Linkage exclusion and mutational analysis of the noggin gene in patients with fibrodysplasia ossificans progressiva (FOP)

Fibrodysplasia ossificans progressiva (FOP) is an extremely rare and disabling genetic disorder characterized by congenital malformation of the great toes and by progressive heterotopic endochondral ossification in predictable anatomical patterns. Although elevated levels of bone morphogenetic protein 4 (BMP4) occur in lymphoblastoid cells and in lesional cells of patients with FOP, mutations have not been identified in the BMP4 gene, suggesting that the mutation in FOP may reside in a BMP4-interacting factor or in another component of the BMP4 pathway. A powerful antagonist of BMP4 is the secreted polypeptide noggin. A recent case report described a heterozygous 42-bp deletion in the protein-coding region of the noggin gene in a patient with FOP. In order to determine if noggin mutations are a widespread finding in FOP, we examined 31 families with 1 or more FOP patients. Linkage analysis with an array of highly polymorphic microsatellite markers closely linked to the noggin gene was performed in four classically-affected multigenerational FOP families and excluded linkage of the noggin locus to FOP (the multipoint lod score was -2 or less throughout the entire range of markers). We sequenced the noggin gene in affected members of all four families, as well as in 18 patients with sporadic FOP, and failed to detect any mutations. Single-strand conformation polymorphism (SSCP) analysis of 4 of these patients plus an additional 9 patients also failed to reveal any mutations. Among the samples analyzed by SSCP and DNA sequencing was an independently obtained DNA sample from the identical FOP patient previously described with the 42-bp noggin deletion; no mutation was detected. Examination of the DNA sequences of 20 cloned noggin PCR products, undertaken to evaluate the possibility of a somatic mutation in the noggin gene which could be carried by a small subset of white blood cells, also failed to detect the presence of the reported 42-bp deletion. We conclude that mutations in the coding region of noggin are not associated with FOP.     

Identification of a novel NOG gene mutation (P35S) in an Italian family with symphalangism

Symphalangism (SYM or SYM1) is an autosomal dominant disorder characterized by multiple joint fusions. The disease is caused by mutations of the NOG gene, that maps to chromosome 17q22. So far, only six independent NOG mutations have been identified. We have analysed an Italian family in which father and son had bilateral symphalangism and detected a novel NOG mutation (P35S), originated in the father from a c.914C>T transition. A different mutation in the same codon (P35R) has been previously described. Comparison between different noggin gene hortologs shows that codon 35 is conserved. Therefore, this codon should play an important role in NOG gene function. This is the first mutation described for NOG after the initial report of NOG mutations being causative of SYM.     

Who is a carrier? Detection of unsuspected mutations in 21-hydroxylase deficiency

Congenital adrenal hyperplasia due to 21-hydroxylase deficiency is a common autosomal-recessive disorder. During our routine genotyping of affected individuals and their relatives using allele-specific oligonucleotide hybridization and single-strand conformational polymorphism analysis, we identified two families each segregating three mutations. In both families, a mutation known to be associated with 21-hydroxylase deficiency was identified in healthy individuals but was not detected in the propositus. The propositus in family 1 was shown to be a homozygous carrier for G at nucleotide 655, which alters the splice acceptor site at exon 3. The propositus in family 2 carried the same splicing mutation on the maternal allele and a gene deletion/conversion on the paternal allele. In both families, other clinically unaffected relatives carried the Q318X mutation in exon 8. If molecular diagnostic studies had been limited to the mutation carried by the propositi, relatives would have been misinformed regarding their status as carriers or mildly affected individuals. The findings in these two families emphasize the high frequency of alleles causing 21-hydroxylase deficiency in the population. © 1996 Wiley-Liss, Inc.     

双重杂合性突变Arg304Gln和Arg304Trp导致的遗传性凝血因子Ⅶ缺陷症

目的 探讨1例遗传性凝血因子Ⅶ(coagulation factorⅦ，FⅦ)缺陷症及其家系基因突变的类型。方法 检测凝血指标以明确诊断；用DNA直接测序法对先证者及其家庭成员FⅦ基因的全部外显子和其侧翼以及启动子进行分析，寻找基因突变；将含突变序列克隆人pGEM T—easy质粒载体中，对所得两条染色体相应序列分别测序，以确定不同突变在染色体上的分布。应用限制性内切酶Msp Ⅰ对先证者及家系成员相应基因片段进行酶切分析，证实测序所发现的突变。结果 先证者在第8外显子上有两种基因突变：11348位C→T突变和11349位G→A突变。pGEM T—easy质粒克隆测序结果显示上述两种突变位于不同的染色体上。为不同染色体同一编码区Arg(CGG)304Trp(TGG)和Arg(CGG)304Gln(CAG)双重杂合性突变。其父亲、母亲分别为11349位G→A和11348位C→T杂合突变；其弟弟FⅦ基因为正常野生型；其哥哥和先证者的3个子女均为杂合性突变。聚合酶链反应辅助限制性酶切证实了先证者及其家系成员的基因突变。结论 先证者FⅦ基因突变为不同染色体同一编码区Arg304Trp和Arg304Gln双重杂合性突变，此种突变类型的组合尚属首例。     

