozygous variants in the MYO7A gene are the disease-causing mutations in these families. Furthermore, given the absence of retinal disease in all affected patients examined, particularly a 28 year old patient, suggests that at least one family may segregate a DFNB2 presentation rather than USH1B. This finding further supports the premise that the MYO7A gene is responsible for two distinct diseases and gives evidence that the p.P1887L mutation in a homozygous state may be responsible for nonsyndromic hearing loss.     

A novel mutation of the doublecortin gene in Japanese patients with X-linked lissencephaly and subcortical band heterotopia

The doublecortin (DCX) gene was recently found to be involved in patients with X-linked lissencephaly and subcortical band heterotopia or double cortex syndrome. We have studied the coding regions of the DCX gene in 11 Japanese patients with cortical dysplasia and have identified three different mutations (R186C in exon 3, R272X and R303X in exon 5) in four sporadic female cases. R272X, which has been detected in two unrelated cases, is a novel mutation. Although the number of cases studied remains limited, exon 5 may be a common mutational site in Japanese patients in contrast to many previus reports concerning exons 2 and 3. Received: 28 October 1998 / Accepted: 26 February 1999     

Hypobetalipoproteinemia with an apparently recessive inheritance due to a "de novo" mutation of apolipoprotein B

Familial hypobetalipoproteinemia (FHBL) is a co-dominant disorder either linked or not linked to apolipoprotein (apo) B gene. Abetalipoproteinemia (ABL) is a recessive disorder due to mutations of microsomal triglyceride transfer protein (MTP) gene. We investigated a patient with apparently recessive hypobetalipoproteinemia consistent with symptomatic heterozygous FHBL or a "mild" form of ABL. The proband had fatty liver associated with LDL-cholesterol (LDL-C) and apo B levels <5th percentile but no truncated apo B forms detectable in plasma. MTP gene sequence revealed that he was a carrier of the I128T polymorphism and an unreported amino acid substitution (V168I) unlikely to be the cause of hypobetalipoproteinemia. Apo B gene sequence showed that he was heterozygous for two single base substitutions in exon 9 and 22 resulting in a nonsense (Q294X) and a missense (R1101H) mutation, respectively. Neither of his parents carried the Q294X; his father and paternal grandmother carried the R1101H mutation. Analysis of polymorphic genetic markers excluded non-paternity. In conclusion, the proband has a "de novo" mutation of apo B gene resulting in a short truncated apo B form (apo B-6.46). Sporadic cases of FHBL with an apparently recessive transmission may be caused by "de novo" mutations of apo B gene.     

Three children with triple A syndrome due to a mutation (R478X) in the AAAS gene

OBJECTIVE: To investigate the phenotype and genotype of 3 unrelated children with triple A syndrome from southern Turkey. METHODS: The coding sequence of the AAAS gene was sequenced including exon-intron boundaries. Haplotype analysis using markers from AAAS region was performed in order to assess potential founder effects. RESULTS: In all 3 patients, the identical nonsense mutation (R478X) in exon 16 of the AAAS gene was identified. The patients who may be distantly related appeared phenotypically similar with the classical triad of the triple A syndrome (adrenal insufficiency, alacrima and achalasia) with dermatological manifestations while lacking neurological features except for mild mental retardation. CONCLUSION: The R478X mutation tends to result in a rather severe phenotype although genotype-phenotype relationships cannot be drawn due to the small number of patients.     

Novel and recurrent tyrosine aminotransferase gene mutations in tyrosinemia typeII

Tyrosinemia typeII (Richner-Hanhart syndrome, RHS) is a disorder of autosomal recessive inheritance characterized by keratitis, palmoplantar hyperkeratosis, mental retardation, and elevated blood tyrosine levels. The disease results from deficiency in hepatic tyrosine aminotransferase (TAT). We have previously described one deletion and six different point mutations in four RHS patients. We have now analyzed the TAT genes in a further seven unrelated RHS families from Italy, France, the United Kingdom, and the United States. We have established PCR conditions for the amplification of all twelve TAT exons and have screened the products for mutations by direct sequence analysis or by first performing single-strand conformation polymorphism analysis. We have thus identified the presumably pathological mutations in eight RHS alleles, including two nonsense mutations (R57X, E411X) and four amino acid substitutions (R119W, L201R, R433Q, R433W). Only the R57X mutation, which was found in one Scottish and two Italian families, has been previously reported in another Italian family. Haplotype analysis indicates that this mutation, which involves a CpG dinucleotide hot spot, has a common origin in the three Italian families but arose independently in the Scottish family. Two polymorphisms have also been detected, viz., a protein polymorphism, P15S, and a silent substitution S103S (TCG→TCA). Expression of R433Q and R433W demonstrate reduced activity of the mutant proteins. In all, twelve different TAT gene mutations have now been identified in tyrosinemia typeII. Received: 8 October 1997 / Accepted: 29 October 1997     

A novel AAAS gene mutation (p.R194X) in a patient with triple A syndrome

OBJECTIVE: The clinical and molecular data of a patient with triple A syndrome are reported. PATIENT: A 21-year-old male who was diagnosed for adrenal insufficiency at the age of 2 years after a severe attack of adrenal crisis. At the age of 4 years, achalasia and alacrima were diagnosed. Puberty started at the age of 17 years. At the same time, symptoms of central, peripheral, and autonomic nervous system dysfunction were noted. Later on, at the age of 20 years, a bone age delay of 6 years and severe osteoporosis was diagnosed. RESULTS: A compound heterozygous AAAS mutation consisting of two mutations was found: a C > T transition in exon 7 resulting in a change of arginine at amino acid position 194 into a stop codon (Arg194X) at one allele, and a C > T transition in exon 12 resulting in a change of glutamine at amino acid position 387 into a stop codon (Gln387X) on the other allele. CONCLUSION: The mutation in exon 7 (p.R194X) of the AAAS gene is a novel mutation which has not been found in any other family so far, whereas the second was already found in some other families. This case adds to the clinical and molecular spectrum of triple A syndrome and may provide a new insight into the functions of AAAS gene.     

