A naturally occurring mutation of insulin receptor alanine 1134 impairs tyrosine kinase function and is associated with dominantly inherited insulin resistance

We have identified a previously undescribed genetic variant of the insulin receptor (Ala1134----Thr1134) in a family with the Type A syndrome of insulin resistance. Using the polymerase chain reaction to amplify insulin receptor cDNA and genomic DNA (exon 19), this mutation was detected in 1/2 alleles in the proband, her two affected sisters, and her affected father. Two normal alleles were present in the unaffected mother. No additional structural changes were encoded by the remainder of the proband's receptor cDNA. The Ala1134 mutant receptor was expressed in Chinese hamster ovary cells. The expressed mutant receptors were processed normally and displayed normal affinity of insulin binding but were markedly deficient in insulin-stimulated autophosphorylation. The mutant receptor was unable to catalyze the phosphorylation of the endogenous substrate, pp185, and insulin-stimulated kinase activity toward an exogenous substrate in vitro also was markedly impaired. Ala1134 is a highly conserved residue located in a consensus sequence found in most tyrosine kinases. It is likely that this previously uncharacterized residue and/or the immediate region surrounding it are important for normal kinase function in other members of this receptor family. This study also demonstrates that severe insulin resistance with dominant inheritance may be caused by a missense mutation in one allele of the insulin receptor gene.     

Molecular genetics of severe insulin resistance

Leprechaunism and type A diabetes represent inborn errors of insulin resistance whose phenotypes suggested causation by mutations in the insulin receptor gene. Cells cultured from patients with leprechaunism specifically lacked high-affinity insulin binding. Partial but different degrees of impairment were observed in cells cultured from first-degree relatives. Different mutations in the insulin receptor's alpha subunit were proposed in different families (Ark-1, Atl, Minn, Mount Sinai) based on phenotype, cellular insulin binding, and insulin receptor structure. Molecular cloning and sequencing of mutant insulin receptor cDNA from family Ark-1 confirmed that the proband inherited a maternal missense and a paternal nonsense mutation in the alpha subunit and was a compound heterozygote. The insulin receptor was immunologically present on the plasma membrane of fibroblasts cultured from patients Ark-1 and Atl but was markedly reduced in cells from patients Minn and Mount Sinai. In cells from patient Minn, but not from patient Mount Sinai, the decreased number of insulin receptors was associated with reduced insulin receptor mRNA. In two families with the less severe form of insulin resistance, type A diabetes, mutations altered post-translational processing of the insulin receptor molecule. At a cellular level, these mutations of the alpha subunit of the insulin receptor shared defective binding and impaired stimulation of sugar transport by insulin. In family Atl, however, glucose uptake was constitutively increased. Thus, genetic variation in the insulin receptor gene causes a spectrum of inherited insulin-resistant syndromes and altered cellular signaling.     

Functional properties of a naturally occurring Trp1200----Ser1200 mutation of the insulin receptor

Based on the sequence of cDNA encoding the intracellular domain of the insulin receptor beta-subunit, we recently defined a heterozygous point mutation causing a Ser for Trp substitution at position 1200 in the tyrosine kinase domain of a patient (BI-2) with the type A syndrome of insulin resistance. We have now sequenced the remainder of BI-2's insulin receptor cDNA-coding region and find no additional alterations in the encoded proreceptor protein. The nucleotide sequence of cDNA encoding the portion of the beta-subunit which includes Trp1200 was normal in BI-2's unaffected mother. Hybridization of a mutant allele-specific oligonucleotide to polymerase chain reaction-amplified cDNA confirmed the presence of the mutant allele in the proband and excluded it in her unaffected sister and mother, 18 normal control subjects, and six other subjects with insulin resistance. To determine whether this mutation had functional consequences for receptor signalling, we reconstructed it into a full-length insulin receptor cDNA expression vector. Chinese hamster ovary cells were transfected with mutant cDNA, and the expressed insulin receptors were compared to receptors expressed by cells transfected with wild-type receptor cDNA. Both mutant and wild-type receptors were properly processed into receptor alpha- and beta-subunits, were expressed on the cell surface, and displayed similar insulin-binding affinity. In contrast, insulin-stimulated autophosphorylation of the mutant receptors was severely impaired, whether assessed in intact cells or with a partially purified receptor preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alternative splicing of exon 17 and a missense mutation in exon 20 of the insulin receptor gene in two brothers with a novel syndrome of insulin resistance (congenital fiber-type disproportion myopathy)