A novel cis-AB allele derived from the A transferase gene by nucleotide substitution C796A

The cis-AB is a very rare phenotype in the ABO blood group system. It corresponds to a special ABO allele encoding a glycosyltransferase that is capable of synthesizing both A and B antigens. Until now, gene sequences of only 3 cis-AB alleles were characterized. One was the A(1v) allele with a nucleotide substitution G803C at codon 268; the second was the B allele with a nucleotide substitution A796C at codon 266; and the third arose from a point mutation C700T at codon 234 in exon 7 of the B transferase gene. In this study, we found a novel cis-AB allele when performing paternity tests in Chang Gung Memorial Hospital in Taiwan. Although his father was O blood type, a serologically AB blood type child was confirmed as being his father's offspring on the basis of 16 microsatellite markers (99.97% plausibility for the child and father). Exons 6 and 7 of the child's ABO alleles were characterized by direct sequencing and gene cloning. The results showed that the child has one O(1) allele and the second allele is almost identical to A(1*02) allele except for a single point mutation at nucleotide position 796, where an A replaces a C and leads to a change of leucine to methionine at amino acid 266. This implies that the child's O(1) allele was inherited from his father and the other allele was inherited from his mother. In conclusion, the novel cis-AB allele reported here is derived from the A transferase gene through a nucleotide substitution C796A, which differs from the 3 previously reported cis-AB alleles.     

Hereditary coproporphyria: Exon screening by heteroduplex analysis detects three novel mutations in the coproporphyrinogen oxidase gene

Hereditary coproporphyria is a dominantly inherited disorder of porphyrin metabolism caused by a partial deficiency of coproporphyrinogen oxidase, the sixth enzyme in the heme synthetic pathway. We investigated the molecular basis of hereditary coproporphyria in three unrelated patients, amplifying each exon of the coproporphyrinogen oxidase gene and performing heteroduplex analysis to look for mutations. Unique heteroduplex patterns were noted in exons 2, 3, and 6. Sequencing revealed different mutations in each patient: a G→A point mutation encoding a glutamic acid to lysine substitution at codon 101 (E101K), a C→T point mutation encoding a proline to serine substitution at codon 149 (P149S), and a one base-pair insertion in exon 6 (968insT). No other mutations were found on sequencing the remaining exons and their intron-exon junctions. The two point mutations affect amino acids that are conserved in all species studied to date. The one base-pair insertion in exon 6 is the first frameshift mutation to be described in the coproporphyrinogen oxidase gene. This study adds three new mutations to those that have been previously reported, and all have been restricted to single families. These results indicate that hereditary coproporphyria is a genetically heterogeneous disease. Hum Mutat 10:196–200, 1997. © 1997 Wiley-Liss, Inc.     

A comprehensive review of reported heritable noggin-associated syndromes and proposed clinical utility of one broadly inclusive diagnostic term: NOG-related-symphalangism spectrum disorder (NOG-SSD)

The NOG gene encodes noggin, a secreted polypeptide that is important for regulating multiple signaling pathways during human development, particularly in cartilage and bone. The hallmark of NOG-related syndromes is proximal symphalangism, defined by abnormal fusion of the proximal interphalangeal joints of the hands and feet. Many additional features secondary to NOG mutations are commonly but inconsistently observed, including a characteristic facies with a hemicylindrical nose, congenital conductive hearing loss due to stapes fixation, and hyperopia. The variable clinical presentations led to the designation of five different autosomal dominant syndromes, all subsequently found to have resulted from NOG mutations. These include (1) proximal symphalangism; (2) multiple synostoses syndrome 1; (3) stapes ankylosis with broad thumbs and toes; (4) tarsal-carpal coalition syndrome; and (5) brachydactyly type B2. Herein, we review the phenotypic features associated with mutations in the NOG gene, demonstrating the overlapping characteristics of these syndromes. Due to the variable phenotypic spectrum within families and among families with the same mutation, we propose a unifying term, NOG-related symphalangism spectrum disorder (NOG-SSD), to aid in the clinical recognition and evaluation of all affected individuals with these phenotypes. These NOG gene variants are available in a new locus-specific database (https://NOG.lovd.nl).     

