Cervicitis aetiology and case definition: a study in Australian women attending sexually transmitted infection clinics.

OBJECTIVES: Studies examining cervicitis aetiology and prevalence lack comparability due to varying criteria for cervicitis. We aimed to outline cervicitis associations and suggest a best case definition. METHODS: A cross-sectional study of 558 women at three sexually transmitted infection clinics in Sydney, Australia, 2006-2010, examined pathogen and behavioural associations of cervicitis using three cervicitis definitions: 'microscopy' (>30 pmnl/hpf (polymorphonuclear leucocytes per high-powered field on cervical Gram stain)), 'cervical discharge' (yellow and/or mucopurulent cervical discharge) or 'micro+cervical discharge' (combined 'microscopy' and 'cervical discharge'). RESULTS: Chlamydia trachomatis (CT), Mycoplasma genitalium (MG), Trichomonas vaginalis (TV) and Neisseria gonorrhoeae (NG) had the strongest associations with cervicitis definitions 'micro+cervical discharge': CT adjusted prevalence ratio (APR)=2.13 (95% CI 1.38 to 3.30) p=0.0006, MG APR=2.21 (1.33 to 3.69) p=0.002, TV APR=2.37 (1.44 to 3.90) p=0.0007 NG PR=4.42 (3.79 to 5.15) p<0.0001 and 'cervical discharge': CT APR=1.90 (1.25 to 2.89) p=0.003, MG APR=1.93 (1.17 to 3.19) p=0.011, TV APR=2.02 (1.24 to 3.31) p=0.005 NG PR=3.88 (3.36 to 4.48) p<0.0001. Condom use for vaginal sex 'always/sometimes' reduced cervicitis risk: ('micro+cervical discharge') APR=0.69 (0.51 to 0.93) p=0.016. Combined population attributable risk % (PAR%) of these four pathogens was only 18.0% with a protective PAR% of condoms of 25.7%. Exposures not associated with cervicitis included bacterial vaginosis, Mycoplasma hominis, Ureaplasma urealyticum, herpes simplex virus 1&2, cytomegalovirus, Candida, age, smoking and hormonal contraception. CONCLUSIONS: Cervicitis was associated with CT, MG, TV and NG with combined PAR% of these pathogens only 18% in this setting, suggesting other factors are involved. Condoms significantly reduced cervicitis risk. Cervicitis definitions with best clinical utility and pathogen prediction were 'cervical discharge' and 'micro+cervical discharge'.

Prolonged second diagnostic window for human immunodeficiency virus type 1 in a fourth-generation immunoassay: are alternative testing strategies required?

Diagnosis of acute HIV is done by patient history and examination and testing of RNA, proviral DNA, and serology using fourth-generation antigen/antibody detection assays. We describe an HIV-1 primary infection with a second diagnostic window of 18 to 34 days on a fourth-generation immunoassay, which would have been missed using some current algorithms. Caution must be exercised when fourth-generation HIV-1 immunoassays are interpreted in isolation, and additional testing should be considered depending on patient risk assessment.

Mycoplasma genitalium is associated with cervicitis and HIV infection in an urban Australian STI clinic population.

OBJECTIVES: To investigate the prevalence of the genital mollicutes, Mycoplasma genitalium (MG), Mycoplasma hominis (MH), Ureaplasma urealyticum (UU) and Ureaplasma parvum (UP), and their associations with cervicitis in a sexually transmitted infection (STI) clinic population. Clinical correlates of MG infection were also assessed. METHODS: 527 women were enrolled in a cross-sectional study at two STI clinics in Sydney between June 2006 and January 2010. Genital mollicutes were detected by multiplex PCR testing of cervical swabs, and associations with cervicitis were analysed. Cervicitis was defined as >30 polymorphonuclear cells per high-power field in at least three non-adjacent fields of cervical mucus on Gram stain. RESULTS: MG was found in 4.0% of women, MH in 17.1%, UU in 14.1%, and UP in 51.8%. MG was the only mollicute associated with cervicitis (unadjusted prevalence ratio (PR) 1.85, 95% CI 1.52 to 2.26, p<0.0001), and this association remained after adjustment for Chlamydia trachomatis (CT) infection (adjusted PR 1.24 (95% CI 1.04 to 1.48), p=0.02). MG was significantly associated with women being HIV positive (p=0.03), but not with age, vaginal discharge, commercial sex work, being of culturally and linguistically diverse background, or concurrent CT infection. Two of the 21 women with MG had ectopic pregnancies. CONCLUSIONS: The authors recommend wider application of PCR testing for MG in STI services, particularly in high-risk women and those with cervicitis or HIV infection.

