Twenty-eight recA mutants, isolated after spontaneous mutagenesis generated by the combined action of RecA1202(Prtc) and UmuDC proteins, were characterized and sequenced. The mutations are intragenic suppressors of the recA1202 allele and were detected by the reduced coprotease activity of the gene product. Twenty distinct mutation sites were found, among which two mutations, recA1620 (V-275-->D) and recA1631 (I-284-->N), were mapped in the C-terminal portion of the interfilament contact region (IFCR) in the RecA crystal. An interaction of this region with the part of the IFCR in which the recA1202 mutation (Q-184-->K) is mapped could occur only intermolecularly. Thus, altered IFCR and the likely resulting change in interfilament association appear to be important aspects of the formation of a constitutively active RecA coprotease. This observation is consistent with the filament-bundle theory (R. M. Story, I. T. Weber, and T. A. Steitz, Nature (London) 335:318-325, 1992). Furthermore, we found that among the 20 suppressor mutations, 3 missense mutations that lead to recombination-defective (Rec-) phenotypes also mapped in the IFCR, suggesting that the IFCR, with its putative function in interfilament association, is required for the recombinase activity of RecA. We propose that RecA-DNA complexes may form bundles analogous to the RecA bundles (lacking DNA) described by Story et al. and that these RecA-DNA bundles play a role in homologous recombination.

A polypeptide chain-terminating mutation in the yeast mitochondrial oxi 1 gene has been shown to be an ochre (TAA) mutation by DNA sequence analysis. Mitochondrially inherited revertants of this mutation include two types: In the first, the ochre codon has been changed to a sense codon by further mutation in the oxi 1 gene while, in the second, the ochre codon is still present, indicating the occurrence of an extrageneic ochre suppressor mutation. This mitochondrial ochre suppressor, termed MSU1, has been "cloned" in rho- strains of yeast and tested against other oxi 1 mutations. Several additional mutations are also suppressible, and those examined so far are also ochre mutations. MSU1 does not suppress known frameshift or missense mutations at oxi 1. Isoelectric focusing of the gene product (cytochrome oxidase subunit II) from a suppressed-mutant strain indicates that suppression does not involve insertion of charged amino acids. Physical mapping of the mtDNA retained in the MSU1-carrying rho- clones localizes the suppressor mutation to the gene coding the 15S rRNA or a site not more than 300 base pairs from it. No known tRNA genes occur this close to the 15S rRNA gene, and mtDNA from a suppressor-carrying rho- does not hybridize detectably to mitochondrial tRNAs. These results suggest that MSU1 may be an alteration in the 15S rRNA.

The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that plays a key role in extracellular calcium ion homeostasis. We have engineered 11 CaR mutants that have been described in the disorders familial benign hypercalcemia (FBH), neonatal severe hyperparathyroidism (NSHPT), and autosomal dominant hypocalcaemia (ADH), and studied their function by characterizing intracellular calcium [Ca2+]i transients in response to varying concentrations of extracellular calcium [Ca2+]o or gadolinium [Gd3+]o. The wild type receptor had an EC50 for calcium (EC50[Ca2+]o) (the value of [Ca2+]o producing half of the maximal increase in [Ca2+]i) of 4.0 mM (+/- 0.1 SEM). However, five missense mutations associated with FBH or NSHPT, (P55L, N178D, P221S, R227L, and V817I) had significantly higher EC50[Ca2+]os of between 5.5 and 9.3 mM (all P < 0.01). Another FBH mutation, Y218S, had an EC50[Ca2+]o of > 50 mM but had only a mildly attenuated response to gadolinium, while the FBH mutations, R680C and P747fs, were unresponsive to either calcium or gadolinium. In contrast, three mutations associated with ADH, (F128L, T151M, and E191K), showed significantly reduced EC50[Ca2+]os of between 2.2 and 2.8 mM (all P < 0.01). These findings provide insights into the functional domains of the CaR and demonstrate that mutations which enhance or reduce the responsiveness of the CaR to [Ca2+]o cause the disorders ADH...