Mutations of the NOG gene in individuals with proximal symphalangism and multiple synostosis syndrome

Proximal symphalangism is an autosomal-dominant disorder with ankylosis of the proximal interphalangeal joints, carpal and tarsal bone fusion, and conductive deafness. These symptoms are shared by another disorder of joint morphogenesis, multiple synostoses syndrome. Recently, it was reported that both disorders were caused by heterozygous mutations of the human noggin gene (NOG). To date, seven mutations of NOG have been identified from unrelated families affected with joint morphogenesis. To characterize the molecular lesions of proximal symphalangism, we performed analyses of NOG in three Japanese individuals with proximal symphalangism. We found three novel mutations: g.551G>A (C184Y) in a sporadic case of symphalangism, g.386T>A (L129X) in a familial case of symphalangism, and a g.58delC (frameshift) in a family with multiple synostosis syndrome. Characteristic genotype-phenotype correlations have not been recognized from the mutations in the NOG gene.     

Compound heterozygous mutations in KvLQT1 cause Jervell and Lange-Nielsen syndrome

Jervell and Lange-Nielsen syndrome (JLNS) is characterized by sensorineural deafness, QT prolongation, abnormal T waves, ventricular tachyarrhythmias, and autosomal recessive inheritance. Previously homozygous mutations in the potassium channel-encoding genes, KvLQT1 (alpha-subunit) and KCNE1 (beta-subunit), have been described in consanguineous families with JLNS. We screened two nonconsanguineous families with JLNS for mutations in KvLQT1, using single-strand conformation polymorphism analysis, denaturing high-performance liquid chromatography, and DNA sequencing. In one family, a missense mutation was identified in exon 6 of KvLQT1 on the maternal side, resulting in a glycine to aspartic acid substitution at codon 269 (G269D). The apparently normal father had an incompletely penetrant missense mutation in exon 3 of KvLQT1, introducing a premature stop codon at position 171. In the other family, a missense mutation resulting in the substitution of asparagine for aspartic acid at codon 202 (D202N) was identified in the mother and maternal grandmother, who had QTc prolongation (borderline in the mother), while the father and paternal grandfather, who were clinically normal, had a deletion of nucleotide 585, resulting in a frameshift and premature termination. In both families, the proband inherited both mutations. In this report we provide evidence that not only homozygous but also compound heterozygous mutations in KvLQT1 may cause JLNS in nonconsanguineous families. Incomplete penetrance in individuals with mutations appears to be frequent, indicating a higher prevalence of mutations than estimated previously. Interestingly, mutations resulting in truncation of the protein appear to be benign, with heterozygous carriers being asymptomatic.     

Molecular characterization of Complement Factor I deficiency in two Spanish families

Complement Factor I (CFI) is a regulator of the classical and alternative pathways. CFI has enzymatic activity and is able to cleave C3b and C4b. Homozygous Factor I deficiency is associated with infectious and/or autoimmune diseases.

Here we describe the biochemical and genetic characterization in two Spanish families with complete Factor I deficiency. In Family 1, the propositus suffered from several episodes of meningitis for more than a year. Biochemical complement studies showed undetectable Factor I levels in the propositus and in her sister, while their parents and a brother had partial Factor I deficiency and were healthy. In Family 2, three out of five children were homozygous for Factor I deficiency, two of whom suffered from meningitis and the third one from several infections. The parents and the other two siblings were healthy and heterozygous for Factor I deficiency.

Molecular studies showed that the two families had different mutations at exon 5 of the Factor I gene, which codifies for module LDLr1. One mutation corresponds to a 772G>A change at the donor splice site that was originally found in a family from Northern England. The second is a new missense mutation 739T>G, that generates a Cys to Gly change.     

Parental mosaicism can cause recurrent transmission of SCN1A mutations associated with severe myoclonic epilepsy of infancy

De novo mutations in the SCN1A gene, encoding the alpha1-subunit of the neuronal voltage-gated sodium channel Nav1.1, are the most frequent genetic cause of Severe Myoclonic Epilepsy of Infancy known so far. A few mutations inherited from an asymptomatic or mildly affected parent have been reported, suggesting that expression of the mutated gene may be variable in the transmitting parent. In this study, we report two unrelated families in which two children of unaffected parents had deleterious SCN1A mutations, and show evidence of somatic and germline mosaicism in the transmitting parents. In one of these families, direct sequencing of blood cell DNA was not sufficient to the SCN1A mutation in the transmitting asymptomatic parent who was mosaic for the mutation. We therefore developed a real-time PCR assay to selectively amplify and quantify the mutant allele present at low levels in the transmitting parent in both families. The allele-specific PCR technique used in this study will be of use in detecting other such cases. These findings will have major consequences for the genetic counseling of asymptomatic parents with only one affected child.     