The insulin receptor (IR) in two brothers with a rare syndrome of congenital muscle fiber type disproportion myopathy (CFTDM) associated with diabetes and severe insulin resistance was studied. By direct sequencing of Epstein-Barr virus-transformed lymphocytes both patients were found to be compound heterozygotes for mutations in the IR gene. The maternal allele was alternatively spliced in exon 17 due to a point mutation in the -1 donor splice site of the exon. The abnormal skipping of exon 17 shifts the amino acid reading frame and leads to a truncated IR, missing the entire tyrosine kinase domain. In the correct spliced variant, the point mutation is silent and results in a normally translated IR. The paternal allele carries a missense mutation in the tyrosine kinase domain. All three cDNA variants were present in the lymphocytes of the patients. Purified IR from 293 cells overexpressing either of the two mutated receptors lacked basal or stimulated IR beta-subunit autophosphorylation. A third brother who inherited both normal alleles has an normal muscle phenotype and insulin sensitivity, suggesting a direct linkage of these IR mutations with the CFTDM phenotype.     

Reduced mRNA and a nonsense mutation in the insulin-receptor gene produce heritable severe insulin resistance.

Leprechaunism is an autosomal recessive syndrome of severe insulin resistance and is characterized by intrauterine growth restriction, acanthosis nigricans, hirsutism, and loss of glucose homeostasis. Here we report a new female patient of Hispanic and Afro-American descent whose fibroblasts and lymphoblasts had markedly impaired insulin binding (less than 10% of that in controls). Insulin binding to lymphoblasts established from both unrelated parents was partially impaired. Insulin-like growth factor-I (IGF-I) and epidermal growth factor (EGF) binding to the patient's fibroblasts were within the normal range. Insulin stimulation of receptor autophosphorylation and kinase activity was markedly reduced in the patient's fibroblasts. The patient's fibroblasts had both a reduced number of immunoreactive insulin receptor (6% of those in controls) and concomitantly reduced amounts of insulin-receptor mRNA, suggesting that both mutations inherited by the patient reduced insulin-receptor mRNA. Sequencing of the insulin-receptor gene and cDNA indicated that the patient was heterozygous for a paternally derived mutation at bp 1333, converting Arg372 to a STOP codon. This nonsense mutation was observed in the insulin-receptor gene, but not in cDNA, indicating reduced amounts of mRNA for the allele containing this mutation. The coding sequence of the maternally inherited insulin-receptor allele was normal. Both the marked reduction in insulin-receptor mRNA in the compound heterozygous fibroblasts of the proband and the partially reduced insulin binding in maternal cells suggest that the maternally derived mutation is located in an insulin-receptor gene sequence that controls cellular mRNA content.     

Two mutant alleles of the insulin receptor gene in a family with a genetic form of insulin resistance: a 10 base pair deletion in exon 1 and a mutation substituting serine for asparagine-462

