Biallelic frameshift variants in PHLDB1 cause mild-type osteogenesis imperfecta with regressive spondylometaphyseal changes.

BACKGROUND: Osteogenesis imperfecta (OI) is a heterogeneous group of inherited disorders characterised by susceptibility to fractures, primarily due to defects in type 1 collagen. The aim of this study is to present a novel OI phenotype and its causative candidate gene. METHODS: Whole-exome sequencing and clinical evaluation were performed in five patients from two unrelated families. PHLDB1 mRNA expression in blood and fibroblasts was investigated by real-time PCR, and western blot analysis was further performed on skin fibroblasts. RESULTS: The common findings among the five affected children were recurrent fractures and/or osteopaenia, platyspondyly, short and bowed long bones, and widened metaphyses. Metaphyseal and vertebral changes regressed after early childhood, and no fractures occurred under bisphosphonate treatment. We identified biallelic NM_001144758.3:c.2392dup and NM_001144758.3:c.2690_2693del pathogenic variants in PHLDB1 in the affected patients, respectively, in the families; parents were heterozygous for these variants. PHLDB1 encodes pleckstrin homology-like domain family B member-1 (PHLDB1) protein, which has a role in insulin-dependent Akt phosphorylation. Compared with controls, a decrease in the expression levels of PHLDB1 in the blood and skin fibroblast samples was detected. Western blot analysis of cultured fibroblasts further confirmed the loss of PHLDB1. CONCLUSION: Two biallelic frameshift variants in the candidate gene PHLDB1 were identified in independent families with a novel, mild-type, autosomal recessive OI. The demonstration of decreased PHLDB1 mRNA expression levels in blood and fibroblast samples supports the hypothesis that PHLDB1 pathogenic variants are causative for the observed phenotype.

A novel GNAS mutation in pseudohypoparathyroidism type 1a in a Chinese man presented with recurrent seizure: a case report.

BACKGROUND: Pseudohypoparathyroidism is a rare genetic disease characterized by hypocalcaemia and hyperphosphataemia due to the defect to the guanine nucleotide-binding protein alpha subunit (GNAS) gene. Patients with pseudoparathyroidism type 1a and 1c could manifest Albright's hereditary osteodystrophy and multiple hormone resistance including gonadotropin and thyroid stimulating hormone. CASE PRESENTATION: Here we report a Chinese man who presented with fatigue, recurrent seizure and Albright's hereditary osteodystrophy. His genetic study revealed a heterozygote mutation in the GNAS gene [NM_000516.4(GNAS): c2787_2788del (p.Val930AspfsTer12)]. After calcium and calcitriol supplement, his seizures achieved partially remission. CONCLUSIONS: We report a case of PHP1a or 1c with a novel frameshift mutation in GNAS gene in a patient presenting with AHO, as well as TSH and partial gonadotropin resistance. This mutation in this case has not been reported in literature and adds to the spectrum of genetic mutations related to PHP.

Severe clinical manifestation of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase deficiency associated with two novel mutations: a case report.

BACKGROUND: Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mHS) deficiency is an autosomal recessive inborn error of metabolism, which will give rise to failure of ketogenesis in liver during illness or fasting. It is a very rare disease with only a few patients reported worldwide, most of which had a good prognosis after proper therapies. CASE PRESENTATION: We report a 9-month-old boy with mHS deficiency presenting with unusually severe and persistent acidosis after diarrhea and reduced oral food intake. The metabolic acidosis persisted even after supplementation with sugar and alkaline solution. Blood purification and assisted respiration alleviated symptoms, but a second onset induced by respiratory infection several days later led to multiple organ failure and death. Urine organic acid analysis during the acute episode revealed a complex pattern of ketogenic dicarboxylic and 3-hydroxydicarboxylic aciduria with prominent elevation of glutaric acid and adipic acid, which seem to be specific to mHS deficiency. Plasma acylcarnitine analysis revealed elevated 3-hydroxybutyrylcarnitine and acetylcarnitine. This is the first report of elevated 3-hydroxybutyrylcarnitine in mHS deficiency. Whole exome sequencing revealed a novel compound heterozygous mutation in HMGCS2 (c.100C > T and c.1465delA). CONCLUSION: This severe case suggests the need for patients with mHS deficiency to avoid recurrent illness because it can induce severe metabolic crisis, possibly leading to death. Such patients may also require special treatment, such as blood purification. Urine organic acid profile during the acute episode may give a hint to the disease.