The γ-Crystallins and Human Cataracts: A Puzzle Made Clearer

Despite the fact that cataracts constitute the leading cause of blindness worldwide, the mechanisms of lens opacification remain unclear. We recently mapped the aculeiform cataract to the gamma-crystallin locus (CRYG) on chromosome 2q33-35, and mutational analysis of the CRYG-genes cluster identified the aculeiform-cataract mutation in exon 2 of gamma-crystallin D (CRYGD). This mutation occurred in a highly conserved amino acid and could be associated with an impaired folding of CRYGD. During our study, we observed that the previously reported Coppock-like-cataract mutation, the first human cataract mutation, in the pseudogene CRYGE represented a polymorphism seen in 23% of our control population. Further analysis of the original Coppock-like-cataract family identified a missense mutation in a highly conserved segment of exon 2 of CRYGC. These mutations were not seen in a large control population. There is no direct evidence, to date, that up-regulation of a pseudogene causes cataracts. To our knowledge, these findings are the first evidence of an involvement of CRYGC and support the role of CRYGD in human cataract formation.     

Mutations in argininosuccinate synthetase mRNA of Japanese patients,causing classical citrullinemia.

Citrullinemia is an autosomal recessive disease caused by a genetic deficiency of argininosuccinate synthetase. In order to characterize mutations in Japanese patients with classical citrullinemia, RNA isolated from 10 unrelated patients was reverse-transcribed, and cDNA amplified by PCR was cloned and sequenced. The 10 mutations identified included 6 missense mutations (A118T, A192V, R272C, G280R, R304W, and R363L), 2 mutations associated with an absence of an exon 7 or exon 13, 1 mutation with a deletion of the first 7 bp in exon 16 (which might be caused by abnormal splicing), and 1 mutation with an insertion of 37 bp within exons 15 and 16 in cDNA. The insertion mutation and the five missense mutations (R304W being excluded) are new mutations described in the present paper. These are in addition to 14 mutations (9 missense mutations, 4 mutations associated with an absence of an exon in mRNA, and 1 splicing mutation) that we identified previously in mainly American patients with neonatal citrullinemia. Two of these 20 mutations, a deletion of exon 13 sequence and a 7-bp deletion in exon 16, were common to Japanese and American populations from different ethnic backgrounds; however, other mutations were unique to each population. Furthermore, the presence of a frequent mutation--the exon 7 deletion mutation in mRNA, which accounts for 10 of 23 affected alleles--was demonstrated in Japanese citrullinemia. This differs from the situation in the United States, where there was far greater heterogeneity of mutations.     

Identification of a candidate missense mutation in a family with von Willebrand disease type IIC

A screening project to identify candidate molecular defects causing von Willebrand disease type IIC (VWD IIC) in a German family was carried out using polymerase chain reaction (PCR) amplification of all 52 exons of the von Willebrand factor (VWF) gene, subsequent electrophoresis of single and double stranded DNA and direct sequencing of PCR products with aberrant electrophoretic patterns. Only one candidate mutation, G550R, caused by a GA transition, was detected in exon 14 of the pro-VWF gene sequence. This mutation was not found on 200 chromosomes of normal individuals. The propositus was homozygous for the mutation and for an extended intragenic haplotype, composed of eight polymorphic markers. Further family members were heterozygous for the mutation and were phenotypically normal or only mildly affected, in accordance with the recessive pattern of inheritance for VWD type IIC. The mutation could influence one of the presumed active centers for the suspected multimerizing enzymatic activity of pro-VWF localized in the D1 and D2 domain, which corresponds to exon 5 and exon 14 of the VWF gene.     

Three novel germline mutations in MLH1 and MSH2 in families with Lynch syndrome living on Jeju island, Korea

Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominant syndrome characterized by predisposition to early-onset cancers. HNPCC is caused by heterozygous loss-of-function mutations within the mismatch repair genes MLH1, MSH2, MSH6, PMS1, and PMS2. We genotyped the MLH1 and MSH2 genes in patients suffering from Lynch syndrome and in 11 unrelated patients who were diagnosed with colorectal cancer and had subsequently undergone surgery. Five Lynch syndrome patients carried germline mutations in MLH1 or MSH2. Two of these were identified as known mutations in MLH1: deletion of exon 10 and a point mutation (V384D). The remaining three patients exhibited novel mutations: a duplication (937_942dupGAAGTT) in MLH1; deletion of exons 8, 9, and 10; and a point mutation in MLH1 (F396I) combined with multiple missense mutations in MSH2 (D295G, K808E, Q855P, and I884T). The findings underline the importance of efficient pre-screening of conspicuous cases.