Mutations in the insulin receptor gene cause several genetic syndromes associated with extreme insulin resistance. We have studied three insulin resistant siblings with acanthosis nigricans, dental abnormalities, and acral hypertrophy. The female patient also had primary amenorrhea due to hyperandrogenism. All three patients were compound heterozygotes with two mutant alleles of the insulin receptor gene. One allele had a 10-bp deletion in the region of exon 1 encoding the hydrophobic signal peptide; this leads to a frameshift and premature chain termination at codon 61. The deletion occurs at the site of a direct repeat of a hexanucleotide sequence interrupted by a tetranucleotide sequence; the deletion may have resulted from recombination between the upstream and downstream hexanucleotide repeats. In the other mutant allele, there is a missense mutation substituting serine for Asn⁴⁶² — a mutation identified previously in one allele of the insulin receptor gene in a patient with type-A insulin resistance. The Ser⁴⁶² mutation impaired the ability of acidic pH to dissociate insulin from the receptor. Thus, like the previously described Glu⁴⁶⁰ mutation, the Ser⁴⁶² mutation may retard dissociation of insulin from the receptor in the acidic compartment of the endosome and may, as a result, accelerate the rate of receptor degradation.     

Cloning of the human type XVII collagen gene (COL17A1), and detection of novel mutations in generalized atrophic benign epidermolysis bullosa.

Generalized atrophic benign epidermolysis bullosa (GABEB) is a nonlethal variant of junctional epidermolysis bullosa (JEB). Previous findings have suggested that type XVII collagen is the candidate gene for mutations in this disease. We now have cloned the entire human type XVII collagen gene (COL17A1) and have elucidated its intron-exon organization. The gene comprises 56 distinct exons, which span approximately 52 kb of the genome, on the long arm of chromosome 10. It encodes a polypeptide, the alpha1(XVII) chain, consisting of an intracellular globular domain, a transmembrane segment, and an extracellular domain that contains 15 separate collagenous subdomains, the largest consisting of 242 amino acids. We also have developed a strategy to identify mutations in COL17A1 by use of PCR amplification of genomic DNA, using primers placed on the flanking introns. The PCR products are scanned for sequence variants by heteroduplex analysis using conformation-sensitive gel electrophoresis and then are subjected to direct automated sequencing. We have identified several intragenic polymorphisms in COL17A1, as well as mutations, in both alleles, in two Finnish families with GABEB. The probands in both families showed negative immunofluorescence staining with an anti-type XVII collagen antibody. In one family, the proband was homozygous for a 5-bp deletion, 2944del5, which resulted in frameshift and a premature termination codon of translation. The proband in the other family was a compound heterozygote, with one allele containing the 2944del5 mutation and the other containing a nonsense mutation, Q1023X. These results expand the mutation database in different variants of JEB, and they attest to the functional importance of type XVII collagen as a transmembrane component of the hemidesmosomes at the dermal/epidermal junction.     

Identification and expression of five mutations in the human acid sphingomyelinase gene causing types A and B Niemann-Pick disease. Molecular evidence for genetic heterogeneity in the neuronopathic and non-neuronopathic forms.

The deficient activity of the human lysosomal hydrolase, acid sphingomyelinase (ASM, EC 3.1.4.12), results in the neuronopathic (Type A) and non-neuronopathic (Type B) forms of Niemann-Pick disease (NPD). To investigate the genetic basis of the phenotypic heterogeneity in NPD, the molecular lesions in the ASM gene were determined from three unrelated NPD patients and evaluated by transient expression in COS-1 cells. A Type A NPD patient of Asian Indian ancestry (proband 1) was homoallelic for a T to A transversion in exon 2 of the ASM gene which predicted a premature stop at codon 261 of the ASM polypeptide (designated L261X). In contrast, an unrelated Type A patient of European ancestry (proband 2) was heteroallelic for a two-base (TT) deletion in exon 2 which caused a frame-shift mutation at ASM codon 178 (designated fsL178), leading to a premature stop at codon 190, and a G to A transition in exon 3 which caused a methionine to isoleucine substitution at codon 382 (designated M382I). Transient expression of the fsL178, L261X, and M382I mutations in COS-1 cells demonstrated that these lesions did not produce catalytically active ASM, consistent with the severe neuronopathic Type A NPD phenotype. In contrast, an unrelated Type B patient of European descent (proband 3) was heteroallelic for two missense mutations, a G to A transition in exon 2 which predicted a glycine to arginine substitution at ASM codon 242 (designated G242R), and an A to G transition in exon 3 which resulted in an asparagine to serine substitution at codon 383 (designated N383S). Interestingly, the G242R allele produced ASM activity in COS-1 cells at levels about 40% of that expressed by the normal allele, thereby explaining the mild Type B phenotype of proband 3 and the high residual activity (i.e. approximately 15% of normal) in cultured lymphoblasts. In contrast, the N383S allele did not produce catalytically active enzyme. None of these five ASM mutations was detected in over 60 other unrelated NPD patients analyzed, nor were these mutations found in over 100 normal ASM alleles. Thus, small deletions or nonsense mutations which trunctated the ASM polypeptide, or missense mutations that rendered the enzyme noncatalytic, resulted in Type A NPD disease, whereas a missense mutation that produced a defective enzyme with residual catalytic activity caused the milder nonneuronopathic Type B phenotype. These findings have facilitated genotype/phenotype correlations for this lysosomal storage disease and provided insights into the functional organization of the ASM polypeptide.     