A Carbonic Anhydrase Pseudogene Sensitizes Select Brucella Lineages to Low CO2 Tension.

Brucella spp. are intracellular pathogens that cause a disease known as brucellosis. Though the genus is highly monomorphic at the genetic level, species have animal host preferences and some defining physiologic characteristics. Of note is the requirement for CO2 supplementation to cultivate particular species, which confounded early efforts to isolate B. abortus from diseased cattle. Differences in the capacity of Brucella species to assimilate CO2 are determined by mutations in the carbonic anhydrase gene, bcaA Ancestral single-nucleotide insertions in bcaA have resulted in frameshifted pseudogenes in B. abortus and B. ovis lineages, which underlie their inability to grow under the low CO2 tension of a standard atmosphere. Incubation of wild-type B. ovis in air selects for mutations that "rescue" a functional bcaA reading frame, which enables growth under low CO2 and enhances the growth rate under high CO2 Accordingly, we show that heterologous expression of functional Escherichia coli carbonic anhydrases enables B. ovis growth in air. Growth of B. ovis is acutely sensitive to a reduction in CO2 tension, while frame-rescued B. ovis mutants are insensitive to CO2 shifts. B. ovis initiates a gene expression program upon CO2 downshift that resembles the stringent response and results in transcriptional activation of its type IV secretion system. Our study provides evidence that loss-of-function insertion mutations in bcaA sensitize the response of B. ovis and B. abortus to reduced CO2 tension relative to that of other Brucella lineages. CO2-dependent starvation and virulence gene expression programs in these species may influence persistence or transmission in natural hosts.IMPORTANCEBrucella spp. are highly related, but they exhibit differences in animal host preference that must be determined by genome sequence differences. B. ovis and the majority of B. abortus strains require high CO2 tension to be cultivated in vitro and harbor conserved insertional mutations in the carbonic anhydrase gene, bcaA, which underlie this trait. Mutants that grow in a standard atmosphere, first reported nearly a century ago, are easily selected in the laboratory. These mutants harbor varied indel polymorphisms in bcaA that restore its consensus reading frame and rescue its function. Loss of bcaA function has evolved independently in the B. ovis and B. abortus lineages and results in a dramatically increased sensitivity to CO2 limitation.

Heterozygous Truncating Variants in POMP Escape Nonsense-Mediated Decay and Cause a Unique Immune Dysregulatory Syndrome.

The proteasome processes proteins to facilitate immune recognition and host defense. When inherently defective, it can lead to aberrant immunity resulting in a dysregulated response that can cause autoimmunity and/or autoinflammation. Biallelic or digenic loss-of-function variants in some of the proteasome subunits have been described as causing a primary immunodeficiency disease that manifests as a severe dysregulatory syndrome: chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE). Proteasome maturation protein (POMP) is a chaperone for proteasome assembly and is critical for the incorporation of catalytic subunits into the proteasome. Here, we characterize and describe POMP-related autoinflammation and immune dysregulation disease (PRAID) discovered in two unrelated individuals with a unique constellation of early-onset combined immunodeficiency, inflammatory neutrophilic dermatosis, and autoimmunity. We also begin to delineate a complex genetic mechanism whereby de novo heterozygous frameshift variants in the penultimate exon of POMP escape nonsense-mediated mRNA decay (NMD) and result in a truncated protein that perturbs proteasome assembly by a dominant-negative mechanism. To our knowledge, this mechanism has not been reported in any primary immunodeficiencies, autoinflammatory syndromes, or autoimmune diseases. Here, we define a unique hypo- and hyper-immune phenotype and report an immune dysregulation syndrome caused by frameshift mutations that escape NMD.