Two novel mutations in NOTCH3 gene causes cerebral autosomal dominant arteriopathy with subcritical infarct and leucoencephalopathy in two Chinese families

Objective: To investigate the genetic pathogenic causes of cerebral autosomal dominant arteriopathy with subcritical infarct and leucoencephalopathy (CADASIL) in two Chinese families, to provide the molecular basis for genetic counseling and antenatal diagnosis. Methods: The genetic mutation of gene NOTCH3 of propositus and family members was analyzed in these two CADASIL families by polymerase chain reaction and DNA sequencing technology directly. At the same time, the NOTCH3 gene mutation point of 100 healthy collators was detected, to explicit the pathogenic mutation by function prediction with Polyphen-2 and SIFT. Results: Both propositus of the two families and patients with symptom were all accorded with the clinical features of CADASIL. It was shown by DNA sequencing that the 19^th exon [c. 3043 T > A (p.Cys1015Ser)] in gene NOTCH3 of propositus, 2 patients (II3, III7), and a presymptomatic patient (IV1) in Family I all had heterozygosity missense mutation; and the 3^rd exon [c.316T > G, p. (Cys106Gly)] in gene NOTCH3 of the propositus, a patient (IV3) and two presymptomatic patients (IV5, 6) in Family II all had heterozygosity missense mutation; and no mutations were detected in the 100 healthy collators. It was indicated by analyzing the function prediction that the mutation of [c. 3043 T > A (p.Cys1015Ser)] and [c.316T > G, p. (Cys106Gly)] may both influence encoding protein in NOTCH3. By analysis of the conservatism of mutation point in each species, these two basic groups were highly conserved. Conclusion: The heterozygosity missense mutation of 19^th exon [c. 3043 T > A (p.Cys1015Ser)] and the 3^rd exon [c.316T > G, p. (Cys106Gly)] in NOTCH3 gene are the new pathogenic mutations of CADASIL, and enriches the mutation spectrum of NOTCH3 gene.     

A homozygous HAMP mutation in a multiply consanguineous family with pseudo-dominant juvenile hemochromatosis

Juvenile hemochromatosis (JH) is an autosomal recessive condition that leads to significant morbidity due to early onset systemic iron overload. The majority of families with JH link to chromosome 1q and were recently found to have mutations in the HFE2 gene encoding hemojuvelin; however, several JH families have been reported to have mutations in the HAMP gene encoding hepcidin. Here, we report a multiply consanguineous family with a father and daughter showing iron overload consistent with JH. Sequence analysis of HAMP revealed homozygosity for amino acid substitution C78T due to a c.233G > A mutation. This mutation disrupts one of eight highly conserved cysteines that are believed to be critical for the function of the active enzyme. This finding adds support to the importance of the role of these conserved cysteines in the activity of hepcidin.     

Mutations in the protoporphyrinogen oxidase gene in patients with variegate porphyria

Variegate porphyria (VP) is an acute hepatic porphyria with autosomal dominant inheritance due to a partial deficiency of protoporphyrinogen oxidase (PPOX) activity. The molecular defect responsible for VP was investigated by sequencing PPOX gene coding sequence from four patients in three unrelated VP families of French Caucasian origin. In a first patient, a point insertion of a G at position 1022 of the cDNA, produced a frameshift resulting in a premature stop codon. In three other patients from two unrelated families we found a missense point mutation leading to glycine to arginine substitution (G232R) in exon 7. This Gly232 appears to be a strictly conserved residue through evolution. In one VP family, we observed the cosegregation of the G232R missense mutation and the deficient PPOX activity. The mutations reported here are the first to be described in patients with VP and support the conclusion that PPOX gene defects are disease causing mutations in human variegate porphyria.      