Trichomonas vaginalis: underdiagnosis in urban Australia could facilitate re-emergence.

OBJECTIVES: Trichomonas vaginalis (TV) has a low profile in urban sexually transmitted infection (STI) clinics in many developed countries. The objective of this study was to determine the true prevalence of TV in an Australian urban sexual health setting using sensitive molecular diagnostic techniques. METHODS: A cross-sectional study investigating the aetiology of cervicitis in women attending two urban sexual health clinics in Sydney, Australia, enrolled 356 consecutive eligible women from 2006 to 2008. The diagnostic yield from the standard clinical practice of discretionary high vaginal wet preparation microscopy in women with suspicious vaginal discharge was compared with universal use of nested PCR for TV of cervical samples. RESULTS: TV was detected by PCR in 17/356 women (4.8%, 95% CI 2.8 to 7.5%), whereas only four cases (1.1%, 95% CI 0.3 to 2.8%) were detected by discretionary wet preparation microscopy. Eleven of the 17 women (p=0.003) were of culturally and linguistically diverse background. Additionally, cervicitis was found to be significantly associated with TV, RR 1.66 (1.14 to 2.42), p=0.034. CONCLUSIONS: Traditional TV-detection methods underestimate TV prevalence in urban Australia. The TV prevalence of 4.8% by PCR testing in this study exceeds previously reported urban Australian TV rates of <1%. An increase in trichomoniasis-associated adverse reproductive outcomes and enhanced HIV transmission poses a salient public health threat. Accordingly, TV warrants a higher profile in urban STI clinic settings in developed countries, and we suggest that priority be given to development of standardised molecular TV detection techniques and that these become part of routine STI testing.

Multiplex PCR testing detection of higher-than-expected rates of cervical mycoplasma, ureaplasma, and trichomonas and viral agent infections in sexually active australian women.

Knowing the prevalence of potential etiologic agents of nongonococcal and nonchlamydial cervicitis is important for improving the efficacy of empirical treatments for this commonly encountered condition. We describe four multiplex PCRs (mPCRs), designated VDL05, VDL06, VDL07, and VDL09, which facilitate the detection of a wide range of agents either known to be or putatively associated with cervicitis, including cytomegalovirus (CMV), enterovirus (EV), Epstein-Barr virus (EBV), varicella-zoster virus (VZV), herpes simplex virus type 1 (HSV-1), and herpes simplex virus type 2 (HSV-2) (VDL05); Ureaplasma parvum, Ureaplasma urealyticum, Mycoplasma genitalium, and Mycoplasma hominis (VDL06); Chlamydia trachomatis, Trichomonas vaginalis, Treponema pallidum, and group B streptococci (VDL07); and adenovirus species A to E (VDL09). The mPCRs were used to test 233 cervical swabs from 175 women attending a sexual-health clinic in Sydney, Australia, during 2006 and 2007. The agents detected alone or in combination in all cervical swabs (percentage of total swabs) included CMV (6.0), EV (2.1), EBV (2.6), VZV (4.7), HSV-1 (2.6), HSV-2 (0.8), HSV-2 and VZV (0.4), U. parvum (57.0), U. urealyticum (6.1), M. genitalium (1.3), M. hominis (13.7), C. trachomatis (0.4), T. vaginalis (3.4), and group B streptococci (0.4). Adenovirus species A to E and T. pallidum were not detected. These assays are adaptable for routine diagnostic laboratories and provide an opportunity to measure the true prevalence of microorganisms potentially associated with cervicitis and other genital infections.