Autosomal-recessive cerebellar ataxia caused by a novel ADCK3 mutation that elongates the protein: clinical, genetic and biochemical characterisation.

BACKGROUND: The autosomal-recessive cerebellar ataxias (ARCA) are a clinically and genetically heterogeneous group of neurodegenerative disorders. The large number of ARCA genes leads to delay and difficulties obtaining an exact diagnosis in many patients and families. Ubiquinone (CoQ10) deficiency is one of the potentially treatable causes of ARCAs as some patients respond to CoQ10 supplementation. The AarF domain containing kinase 3 gene (ADCK3) is one of several genes associated with CoQ10 deficiency. ADCK3 encodes a mitochondrial protein which functions as an electron-transfer membrane protein complex in the mitochondrial respiratory chain (MRC). METHODS: We report two siblings from a consanguineous Pakistani family who presented with cerebellar ataxia and severe myoclonus from adolescence. Whole exome sequencing and biochemical assessment of fibroblasts were performed in the index patient. RESULTS: A novel homozygous frameshift mutation in ADCK3 (p.Ser616Leufs*114), was identified in both siblings. This frameshift mutation results in the loss of the stop codon, extending the coding protein by 81 amino acids. Significant CoQ10 deficiency and reduced MRC enzyme activities in the index patient's fibroblasts suggested that the mutant protein may reduce the efficiency of mitochondrial electron transfer. CoQ10 supplementation was initiated following these genetic and biochemical analyses. She gained substantial improvement in myoclonic movements, ataxic gait and dysarthric speech after treatment. CONCLUSION: This study highlights the importance of diagnosing ADCK3 mutations and the potential benefit of treatment for patients. The identification of this new mutation broadens the phenotypic spectrum associated with ADCK3 mutations and provides further understanding of their pathogenic mechanism.

Malonyl coenzyme A decarboxylase deficiency: early dietary restriction and time course of cardiomyopathy.

Malonyl coenzyme A (CoA) decarboxylase (MCD) deficiency is a rare autosomal recessive organic acidemia characterized by varying degrees of organ involvement and severity. MCD regulates fatty acid biosynthesis and converts malonyl-CoA to acetyl-CoA. Cardiomyopathy is 1 of the leading causes of morbidity and mortality in this disorder. It is unknown if diet alone prevents cardiomyopathy development based in published literature. We report a 10-month-old infant girl identified by newborn screening and confirmed MCD deficiency with a novel homozygous MLYCD mutation. She had normal echocardiogram measurements before transition to high medium-chain triglycerides and low long-chain triglycerides diet. Left ventricular noncompaction development was not prevented by dietary interventions. Further restriction of long-chain triglycerides and medium-chain triglycerides supplementation in combination with angiotensin-converting enzyme inhibitors helped to improve echocardiogram findings. Patient remained asymptomatic, with normal development and growth. Our case emphasizes the need for ongoing cardiac disease screening in patients with MCD deficiency and the benefits and limitations of current dietary interventions.

Hereditary dentine diseases resulting from mutations in DSPP gene.