Molecular and structural characterization of five novel mutations in the Bruton's tyrosine kinase gene from patients with X-linked agammaglobulinemia.

BACKGROUND: The Btk (Bruton's tyrosine kinase) gene has been shown to be mutated in the human immunodeficiency disease, XLA (X-linked agammaglobulinemia). Btk is a member of the Tec family of cytosolic protein tyrosine kinases with distinct functional domains PH, TH, SH3, SH2, and kinase. Mutations have been observed in each of the Btk subdomains in XLA. We have analyzed the Btk gene in six XLA patients from five unrelated families. MATERIALS AND METHODS: DNA was prepared from the patients peripheral blood. The Btk exons including the junctional sequences were analyzed by single-strand conformation polymorphism (SSCP) followed by direct nucleotide sequencing after PCR-amplification. For structural analysis, the missense mutations were introduced into three-dimensional models of the PH and kinase domains of Btk and the outcome was predicted based on the knowledge of the protein function. RESULTS: Five novel mutations and two novel polymorphisms, all of which resulted from single-base alterations, were identified. Three of the five mutations were in the PH domain and two were in the kinase domain of Btk. Three of these mutations were of the missense type, two of which altered the same codon in the PH domain; the third one was located in the kinase domain. The fourth mutation was a point deletion in the PH domain causing a frameshift followed by premature termination. The fifth mutation was a splice donor-site mutation within the kinase domain which could result in an exon skipping. In four of the five instances, mothers of the patients were shown to be obligate carriers. In one instance, a sibling sister was identified as a heterozygote establishing her as a carrier. CONCLUSIONS: Functional consequences of the mutations causing frameshifts and altered splicing can be inferred directly. Functional consequences of the missense mutations were interpreted by 3-dimensional structural modeling of Btk domains. It is proposed that the two PH domain mutations will interfere with membrane localization while the kinase domain mutation will interfere with the enzymatic function of Btk. This study provides further insight into the role of Btk in XLA.     

Dominant and recessive compound heterozygous mutations in epidermolysis bullosa simplex demonstrate the role of the stutter region in keratin intermediate filament assembly

Keratin intermediate filaments are important cytoskeletal structural proteins involved in maintaining cell shape and function. Mutations in the epidermal keratin genes, keratin 5 or keratin 14 lead to the disruption of keratin filament assembly, resulting in an autosomal dominant inherited blistering skin disease, epidermolysis bullosa simplex (EBS). We investigated a large EBS kindred who exhibited a markedly heterogeneous clinical presentation and detected two distinct keratin 5 mutations in the proband, the most severely affected. One missense mutation (E170K) in the highly conserved helix initiation peptide sequence of the 1A rod domain was found in all the affected family members. In contrast, the other missense mutation (E418K) was found only in the proband. The E418K mutation was located in the stutter region, an interruption in the heptad repeat regularity, whose function as yet remains unclear. We hypothesized that this mutated stutter allele was clinically silent when combined with the wild type allele but aggravates the clinical severity of EBS caused by the E170K mutation on the other allele. To confirm this in vitro, we transfected mutant keratin 5 cDNA into cultured cells. Although only 12.7% of the cells transfected with the E170K mutation alone showed disrupted keratin filament aggregations, significantly more cells (30.0%) cotransfected with both E170K and E418K mutations demonstrated keratin aggregation (p < 0.05). These transfection assay results corresponded to the heterogeneous clinical findings of the EBS patient in this kindred. We have identified the first case of both compound heterozygous dominant (E170K) and recessive (E418K) mutations in any keratin gene and confirmed the significant involvement of the stutter region in the assembly and organization of the keratin intermediate filament network in vitro.     