Cervicitis: a prospective observational study of empiric azithromycin treatment in women with cervicitis and non-specific cervicitis.

Treatment with 1 G azithromycin was observed prospectively in 130 women with cervicitis (>30 polymorphonuclear leucocytes/high-powered field) enrolled in a cervicitis aetiology study of 558 women at three sexually transmitted infection clinics in Sydney, Australia. Two overlapping groups of women with cervicitis were considered: 'cervicitis group 1' (n = 116) excluded women with Trichomonas vaginalis and a subgroup of this, 'cervicitis group 2' (non-specific cervicitis) (n = 96) further excluded women with Neisseria gonorrhoea, Chlamydia trachomatis and Mycoplasma genitalium at enrolment. Testing for Chlamydia trachomatis, Mycoplasma genitalium and Trichomonas vaginalis was by PCR and Neisseria gonorrhoea by PCR and culture. Treatment outcomes were cervicitis or vaginal symptoms at follow-up. Effect on cervicitis at follow-up was also assessed after additional reported partner treatment. In 'cervicitis group 1' where prevalence of Mycoplasma genitalium and/or Chlamydia trachomatis was 23/116 (19.8%), azithromycin reduced cervicitis at follow-up (RR = 0.62 (95% CI 0.39-0.97) p = 0.035), but there was no significant effect in non-specific cervicitis ('cervicitis group 2') (RR = 0.60 (95% CI 0.35-1.01) p = 0.056). Empiric treatment did not reduce vaginal symptoms at follow-up in either group. No effect of empiric partner treatment was seen. The conclusion was that empiric azithromycin treatment of cervicitis reduces cervicitis at follow-up in populations with high prevalence of Chlamydia trachomatis and/or Mycoplasma genitalium. There are no benefits of empiric azithromycin for non-specific cervicitis or empiric partner treatment.

Deficiency of dihydrolipoamide dehydrogenase due to two mutant alleles (E340K and G101del). Analysis of a family and prenatal testing.

A male child with metabolic acidosis was diagnosed as having dihydrolipoamide dehydrogenase (E3) deficiency. E3 activity of the proband's cultured fibroblasts and blood lymphocytes was 3-9% of normal, while in the parent's lymphocytes it was about 60% of normal. The proband's pyruvate dehydrogenase complex (PDC) and the alpha-ketoglutarate dehydrogenase complex activities from cultured skin fibroblasts were 12% and 6% of normal, respectively. PDC activity in the parents cultured fibroblasts was 25-31% of normal. Western and Northern blot analyses showed similar quantities of E3 protein and mRNA in cultured fibroblasts from the proband and his parents. DNA sequencing of cloned full-length E3 cDNAs, from the proband and the parents, showed two mutations on different alleles of proband were inherited from the parents. One mutation is a three nucleotide (AGG) deletion, from the mother, resulting in deletion of Gly101 in the FAD binding domain. The other mutation is a nucleotide substitution (G to A), from the father, leading to substitution of Lys for Glu340 in the central domain. The same deletion mutation was found in E3 cDNA from a chorionic villus sample and cultured fibroblasts obtained from the mother's subsequent offspring. This finding illustrates the possibility of successful prenatal diagnosis of E3 deficiency utilizing mutations characterized prior to initiation of pregnancy.

Tumors of the brachial plexus.