OBJECTIVES: This review groups the newest results of molecular analyses of DSPP gene for patients diagnosed either with dentinogenesis imperfecta type II/III or dentine dysplasia and tries to link the phenotypes with specific mutations in the DSPP gene. DATA: The review includes biochemical data introducing a specificity of DSPP protein which justifies it as a critical factor for dentine mineralization and maturation. The majority of the review analyzes mutations in the DSPP gene which result in phenotypes of dentinogenesis imperfecta types II or/and III or dentine dysplasia. SOURCES: An electronic search was conducted in the databases of Pub Med and supplemented by manual study of relevant references. STUDY SELECTION: 52 out of 108 references were finally selected for the review based on the novelty and/or originality of data. CONCLUSION: Hereditary dentine disorders dentinogenesis imperfecta type II/III and dentine dysplasia are currently proposed to be one disease with distinct clinical manifestations reflecting various mutations in the same DSPP gene. For years both disorders were linked exclusively to mutations in the DSP code but a growing number of papers describe mutations which manifest a similar phenotype but are localized in the strongly repetitive sequence of the 3' terminus of the DSPP which codes DPP protein. Our search suggests that the localization of mutation in the sequence of the DSPP gene might result in a different phenotype due to the diverse cellular fate of the mutated protein. Thus comprehensive research on the cellular fate and processing of both normal and mutated DSPP is still required.

Mutations in SLC30A10 cause parkinsonism and dystonia with hypermanganesemia, polycythemia, and chronic liver disease.

Manganese is essential for several metabolic pathways but becomes toxic in excessive amounts. Manganese levels in the body are therefore tightly regulated, but the responsible protein(s) remain incompletely known. We studied two consanguineous families with neurologic disorders including juvenile-onset dystonia, adult-onset parkinsonism, severe hypermanganesemia, polycythemia, and chronic hepatic disease, including steatosis and cirrhosis. We localized the genetic defect by homozygosity mapping and then identified two different homozygous frameshift SLC30A10 mutations, segregating with disease. SLC30A10 is highly expressed in the liver and brain, including in the basal ganglia. Its encoded protein belongs to a large family of membrane transporters, mediating the efflux of divalent cations from the cytosol. We show the localization of SLC30A10 in normal human liver and nervous system, and its depletion in liver from one affected individual. Our in silico analyses suggest that SLC30A10 possesses substrate specificity different from its closest (zinc-transporting) homologs. We also show that the expression of SLC30A10 and the levels of the encoded protein are markedly induced by manganese in vitro. The phenotype associated with SLC30A10 mutations is broad, including neurologic, hepatic, and hematologic disturbances. Intrafamilial phenotypic variability is also present. Chelation therapy can normalize the manganesemia, leading to marked clinical improvements. In conclusion, we show that SLC30A10 mutations cause a treatable recessive disease with pleomorphic phenotype, and provide compelling evidence that SLC30A10 plays a pivotal role in manganese transport. This work has broad implications for understanding of the manganese biology and pathophysiology in multiple human organs.

Novel mutations in pyridoxine-dependent epilepsy.

PURPOSE: Pyridoxine-Dependent Epilepsy (PDE) is a rare autosomal recessive disease with neonatal seizures resistant to conventional anti-epileptic drugs. This metabolic disease has to be diagnosed early and treated to improve outcome. We report on two new mutations that open new prenatal prospects and suggest a new diagnostic procedure. CASE REPORT: We describe PDE in a neonate carrying two novel mutations in the ALDH7A1 gene: c.[852_856delCTTAG] + [1230C > A]; p.[(Phe410Leu)] + p.[(Leu285CysfsX26)]. This case also illustrates that diagnosis could have been made without any pyridoxine withdrawal, thanks to the measurement of biomarkers. The patient was successfully treated with pyridoxine supplementation and currently shows normal neurological development.

Absence of CHEK2*1100delC mutation in families with hereditary breast cancer in North America.

The CHEK2*1100delC mutation has been reported to confer a twofold increased risk of breast cancer among carriers. The frequency of the mutation varies among populations. The highest frequency has been described in Northern and Eastern European countries; the frequency may be much lower in North America. In this study, our aim was to determine the frequency of CHEK2*1100delC in members of breast cancer families who tested negative for a deleterious mutation in BRCA1/2 at the University of Michigan Comprehensive Cancer Center. We genotyped 102 members from 90 families for CHEK2*1100delC. Most of these families had several cases of breast cancer or ovarian cancer (or both), as well as multiple members with other cancer types in a single lineage. No CHEK2*1100delC mutations were detected in any of the 102 individuals, including 51 women diagnosed with breast cancer at an early age (<45 years), 8 women with bilateral breast cancer, 3 men with breast cancer, and 8 women with ovarian cancer. Our data are consistent with the reported very low frequency of CHEK2*1100delC mutations in North American populations (compared with Northern Europe), rendering CHEK2*1100delC such an unlikely culprit in BRCA1/2 negative families that routine testing of these families appears unwarranted.