Substitution of isoleucine for methionine at position 1153 in the beta-subunit of the human insulin receptor. A mutation that impairs receptor tyrosine kinase activity, receptor endocytosis, and insulin action.

The intracellular domain of the insulin receptor possesses activity as a tyrosine-specific protein kinase. The receptor tyrosine kinase is stimulated by insulin binding to the extracellular domain of the receptor. Previously, we have identified a patient with a genetic form of insulin resistance who is heterozygous for a mutation substituting Ile for Met1153 in the tyrosine kinase domain of the receptor near the cluster of the three major autophosphorylation sites (Tyr1158, Tyr1162, and Tyr1163). In this investigation, the Ile1153 mutant receptor was expressed by transfection of mutant cDNA into NIH-3T3 cells. The mutation impairs receptor tyrosine kinase activity and also inhibits the ability of insulin to stimulate 2-deoxyglucose uptake and thymidine incorporation. These data support the hypothesis that the receptor tyrosine activity plays a necessary role in the ability of the receptor to mediate insulin action in vivo. Furthermore, expression of the Ile1153 mutant receptor exerted a dominant negative effect to inhibit the ability of endogenous murine receptors for insulin and insulin-like growth factor I to mediate their actions upon the cell. This observation is consistent with previous suggestions that mutant receptors dimerize with wild type receptors, thereby creating hybrid molecules which lack biological activity. The dominant negative effect of the mutant receptor may explain the dominant mode of inheritance of insulin resistance caused by the Ile1153 mutation. Finally, the mutation inhibits the ability of insulin to stimulate receptor endocytosis. This may explain the normal number of insulin receptors on the surface of the patient's cells in vivo. Despite the presence of markedly elevated levels of insulin in the patient's plasma, the receptors were resistant to down-regulation.     

A leucine-to-proline mutation in the insulin receptor in a family with insulin resistance.

We have determined the primary structure of a mutant insulin receptor of a leprechaun patient born from a consanguineous marriage. A characteristic feature of leprechaunism is an extreme resistance to insulin. In this patient the insulin resistance seems to result from an observed lack of insulin binding to intact cells. Solubilization of cells in non-ionic detergents leads to the appearance of insulin receptors which can bind insulin. However, the insulin-stimulated autophosphorylation of the receptor's beta subunit is markedly reduced. Cloning and sequencing of cDNA derived from insulin receptor mRNA of this patient revealed a leucine-to-proline mutation at position 233 in the alpha subunit. By means of DNA amplification we found that the patient is homozygous for this mutation and that the parents and two grandparents from the consanguineous line are heterozygous. The heterozygous individuals all show decreased insulin binding to cultured fibroblasts. In addition, they are mildly insulin resistant in vivo. These observations show a linkage between the leucine-to-proline mutation and the observed insulin resistance in this family. We therefore conclude that the mutation in the homozygous form is responsible for the extreme insulin resistance in the leprechaun patient. The mutation for the first time characterizes a region in the insulin receptor which seems to be involved in transmitting the insulin binding signal to the tyrosine kinase domain.     

A frequent tyrosinase gene mutation associated with type I-A (tyrosinase-negative) oculocutaneous albinism in Puerto Rico.