眼皮肤白化病Ⅰ型产前基因诊断

目的对已生育过眼皮肤白化病Ⅰ型（oculocutaneous albinism type Ⅰ，OCA1）患儿的两个家系进行酪氨酸酶（tyrosinase，TYR）基因TYR的突变研究和产前基因诊断。方法应用PCR技术扩增TYR基因各外显子、外显子．内含子交界区及启动子区，直接以DNA序列测定技术分析先证者或其父母的基因突变，明确致病性突变后，检测胎儿TYR基因相应位点的DNA序列，获知胎儿的基因型。结果家系1的先证者为R278X和929insC突变复合杂合子；胎儿未获得这2种突变等位基因，基因型和表型均正常。家系2先证者的父母分别为IVS4＋3A→T和G253E突变的杂合子，胎儿只获得了父源性的IVS4＋3A→T突变等位基因，未获得母源性G253E突变等位基因，胎儿为表型正常的致病基因携带者。结论此为中国大陆首次真正意义上的OCA1产前基因诊断；应用上述基因分析方法进行OCA1产前基因诊断是可行的。     

3家系成骨不全基因突变及临床表型分析

目的对3家系成骨不全(OI)测序及其临床表型和突变分析,提高对成骨不全的诊断和认识。方法收集临床资料,提取患者及家属血标本;高通量测序,一代测序验证和结果分析。结果先证者1,FKBP10,EXON6,c.1016GA,p.R339Q,纯合突变,父、母分别为此位点杂合突变。先证者2,COL1A1,EXON9,c.671GA,p.G224D,杂合突变,父亲为此位点杂合突变,母亲基因无变异。先证者3,COL1A1,EXON30,c.2010del T,p.P670fs,杂合突变,母亲此位点杂合突变,父亲基因无突变。结论 FKBP10新位点突变可能导致了XI型成骨不全及其新表型的发生;证实了COL1A1两位点突变和成骨不全之间的关系。     

Ile225Thr loop mutation in the lipoprotein lipase (LPL) gene is a de novo event

Mutations in the lipoprotein lipase (LPL) gene are the most important cause of familial chylomicronemia with over 70 mutations being recorded to date. Thus far de novo mutations have not been described. Here we report on the molecular analysis of the family of a patient previously reported to be LPL deficient on the basis of compound heterozygosity for the Arg²⁴³His and Ile²²⁵Thr mutations, the latter being the first and only mutation identified in the loop region of LPL. Both parents of the propositus were screened for the presence of these two mutations to confirm their status as obligate heterozygotes and to determine the mutation allocation. Although paternal inheritance of the Arg²⁴³His allele could be established, maternal DNA did not show carrier status for the Ile²²⁵Thr substitution. An examination of maternity, using LPL restriction fragment length polymorphisms four polymorphic CA repeats and ApoE genotypes, was consistent with correct biological parentage for the propositus. Therefore, we conclude that the Ile²²⁵Thr mutation constitutes a de novo event, the first to be reported in the LPL gene. Am. J. Med. Genet. 78:313–316, 1998. © 1998 Wiley-Liss, Inc.     

De novo 617G–A nucleotide mutation in the ACVR1 gene in a Taiwanese patient with fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare congenital disease with autosomal dominant transmission characterized by the presence of malformations of the big toes and of postnatal progressive heterotopic endochondral osteogenesis. We report the case of 3-year-old girl with dysplasia of the first metatarsal bones and progressive heterotopic ossificans of the right thigh due to previous diphtheria–tetanus–pertussis immunizations and several inappropriate surgical interventions. Direct sequence analysis identified a 617G–A nucleotide mutation in the patient but not in her parents or brother. Pedigree analysis suggests that a de novo mutation in the ACVR1 gene is responsible for the disease in this family. This is the first report of the results of a mutation analysis in a sporadic case of FOP in a Taiwanese patient.     

A one-base deletion (183delC) and a missense mutation (D276H) in the T-protein gene from a Japanese family with nonketotic hyperglycinemia

Two novel mutations in the gene encoding T-protein, a component of the glycine cleavage system, were identified in a Japanese family with nonketotic hyperglycinemia. The proband had two affected sibs, and enzymatic analysis of the liver sample from the proband revealed the T-protein deficiency. The first mutation, 183delC, was found in exon 1. One of six cytidine residues (base position 183–188) was deleted. The deletion was located in a coding region of the mitochondrial leader peptide and was deduced to create a truncated peptide with 94 amino acids. The second mutation was a base substitution from G to C at position 955 in exon 7. The G955C substitution caused an amino acid change from aspartate to histidine at position 276 (D276H). Aspartic acid at position 276 is evolutionarily conserved among human, bovine, chicken, and pea genes, and replaced by glutamic acid in Escherichia coli, suggesting that the presence of an acidic amino acid at 276 may be crucial for the enzymatic function. No base change other than the 183delC and the G955C was observed in the sequencing analysis. Familial analysis revealed that the 183delC and the D276H mutations were inherited from the father and the mother, respectively. This is the first report of T-protein gene mutation in Oriental patients with nonketotic hyperglycinemia. Received: December 12, 1997 / Accepted: January 16, 1998