Presence of a deletion mutation (c.716delA) in the ligand binding domain of the vitamin D receptor in an Indian patient with vitamin D-dependent rickets type II.

Vitamin D-dependent rickets type II (VDDR-type II) is a rare disorder caused by mutations in the vitamin D receptor (VDR) gene. Here, we describe a patient with VDDR-type II with severe alopecia and rickets. She had hypocalcemia, hypophosphatemia, secondary hyperparathyroidism, and elevated serum alkaline phosphatase and 1,25-dihydroxyvitamin D(3). Sequence analysis of the lymphocyte VDR cDNA revealed deletion mutation c.716delA. Sequence analysis of her genomic DNA fragment amplified from exon 6 of the VDR gene incorporating this mutation confirmed the presence of the mutation in homozygous form. This frameshift mutation in the ligand binding domain (LBD) resulted in premature termination (p.Lys240Argfs) of the VDR protein. The mutant protein contained 246 amino acids, with 239 normal amino acids at the N terminus, followed by seven changed amino acids resulting in complete loss of its LBD. The mutant VDR protein showed evidence of 50% reduced binding with VDR response elements on electrophoretic mobility assay in comparison to the wild-type VDR protein. She was treated with high-dose calcium infusion and oral phosphate. After 18 months of treatment, she gained 6 cm of height, serum calcium and phosphorus improved, alkaline phosphatase levels decreased, and intact PTH normalized. Radiologically, there were signs of healing of rickets. Her parents and one of her siblings had the same c.716delA mutation in heterozygous form. Despite the complete absence of LBD, the rickets showed signs of healing with intravenous calcium.

Loss of pollen-S function in two self-compatible selections of Prunus avium is associated with deletion/mutation of an S haplotype-specific F-box gene.

Recently, an S haplotype-specific F-box (SFB) gene has been proposed as a candidate for the pollen-S specificity gene of RNase-mediated gametophytic self-incompatibility in Prunus (Rosaceae). We have examined two pollen-part mutant haplotypes of sweet cherry (Prunus avium). Both were found to retain the S-RNase, which determines stylar specificity, but one (S3' in JI 2434) has a deletion including the haplotype-specific SFB gene, and the other (S4' in JI 2420) has a frame-shift mutation of the haplotype-specific SFB gene, causing amino acid substitutions and premature termination of the protein. The loss or significant alteration of this highly polymorphic gene and the concomitant loss of pollen self-incompatibility function provides compelling evidence that the SFB gene encodes the pollen specificity component of self-incompatibility in Prunus. These loss-of-function mutations are inconsistent with SFB being the inactivator of non-self S-RNases and indicate the presence of a general inactivation mechanism, with SFB conferring specificity by protecting self S-RNases from inactivation.

Determination of sediment mutagenicity and cytotoxicity in an area subjected to petrochemical contamination.