More than 30 missense mutations in the beta-cardiac myosin heavy chain gene have been shown to be responsible for familial hypertrophic cardiomyopathy. To clarify the effects of these point mutations on myosin motor function, we expressed wild-type and mutant human beta-cardiac myosin heavy chains in insect cells with human cardiac light chains. The wild-type myosin was well purified with similar enzymatic and motor activities to those of the naturally isolated V3 cardiac myosin. Arg249-->Gln and Arg453-->Cys mutations resulted in decreased actin translocating activity (61 and 23% of the wild-type, respectively) with decreased intrinsic ATPase activity. Arg403-->Gln mutation greatly decreased actin translocating activity (27% of wild type) with a 3.3-fold increased dissociation constant for actin, while intrinsic ATPase activity was unchanged. Val606-->Met mutation only mildly affected the actin translocating activity as well as ATPase activity of myosin. The degree of deterioration by each mutation was closely correlated with the prognosis of the affected kindreds, indicating that myosin dysfunction caused by the point mutations is responsible for the pathogenesis of the disease. Structure/function relationship of myosin is discussed.

Five novel mutations have been identified in the gene encoding L1CAM, a neural cell adhesion protein, in families with X linked hydrocephalus (XHC). Interestingly, all five mutations are in the evolutionarily highly conserved Ig-like domains of the protein. The two frameshift mutations (52insC and 955delG) and the nonsense mutation (Trp276Ter) most probably result in functional null alleles and complete absence of L1CAM at the cell surface. The two missense mutations (Tyr194Cys and Pro240Leu) may considerably alter the structure of the L1CAM protein. These data provide convincing evidence that XHC is genetically extremely heterogeneous.

Harlequin ichthyosis (HI) is a devastating skin disorder with an unknown underlying cause. Abnormal keratinocyte lamellar granules (LGs) are a hallmark of HI skin. ABCA12 is a member of the ATP-binding cassette transporter family, and members of the ABCA subfamily are known to have closely related functions as lipid transporters. ABCA3 is involved in lipid secretion via LGs from alveolar type II cells, and missense mutations in ABCA12 have been reported to cause lamellar ichthyosis type 2, a milder form of ichthyosis. Therefore, we hypothesized that HI might be caused by mutations that lead to serious ABCA12 defects. We identify 5 distinct ABCA12 mutations, either in a compound heterozygous or homozygous state, in patients from 4 HI families. All the mutations resulted in truncation or deletion of highly conserved regions of ABCA12. Immunoelectron microscopy revealed that ABCA12 localized to LGs in normal epidermal keratinocytes. We confirmed that ABCA12 defects cause congested lipid secretion in cultured HI keratinocytes and succeeded in obtaining the recovery of LG lipid secretion after corrective gene transfer of ABCA12. We concluded that ABCA12 works as an epidermal keratinocyte lipid transporter and that defective ABCA12 results in a loss of the skin lipid barrier...

Craniofrontonasal syndrome (CFNS) is an X-linked craniofacial disorder with an unusual manifestation pattern, in which affected females show multiple skeletal malformations, whereas the genetic defect causes no or only mild abnormalities in male carriers. Recently, we have mapped a gene for CFNS in the pericentromeric region of the X chromosome that contains the EFNB1 gene, which encodes the ephrin-B1 ligand for Eph receptors. Since Efnb1 mutant mice display a spectrum of malformations and an unusual inheritance reminiscent of CFNS, we analyzed the EFNB1 gene in three families with CFNS. In one family, a deletion of exons 2–5 was identified in an obligate carrier male, his mildly affected brother, and in the affected females. In the two other families, missense mutations in EFNB1 were detected that lead to amino acid exchanges P54L and T111I. Both mutations are located in multimerization and receptor-interaction motifs found within the ephrin-B1 extracellular domain. In all cases, mutations were found consistently in obligate male carriers, clinically affected males, and affected heterozygous females. We conclude that mutations in EFNB1 cause CFNS.

Split-hand/split-foot malformation (SHFM), a limb malformation involving the central rays of the autopod and presenting with syndactyly, median clefts of the hands and feet, and aplasia and/or hypoplasia of the phalanges, metacarpals, and metatarsals, is phenotypically analogous to the naturally occurring murine Dactylaplasia mutant (Dac). Results of recent studies have shown that, in heterozygous Dac embryos, the central segment of the apical ectodermal ridge (AER) degenerates, leaving the anterior and posterior segments intact; this finding suggests that localized failure of ridge maintenance activity is the fundamental developmental defect in Dac and, by inference, in SHFM. Results of gene-targeting studies have demonstrated that p63, a homologue of the cell-cycle regulator TP53, plays a critically important role in regulation of the formation and differentiation of the AER. Two missense mutations, 724A→G, which predicts amino acid substitution K194E, and 982T→C, which predicts amino acid substitution R280C, were identified in exons 5 and 7, respectively, of the p63 gene in two families with SHFM. Two additional mutations (279R→H and 304R→Q) were identified in families with EEC (ectrodactyly, ectodermal dysplasia, and facial cleft) syndrome. All four mutations are found in exons that fall within the DNA-binding domain of p63. The two amino acids mutated in the families with SHFM appear to be primarily involved in maintenance of the overall structure of the domain...