We have determined the mutations in the tyrosinase gene from 12 unrelated Puerto Rican individuals who have type I-A (tyrosinase-negative) oculocutaneous albinism (OCA). All but one individual are of Hispanic descent. Nine individuals were homozygous for a missense mutation (G47D) in exon I at codon 47. Two individuals were heterozygous for the G47D mutation, with one having a missense mutation at codon 373 (T373K) in the homologous allele and the other having an undetermined mutation in the homologous allele. One individual with negroid features was homozygous for a nonsense mutation (W236X). The population migration between Puerto Rico and the Canary Islands is well recognized. Analysis of three individuals with OCA from the Canary Islands showed that one was a compound heterozygote for the G47D mutation and for a novel missense mutation (L216M), one was homozygous for a missense mutation (P81L), and one was heterozygous for the missense mutation P81L. The G47D and P81L missense mutations have been previously described in extended families in the United States. Haplotypes were determined using four polymorphisms linked to the tyrosinase locus. Haplotype analysis showed that the G47D mutation occurred on a single haplotype, consistent with a common founder for all individuals having this mutation. Two different haplotypes were found associated with the P81L mutation, suggesting that this may be either a recurring mutation for the tyrosinase gene or a recombination between haplotypes.     

Structural and biochemical evidence for an autoinhibitory role for tyrosine 984 in the juxtamembrane region of the insulin receptor

Tyrosine 984 in the juxtamembrane region of the insulin receptor, between the transmembrane helix and the cytoplasmic tyrosine kinase domain, is conserved among all insulin receptor-like proteins from hydra to humans. Crystallographic studies of the tyrosine kinase domain and proximal juxtamembrane region reveal that Tyr-984 interacts with several other conserved residues in the N-terminal lobe of the kinase domain, stabilizing a catalytically nonproductive position of alpha-helix C. Steady-state kinetics measurements on the soluble kinase domain demonstrate that replacement of Tyr-984 with phenylalanine results in a 4-fold increase in kcat in the unphosphorylated (basal state) enzyme. Moreover, mutation of Tyr-984 in the full-length insulin receptor results in significantly elevated receptor phosphorylation levels in cells, both in the absence of insulin and following insulin stimulation. These data demonstrate that Tyr-984 plays an important structural role in maintaining the quiescent, basal state of the insulin receptor. In addition, the structural studies suggest a possible target site for small molecule activators of the insulin receptor, with potential use in the treatment of noninsulin-dependent diabetes mellitus.     

An intron 1 splice mutation and a nonsense mutation (W23X) in CYP21 causing severe congenital adrenal hyperplasia

Direct DNA sequencing of the steroid 21-hydroxylase gene (CYP21) revealed two novel mutations in two patients with severe congenital adrenal hyperplasia. The nonsense mutation Trp23Stop (TGG → TGA) was found in a woman with the simple virilizing form of the disease. She was a compound heterozygote, with the previously described Ile173Asn mutation on her other allele. A boy, who developed salt-wasting in the neonatal period, carried an allele with a novel mutation of the canonical splice acceptor site in intron 1 (AG→GG). He was also a compound heterozygote, with the well-known splice mutation in intron 2 on his other allele. Received: 26 February 1996     

Germline mutations of regulator of telomere elongation helicase 1, RTEL1, in Dyskeratosis congenita