This study is an evaluation of the mutagenic and cytotoxic activity of sediments in Bom Jardim stream, one of the tributaries of the Cai River basin, Rio Grande do Sul, Brazil. This stream receives an indirect contribution of treated effluent from a petrochemical plant. The Salmonella/microsome assay, a microsuspension method, was used to evaluate moderately polar extracts of sediment samples at three points along the stream. The grain size analysis showed a lower mean content of fine particles in the principle face (front) of the complex, and this was also the sampling point with the lowest percentage of extracted organics. Low mutagenic activity was observed at the different sites studied, ranging from 3.3 to 8.3%; cytotoxic activity was more important in this area, ranging from 20 to 40%, adding up the results of assays in the presence and absence of external metabolism. In assays without S9mix there were more frequent mutagenic and cytotoxic responses, with frameshift mutations being the most frequent. The results also showed that there was a gradual, seasonal distribution of the responses as the stream mouth is reached, the most compromised points being in front of and downstream of the complex. Mutagenic and cytotoxic activity in sediment samples has proved important to determine environmental quality, despite the complexity of the chemical composition of the environmental matrix. Furthermore, use of the Salmonella assay to monitor mutagenesis and cytotoxicity helped identify the presence of pollutants. This assay is an important tool, aimed mainly at actions to preserve the genetic heritage of the fauna and flora affected by human activity and to improve environmental quality.

Gold color in onions (Allium cepa): a natural mutation of the chalcone isomerase gene resulting in a premature stop codon.

Unusual gold-colored onions were selected from a F3 family originating from a cross between US-type yellow and Brazilian yellow onions. HPLC analysis showed that the gold onions contained a significantly reduced amount of quercetin, the most abundant flavonoid in onions. This result indicated that an early step in the flavonoid biosynthesis pathway might be abnormal in these onions. The expression of flavonoid synthesis genes isolated from onions was examined in gold onions and compared to that in onions of other colors by RT-PCR. The results showed that all genes were transcribed in gold onions as in red onions. In order to identify any critical mutations in flavonoid synthesis genes encoding enzymes involved in early steps of the pathway, the genomic sequence of chalcone isomerase (CHI) was obtained. A premature stop codon and a subsequent single base-pair addition causing a frameshift were identified in the coding region of the CHI gene in the gold onions. Co-segregation of the mutant allele of the CHI gene and the gold phenotype was investigated in the original F2 segregating population. Genotyping of three color groups (red, yellow and gold) of F2 onions revealed perfect co-segregation of the mutant CHI allele with the gold phenotype. All tested gold F2 onions were homozygous for the mutant CHI allele. This perfect co-segregation implies that the presence of a premature stop codon in the gold CHI gene results in an inactive CHI. Inactivation of CHI results in a block in the flavonoid biosynthesis pathway and the accumulation of chalcone derivatives, including a yellow pigment which might be responsible for the gold color in onions.

Penciclovir susceptibilities of herpes simplex virus isolates from patients using penciclovir cream for treatment of recurrent herpes labialis.

The antiherpesvirus agent penciclovir (PCV) shares an identical activation pathway and a similar mode of action with acyclovir (ACV). However, since PCV represents a relatively recent treatment option, the clinical resistance profile to PCV is less well known. A susceptibility program was established to assess the resistance profile for serial herpes simplex virus isolates from immunocompetent patients with recurrent herpes labialis obtained throughout a 4-day period of treatment with topical PCV (1% cream) or a placebo. Two isolates (2 of 1,035 [0.19%]), representing 0.34% of the patients (2 of 585), were confirmed to be PCV-resistant (Pcv(r)) herpes simplex virus type 1 by a plaque reduction assay in MRC-5 cells. These two viruses were highly resistant to PCV (50% inhibitory concentrations [IC(50)s], >55 micro g/ml) and were isolated less than 17 h after the start of patient-initiated treatment. However, subsequent isolates on days 2 and 3 from these patients were completely susceptible to PCV (IC(50)s, <2.0 micro g/ml). Thus, it is not clear whether the resistance to PCV for these two early-treatment isolates was directly associated with the 17 h of PCV treatment; several possible explanations are discussed. In an analysis of the distribution of IC(50) differences between the first and last isolates, there were three patients with minor IC(50) increases in the PCV-treated population and one in the placebo-treated group, although statistically, only the latter was an outlier. No patients were found to have Pcv(r) virus at the end of acute treatment, regardless of treatment group. Overall, the prevalence of Pcv(r) was found to be similar to the 0.3% Acv(r) reported for immunocompetent, untreated populations.

p53 Mutations and microsatellite instability in ovarian cancer: Yin and yang.