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a monogenic autosomal disease with recessive inheritance. It is characterized by multiple autoimmune endocrinopathies, chronic mucocutaneous candidiasis, and ectodermal dystrophies. The defective gene responsible for this disease was recently isolated, and several different mutations in the novel gene, AIRE, have been identified, by us and by others, in patients with APECED. We have shown that the APECED protein is mainly localized, both in vitro and in vivo, to the cell nucleus, where it forms distinct speckles. This accords with the predicted structural features of the protein, which suggest involvement of AIRE in the regulation of gene transcription. Here, we report the results of mutational analyses of a series of 112 patients with APECED who were from various ethnic backgrounds. A total of 16 different mutations, covering 91% of disease alleles, were observed; of these, 8 were novel. The mutations are spread throughout the coding region of AIRE, yet four evident mutational hotspots were observed. In vitro expression of four different naturally occurring nonsense and missense mutations revealed a dramatically altered subcellular location of the protein in cultured cells. Interestingly...

Problems with diagnosis and genetic counseling occur for patients with autosomal recessive proximal spinal muscular atrophy (SMA) who do not show the most common mutation: homozygous absence of at least exon 7 of the telomeric survival motor neuron gene (SMN1). Here we present molecular genetic data for 42 independent nondeleted SMA patients. A nonradioactive quantitative PCR test showed one SMN1 copy in 19 patients (45%). By sequencing cloned reverse-transcription (RT) PCR products or genomic fragments of SMN1, we identified nine different mutations in 18 of the 19 patients, six described for the first time: three missense mutations (Y272C, T274I, S262I), three frameshift mutations in exons 2a, 2b, and 4 (124insT, 241-242ins4, 591delA), one nonsense mutation in exon 1 (Q15X), one Alu-mediated deletion from intron 4 to intron 6, and one donor splice site mutation in intron 7 (c.922+6T-->G). The most frequent mutation, Y272C, was found in 6 (33%) of 18 patients. Each intragenic mutation found in at least two patients occurred on the same haplotype background, indicating founder mutations. Genotype-phenotype correlation allowed inference of the effect of each mutation on the function of the SMN1 protein and the role of the SMN2 copy number in modulating the SMA phenotype. In 14 of 23 SMA patients with two SMN1 copies...

To test whether missense mutations in the cancer susceptibility gene MLH1 adversely affect meiosis, we examined 14 yeast MLH1 mutations for effects on meiotic DNA transactions and gamete viability in the yeast Saccharomyces cerevisiae. Mutations analogous to those associated with hereditary nonpolyposis colorectal cancer (HNPCC) or those that reduce Mlh1p interactions with ATP or DNA all impair replicative mismatch repair as measured by increased mutation rates. However, their effects on meiotic heteroduplex repair, crossing over, chromosome segregation, and gametogenesis vary from complete loss of meiotic functions to no meiotic defect, and mutants defective in one meiotic process are not necessarily defective in others. DNA binding and ATP binding but not ATP hydrolysis are required for meiotic crossing over. The results reveal clear separation of different Mlh1p functions in mitosis and meiosis, and they suggest that some, but not all, MLH1 mutations may be a source of human infertility.

The homeodomain is a highly conserved DNA-binding motif that is found in numerous transcription factors throughout a large variety of species from yeast to humans. These gene-specific transcription factors play critical roles in development and adult homeostasis, and therefore, any germline mutations associated with these proteins can lead to a number of congenital abnormalities. Although much has been revealed concerning the molecular architecture and the mechanism of homeodomain-DNA interactions, the study of disease-causing mutations can further provide us with instructive information as to the role of particular residues in a conserved mode of action. In this paper, I have compiled the homeodomain missense mutations found in various human diseases and re-examined the functional role of the mutational “hot spot” residues in light of the structures obtained from crystallography. These findings should be useful in understanding the essential components of the homeodomain and in attempts to design agonist or antagonists to modulate their activity and to reverse the effects caused by the mutations.