Over a 17-year period, 56 patients with 57 tumors involving the brachial plexus were operated upon. The 40 neural sheath tumors included 26 neurofibromas, 8 schwannomas, 4 malignant neural sheath tumors, 1 fibrosarcoma, and 1 meningioma. Nine of the neurofibromas were associated with von Recklinghausen's disease (VRD), and 2 others were what was termed regionalized neurofibromatosis characterized by involvement of one limb with extension along the course of one or more plexus elements. Seventeen tumors were not of neural sheath origin; 7 were benign and 10 were metastatic malignant tumors compressing or invading the plexus. Benign neurofibromas and malignant sheath tumors almost always presented with pain or functional neural deficit, whereas schwannomas often presented with a palpable mass as their only initial symptom. Patients with solitary neurofibromas were significantly older, often female, and more likely to have tumor on the right side than patients with schwannomas, malignant neural sheath tumors, or neurofibromas associated with VRD. Solitary neurofibromas could often be totally resected without added deficit by sacrificing fascicles entering and leaving tumor that were determined to be "nonfunctional" by intraoperative nerve action potential recordings. Resection of neurofibromas associated with VRD sometimes but not always resulted in significant loss. Operation is nonetheless recommended, especially when malignancy is suspected because of rapidly increasing size, when severe pain or neural deficit is present, or when compression of adjacent plexus elements is a concern. Schwannomas and benign non-neural sheath tumors can usually be extirpated without damage to plexus elements. Forequarter amputation is advised for malignant intrinsic tumors involving distal plexus elements even though gross total resection seems feasible.

MRI, clinical, and biochemical features of partial pyruvate carboxylase deficiency.

We report a boy with a partial deficiency of pyruvate carboxylase as documented in enzyme assays of skin fibroblasts, lymphocytes, and hepatic tissue. Magnetic resonance imaging at age 20 months demonstrated a leukodystrophic process involving the brain stem and subcortical white matter, which, except for the brain stem, improved after biotin treatment. The lymphocyte pyruvate carboxylase activity of both heterozygous parents slightly increased after receiving oral biotin for 1 month, but a definitive enzymatic response to biotin was not confirmed in our patient. At age 6 years, he is dysarthric with a spastic quadriparesis despite improvements in development and myelination. This is the first demonstration of magnetic resonance imaging changes in this disease.

Numerical simulation of ultrasonic wave propagation in anisotropic and attenuative solid materials.

The axisymmetric elastodynamic finite element code developed is capable of predicting quantitatively accurate displacement fields for elastic wave propagation in isotropic and transversely isotropic materials. The numerical algorithm incorporates viscous damping by adding a time-dependent tensor to Hooke's law. Amplitude comparisons are made between the geometric attenuation in the far field and the corresponding finite element predictions to investigate the quality and validity of the code. Through-transmission experimental measurements made with a 1 MHz L-wave transducer attached to an aluminum sample support the code predictions. The algorithm successfully models geometric beam spreading dispersion and energy absorption due to viscous damping. This numerical model is a viable tool for the study of elastic wave propagation in nondestructive testing applications.

Pharyngeal Gonorrhoea in Women: An Important Reservoir for Increasing Neisseria gonorrhoea Prevalence in Urban Australian Heterosexuals?

We aim to characterize sexual behavioral aspects of heterosexual Neisseria gonorrhoea (NG) acquisition in two Sexually Transmitted Diseases clinics in Sydney, Australia, in 2008-2012. Of 167 NG cases, 102 were heterosexually acquired with a trend of increasing NG prevalence in heterosexuals from 1.1% (95% CI 0.6-2.1) in 2008 to 3.0% (95% CI 2.0-4.0) in 2012 (P = 0.027). Of heterosexual male cases, unprotected fellatio was the likely sexual activity for NG acquisition in 21/69 (30.4%) and commercial sex work (CSW) contact the likely source in 28/69 (40.6%). NG prevalence overall in CSW (2.2%) was not significantly higher than in non-CSW (1.2%) (P = 0.15), but in 2012 there was a significant increase in NG prevalence in CSW (8.6%) compared to non-CSW (1.6%) (P < 0.001). Pharyngeal NG was found in 9/33 (27.3%) female cases. Decreased susceptibility to ceftriaxone (MIC ≥ 0.03 mg/L) occurred in 2.5% NG isolates, none heterosexually acquired. All were azithromycin susceptible. A significant trend of increasing prevalence of heterosexual gonorrhoea in an urban Australian STD clinic setting is reported. We advocate maintenance of NG screening in women, including pharyngeal screening in all women with partner change who report fellatio, as pharyngeal NG may be an important reservoir for heterosexual transmission. Outreach to CSW should be enhanced.