OBJECTIVE: We tested the hypothesis that p53 frameshift mutations in ovarian cancer occur as a result of genomic instability rather than as a proximal cause of this process. STUDY DESIGN: Sequencing of the p53 tumor suppressor gene has been carried out on 305 ovarian, fallopian tube, and peritoneal cancers. Two groups of p53 null mutations were identified: (1) those caused by frameshift insertion or deletion mutations (n = 31) and (2) those caused by nonsense mutations (n = 28). As a control group 59 tumors with p53 missense mutations were selected by matching with the p53 null tumors on the basis of patient age at diagnosis, stage and grade of cancer, cancer site, and year of diagnosis. Microsatellite instability was determined from paired normal and tumor tissue deoxyribonucleic acid by means of the following different markers: D2S123, D5S346, D17S250, BAT25, and BAT26. Amplimers from polymerase chain reactions were evaluated on 7% polyacrylamide gels. RESULTS: The p53 null tumors were more likely to be of higher stage and grade. Fallopian tube cancers were more common (P =.02) in the p53 frameshift group. The overall incidence of microsatellite instability was 39%, 36%, and 25% for tumors with p53 frameshift nonsense and missense mutations (P =.30). Microsatellite instability was seen almost exclusively with ovarian cancer (P =.04). CONCLUSIONS: Microsatellite instability is a relatively common event in ovarian cancer and is dependent on marker selection. The p53 frameshift mutations do not appear to occur as a consequence of genomic instability.

Complete thyroxine-binding globulin (TBG) deficiency produced by a mutation in acceptor splice site causing frameshift and early termination of translation (TBG-Kankakee).

Fourteen T4-binding globulin (TBG) variants have been identified at the gene level. They are all located in the coding region of the gene and 6 produce complete deficiency of TBG (TBG-CD). We now describe the first mutation in a noncoding region producing TBG-CD. The proband was treated for over 20 yr with L-T4 because of fatigue associated with a low concentration of serum total T4. Fifteen family members were studied showing low total T4 inherited as an X chromosome-linked trait, and affected males had undetectable TBG in serum. Sequencing of the entire coding region and promoter of the TBG gene revealed no abnormality. However, an A to G transition was found in the acceptor splice junction of intron II that produced a new HaeIII restriction site cosegregating with the TBG-CD phenotype. Sequencing exon 1 to exon 3 of TBG complementary DNA reverse transcribed from messenger RNA of skin fibroblasts from an affected male, confirmed a shift in the ag acceptor splice site. This results in the insertion of a G in exon 2 and causes a frameshift and a premature stop at codon 195. This early termination of translation predicts a truncated TBG lacking 201 amino acids.

Involvement of histidine permease (Hip1p) in manganese transport in Saccharomyces cerevisiae.

In a search for components involved in Mn2+ homeostasis in the budding yeast Saccharomyces cerevisiae, we isolated a mutant with modifications in Mn2+ transport. The mutation was found to be located in HIP1, a gene known to encode a high-affinity permease for histidine. The mutation, designated hip1-272, caused a frameshift that resulted in a stop codon at position 816 of the 1812-bp ORF. This mutation led to Mn2+ resistance, whereas the corresponding null mutation did not. Both hip1-272 cells and the null mutant exhibited low tolerance to divalent cations such as Co2+, Ni2+, Zn2+, and Cu2+. The Mn2+ phenotype was not influenced by supplementary histidine in either mutant, whereas the sensitivity to other divalent cations was alleviated by the addition of histidine. The cellular Mn2+ content of the hip1-272 mutant was lower than that of wild type or null mutant, due to increased rates of Mn2+ efflux. We propose that Hiplp is involved in Mn2+ transport, carrying out a function related to Mn2+ export.