The mutations underlying Hurler syndrome (mucopolysaccharidosis IH) in Druze and Muslim Israeli Arab patients have been characterized. Four alleles were identified, using a combination of (a) PCR amplification of reverse-transcribed RNA or genomic DNA segments, (b) cycle sequencing of PCR products, and (c) restriction-enzyme analysis. One allele has two amino acid substitutions, Gly409-->Arg in exon 9 and Ter-->Cys in exon 14. The other three alleles have mutations in exon 2 (Tyr64-->Ter), exon 7 (Gln310-->Ter), or exon 8 (Thr366-->Pro). Transfection of mutagenized cDNAs into Cos-1 cells showed that two missense mutations, Thr366-->Pro and Ter-->Cys, permitted the expression of only trace amounts of alpha-L-iduronidase activity, whereas Gly409-->Arg permitted the expression of 60% as much enzyme as did the normal cDNA. The nonsense mutations were associated with abnormalities of RNA processing: (1) both a very low level of mRNA and skipping of exon 2 for Tyr64-->Ter and (2) utilization of a cryptic splice site for Gln310-->Ter. In all instances, the probands were found homozygous, and the parents heterozygous, for the mutant alleles, as anticipated from the consanguinity in each family. The two-mutation allele was identified in a family from Gaza; the other three alleles were found in seven families...

Mucopolysaccharidosis IIIC (MPS IIIC, or Sanfilippo C syndrome) is a lysosomal storage disorder caused by the inherited deficiency of the lysosomal membrane enzyme acetyl–coenzyme A:α-glucosaminide N-acetyltransferase (N-acetyltransferase), which leads to impaired degradation of heparan sulfate. We report the narrowing of the candidate region to a 2.6-cM interval between D8S1051 and D8S1831 and the identification of the transmembrane protein 76 gene (TMEM76), which encodes a 73-kDa protein with predicted multiple transmembrane domains and glycosylation sites, as the gene that causes MPS IIIC when it is mutated. Four nonsense mutations, 3 frameshift mutations due to deletions or a duplication, 6 splice-site mutations, and 14 missense mutations were identified among 30 probands with MPS IIIC. Functional expression of human TMEM76 and the mouse ortholog demonstrates that it is the gene that encodes the lysosomal N-acetyltransferase and suggests that this enzyme belongs to a new structural class of proteins that transport the activated acetyl residues across the cell membrane.

Mucopolysaccharidosis type IIIB (MPS IIIB or Sanfilippo B disease) is an autosomal recessive storage disorder caused by deficiency of the lysosomal enzyme α-N-acetylglucosaminidase. Mutation screening was performed on a group of 22 patients using a combination of SSCP/heteroduplex analysis of amplified genomic fragments and direct sequencing of cDNA fragments. Twenty-one different mutations were identified, 18 of them novel. Together they account for 82% of the disease alleles. The mutation spectrum consists of two small insertions, two small deletions, three nonsense mutations, and 14 different missense mutations, one of them (M1L) affecting the initiation codon. The vast genetic heterogeneity seen in this disorder is reflected by the fact that only three of the mutations were identified in more than one patient.


Keywords: mucopolysaccharidosis type IIIB; Sanfilippo B disease; mutation screening; α-N-acetylglucosaminidase

Cystinuria is an autosomal recessive disease characterized by the development of kidney stones. Guided by the identification of the SLC3A1 amino acid–transport gene on chromosome 2, we recently established genetic linkage of cystinuria to chromosome 2p in 17 families, without evidence for locus heterogeneity. Other authors have independently identified missense mutations in SLC3A1 in cystinuria patients. In this report we describe four additional cystinuria-associated mutations in this gene: a frameshift, a deletion, a transversion inducing a critical amino acid change, and a nonsense mutation. The latter stop codon was found in all of eight Ashkenazi Jewish carrier chromosomes examined. This report brings the number of disease-associated mutations in this gene to 10. We also assess the frequency of these mutations in our 17 cystinuria families.