Dyskeratosis congenita (DC) is an inherited bone marrow failure and cancer predisposition syndrome caused by aberrant telomere biology. The classic triad of dysplastic nails, abnormal skin pigmentation, and oral leukoplakia is diagnostic of DC, but substantial clinical heterogeneity exists; the clinically severe variant Hoyeraal Hreidarsson syndrome (HH) also includes cerebellar hypoplasia, severe immunodeficiency, enteropathy, and intrauterine growth retardation. Germline mutations in telomere biology genes account for approximately one-half of known DC families. Using exome sequencing, we identified mutations in RTEL1, a helicase with critical telomeric functions, in two families with HH. In the first family, two siblings with HH and very short telomeres inherited a premature stop codon from their mother who has short telomeres. The proband from the second family has HH and inherited a premature stop codon in RTEL1 from his father and a missense mutation from his mother, who also has short telomeres. In addition, inheritance of only the missense mutation led to very short telomeres in the proband’s brother. Targeted sequencing identified a different RTEL1 missense mutation in one additional DC proband who has bone marrow failure and short telomeres. Both missense mutations affect the helicase domain of RTEL1, and three in silico prediction algorithms suggest that they are likely deleterious. The nonsense mutations both cause truncation of the RTEL1 protein, resulting in loss of the PIP box; this may abrogate an important protein–protein interaction. These findings implicate a new telomere biology gene, RTEL1, in the etiology of DC.     

Molecular and genetic analysis of a compound heterozygote for dysprothrombinemia of prothrombin Tokushima and hypoprothrombinemia.

The molecular and genetic basis of a compound heterozygote for dys- and hypoprothrombinemia was analyzed. Abnormal nucleotide sequences of the human prothrombin gene were screened by PCR-single-strand conformation polymorphism (PCR-SSCP) with endonuclease digestion and mutated primer-mediated PCR-RFLP. A single nucleotide substitution responsible for dysprothrombinemia of prothrombin Tokushima was detected, as were three polymorphisms. The mutation for hypoprothrombinemia was detected by PCR-single-strand conformation polymorphism (PCR-SSCP) with endonuclease digestion in exon 6, near MboII-RFLP and NcoI-RFLP. Sequencing of PCR-amplified genomic DNA revealed a single base insertion of thymine (T) at position 4177. The resulting frameshift mutation caused both an altered amino acid sequence from codon 114 and a premature termination codon (i.e., TGA) at codon 174 in exon 7. Because exon 7 encodes the kringle 2 domain preceding the thrombin sequence, this frameshift leads to the null prothrombin phenotype. The inheritance of the hypoprothrombinemia gene from the father to the proband was proved by PCR-SSCP with endonuclease digestion and mutated primer-mediated PCR-RFLP.     

The tyrosine kinase activity of the chicken insulin receptor is similar to that of the human insulin receptor

The tyrosine kinase activity of a chimeric insulin receptor composed of the extracellular domain of the human insulin receptor (IR) and the intracellular domain of the chicken IR was compared with wild-type human IR. The degrees of autophosphorylation, phosphorylation of IRS-1, and in vitro phosphorylation of an exogenous substrate after stimulation by human insulin were similar to that seen with the human IR. We conclude that the insulin resistance of chickens is not attributable to a lower level of intrinsic tyrosine kinase activity of IR.     

Molecular basis of group A xeroderma pigmentosum: A missense mutation and two deletions located in a zinc finger consensus sequence of the XPAC gene

Summary The molecular basis of group A xeroderma pigmentosum (XP) was investigated, and 3 mutations located in a zinc finger consensus sequence (nucleotide 313–387) of the XP group A complementing (XPAC) gene were identified in 2 Caucasian patients GM2990 and GM2009 who had typical symptoms of group A XP. The first mutation was a C deletion at nucleotide 374. Patient GM2990 was a homozygote for this mutation. The second mutation was a 5-bp deletion (CTTAT) at nucleotides 349–353. The third mutation was a G to T transversion at nucleotide 323 that alters the Cys-108 codon (TGT) to a Phe codon (TTT). Patient GM2009 was a compound heterozygote for the 5-bp deletion and the missense mutation. Both deletions introduce frameshifts with premature translation terminations resulting in instability of the XPAC mRNA and disruption of the putative zinc finger domain of the XPAC protein. The missense mutation also predicts disruption of the zinc finger domain of the XPAC protein. The expression study showed that the missense mutation does indeed causes loss of repair activity of the XPAC protein. We conclude that these 3 mutations are responsible for group A XP.