Primary health care providers surveyed commonly misinterpret 'first void urine' for chlamydia screening.

An open question survey of general practitioners (GP) and hospital emergency department (ED) doctors revealed that the term 'FVU' (first void urine) used for urine chlamydia testing, is ambiguous, potentially leading to incorrect urine sample collection and barriers to effective screening. The results of this survey indicate that only 4.3% (95% confidence interval [CI] 0.5-14.5%) of GP and 6.9% (95% CI 0.9-22.8%) of ED doctors respectively, correctly interpreted the meaning of FVU. The majority of clinicians surveyed misunderstood 'FVU' to require the first urine void of the day, accounting for 68.1% (95% CI 52.9-80.9%) of GP responses and 37.9% (95% CI 20.7-57.7%) of ED doctors responses. This highlights the need for clarification and standardisation of terminology used in urine chlamydia screening for health care providers, in order to optimise strategies for diagnosis and control of the ongoing chlamydia epidemic.

Cervicitis: a review.

PURPOSE OF REVIEW: Sexually transmitted infections impact significantly on global health. Whereas Chlamydia, Neisseria gonorrhoea and syphilis have been extensively examined, there remains a paucity of knowledge of nonchlamydial and nongonococcal cervicitis, an arguably more prevalent but poorly characterized condition with uncertain clinical implications. With increasing application of molecular diagnostic methods for the detection of sexually transmitted infections and a growing body of literature on cervicitis, a review is timely. RECENT FINDINGS: The number of putative aetiological agents implicated in cervicitis is growing and includes Mycoplasma genitalium, herpes simplex virus, cytomegalovirus, bacterial vaginosis and Trichomonas. The potential role of cervicitis in HIV transmission has been highlighted. Increasing broad-spectrum antibiotic usage with associated emergence of antimicrobial resistance reinforces the need for targeted antibiotic therapies, including the management of cervicitis. SUMMARY: As our understanding of the aetiology and significance of cervicitis, particularly nonspecific cervicitis, improves, management will be refined. Advances in molecular diagnostic testing will facilitate this process, but urinary nucleic acid amplification testing should not replace clinical examination while cervicitis prevalence and significance is not yet established. A standardized approach to cervicitis research, particularly with consensus of case definition, may facilitate outcomes that can be more generally applied in clinical practice.

The psychosocial evaluation of the hospice patient.

A psychosocial evaluation of a patient is an important first step in the provision of care to hospice patients and their families. It serves as a guide for intervention, communicates social work concerns to other caregivers, and articulates important features of the patient/family system from an interdisciplinary perspective. The psychosocial evaluation addresses the patient's problems in a historical framework by identifying important developmental features as they pertain to the tasks of dying. It identifies physical resources and needs and evaluates the patient's psychosocial functioning, with particular emphasis on the patient's roles within a family system. Hospice recognizes the uniqueness of each individual as he or she confronts life's final challenge. An adequate psychosocial evaluation brings that uniqueness into focus.

A deficiency of both subunits of pyruvate dehydrogenase which is not expressed in fibroblasts.

Pyruvate dehydrogenase complex (PDC) deficiency usually has been detected by decreased activity in cultured skin fibroblasts. We investigated two brothers in whom PDC activity was less than 10% of controls in lymphocytes but normal in skin fibroblasts. They both had abnormal neuromuscular development and lactic acidosis which was aggravated by ingestion of carbohydrate. One brother died at age 3 yr and tissues were obtained at autopsy soon after death. The brain was swollen with diffuse acute hemorrhages but without the lesions characteristic of Leigh's disease. PDC activity was virtually undetectable in mitochondria or homogenates of liver, skeletal muscle, and heart, but was about 30% of controls in kidney. The activity of the first component E1 was not detectable in mitochondria from liver, whereas the activities of the second and third components were normal; the activities of all components were normal in fibroblasts. Western immunoblot analysis showed absent to trace amounts of both the E1 alpha and E1 beta subunits in liver, skeletal muscle, and heart, with normal amounts of the second and third components. About one-fourth of control amounts of E1 alpha and E1 beta were present in kidney and normal levels were present in fibroblasts. PDC activity in lymphocytes from the mother was 35% of controls; she had normal PDC activity in her fibroblasts. PDC activity was normal in lymphocytes from the brothers' sister, father, and maternal grandparents and great-grandmother. The mode of inheritance was not established. In conclusion, PDC deficiency may not be detected in skin fibroblasts in some cases; the mechanism of variable tissue expression of E1 remains to be delineated.