Hereditary combined vitamin K–dependent (VKD) coagulation factor deficiency is an autosomal recessive bleeding disorder associated with defects in either the γ-carboxylase, which carboxylates VKD proteins to render them active, or the vitamin K epoxide reductase (VKORC1), which supplies the reduced vitamin K cofactor required for carboxylation. Such deficiencies are rare, and we report the fourth case resulting from mutations in the carboxylase gene, identified in a Tunisian girl who exhibited impaired function in hemostatic VKD factors that was not restored by vitamin K administration. Sequence analysis of the proposita did not identify any mutations in the VKORC1 gene but, remarkably, revealed 3 heterozygous mutations in the carboxylase gene that caused the substitutions Asp31Asn, Trp157Arg, and Thr591Lys. None of these mutations have previously been reported. Family analysis showed that Asp31Asn and Thr591Lys were coallelic and maternally transmitted while Trp157Arg was transmitted by the father, and a genomic screen of 100 healthy individuals ruled out frequent polymorphisms. Mutational analysis indicated wild-type activity for the Asp31Asn carboxylase. In contrast, the respective Trp157Arg and Thr591Lys activities were 8% and 0% that of wild-type carboxylase...

The chromatin-associated protein ATRX was originally identified because mutations in the ATRX gene cause a severe form of syndromal X-linked mental retardation associated with α-thalassemia. Half of all of the disease-associated missense mutations cluster in a cysteine-rich region in the N terminus of ATRX. This region was named the ATRX-DNMT3-DNMT3L (ADD) domain, based on sequence homology with a family of DNA methyltransferases. Here, we report the solution structure of the ADD domain of ATRX, which consists of an N-terminal GATA-like zinc finger, a plant homeodomain finger, and a long C-terminal α-helix that pack together to form a single globular domain. Interestingly, the α-helix of the GATA-like finger is exposed and highly basic, suggesting a DNA-binding function for ATRX. The disease-causing mutations fall into two groups: the majority affect buried residues and hence affect the structural integrity of the ADD domain; another group affects a cluster of surface residues, and these are likely to perturb a potential protein interaction site. The effects of individual point mutations on the folding state and stability of the ADD domain correlate well with the levels of mutant ATRX protein in patients, providing insights into the molecular pathophysiology of ATR-X syndrome.

Arts syndrome is an X-linked disorder characterized by mental retardation, early-onset hypotonia, ataxia, delayed motor development, hearing impairment, and optic atrophy. Linkage analysis in a Dutch family and an Australian family suggested that the candidate gene maps to Xq22.1-q24. Oligonucleotide microarray expression profiling of fibroblasts from two probands of the Dutch family revealed reduced expression levels of the phosphoribosyl pyrophosphate synthetase 1 gene (PRPS1). Subsequent sequencing of PRPS1 led to the identification of two different missense mutations, c.455T→C (p.L152P) in the Dutch family and c.398A→C (p.Q133P) in the Australian family. Both mutations result in a loss of phosphoribosyl pyrophosphate synthetase 1 activity, as was shown in silico by molecular modeling and was shown in vitro by phosphoribosyl pyrophosphate synthetase activity assays in erythrocytes and fibroblasts from patients. This is in contrast to the gain-of-function mutations in PRPS1 that were identified previously in PRPS-related gout. The loss-of-function mutations of PRPS1 likely result in impaired purine biosynthesis, which is supported by the undetectable hypoxanthine in urine and the reduced uric acid levels in serum from patients. To replenish low levels of purines...

NALP proteins, also known as NLRPs, belong to the CATERPILLER protein family involved, like Toll-like receptors, in the recognition of microbial molecules and the subsequent activation of inflammatory and immune responses. Current advances in the function of NALPs support the recently proposed model of a disease continuum bridging autoimmune and autoinflammatory disorders. Among these diseases, hereditary periodic fevers (HPFs) are Mendelian disorders associated with sequence variations in very few genes; these variations are mostly missense mutations whose deleterious effect, which is particularly difficult to assess, is often questionable. The growing number of identified sporadic cases of periodic fever syndrome, together with the lack of discriminatory clinical criteria, has greatly hampered the identification of new disease-causing genes, a step that is, however, essential for appropriate management of these disorders. Using a candidate gene approach, we identified nonambiguous mutations in NALP12 (i.e., nonsense and splice site) in two families with periodic fever syndromes. As shown by means of functional studies, these two NALP12 mutations have a deleterious effect on NF-κB signaling. Overall, these data identify a group of HPFs defined by molecular defects in NALP12...