Identification of two mutations in a compound heterozygous child with dihydrolipoamide dehydrogenase deficiency.

An infant girl with elevated blood lactate, pyruvate, and plasma branched-chain amino acids was diagnosed with dihydrolipoamide dehydrogenase (E3; dihydrolipoamide: NAD+ oxidoreductase, EC 1.8.1.4) deficiency. Activities of the pyruvate dehydrogenase complex and E3 from patient were 26 and 2% of controls in blood lymphocytes, and 11 and 14% in cultured skin fibroblasts, respectively. Western blot analysis demonstrated that the amount of E3 protein in fibroblasts from the patient and her father was about half of controls, while Northern blot analysis showed normal amounts of E3 RNA. DNA sequencing of cloned full-length E3 cDNAs from the patient revealed two mutations in separate alleles. One is a single base insertion of an extra adenine in the last codon of the leader peptide sequence (TAC-->TAAC) leading to a nonsense mutation which results in the premature termination of the precursor E3 polypeptide (Y35X). The other is a missense mutation due to substitution of guanine for adenine, causing an Arg-->Gly substitution at amino acid 460 of the mature protein (R460G) which triggers the loss of E3 activity probably by structural change in the E3 dimer. DNA sequencing of E3 cDNAs from the parents demonstrated that the nonsense mutation was inherited from the father and the missense mutation was inherited from the mother.

Three new mutations of the pyruvate dehydrogenase alpha subunit: a point mutation (M181V), 3 bp deletion (-R282), and 16 bp insertion/frameshift (K358SVS-->TVDQS).

Three novel mutations in the coding region of E1 alpha gene were found in three PDC-deficient male patients, including a missense mutation (M181V), a 3 bp deletion (AGA, corresponding to R282), and a 16 bp insertion (CAGTGGATCAAGTTTA), causing a frameshift starting with lysine 358 and resulting in decrease of both E1 subunits.

Pubertal alterations in growth and body composition. VI. Pubertal insulin resistance: relation to adiposity, body fat distribution and hormone release.

OBJECTIVE: To investigate the independent influence of alterations in fat mass, body fat distribution and hormone release on pubertal increases in fasting serum insulin concentrations and on insulin resistance assessed by the homeostasis model (HOMA). DESIGN AND SUBJECTS: Cross-sectional investigation of pre- (n=11, n=8), mid- (n=10, n=11), and late-pubertal (n=10, n=11) boys and girls with normal body weight and growth velocity. MEASUREMENTS: Body composition (by a four-compartment model), abdominal fat distribution and mid-thigh interfascicular plus intermuscle (extramyocellular) fat (by magnetic resonance imaging), total body subcutaneous fat (by skinfolds), mean nocturnal growth hormone (GH) release and 06:00 h samples of serum insulin, sex steroids, leptin and insulin-like growth factor-I (IGF-I). RESULTS: Pubertal insulin resistance was suggested by greater (P<0.001) fasting serum insulin concentrations in the late-pubertal than pre- and mid-pubertal groups while serum glucose concentrations were unchanged and greater (P<0.001) HOMA values in late-pubertal than pre- and mid-pubertal youth. From univariate correlation fat mass was most related to HOMA (r=0.59, P<0.001). Two hierarchical regression models were developed to predict HOMA. In one approach, subject differences in sex, pubertal maturation, height and weight were held constant by adding these variables as a block in the first step of the model (r(2)=0.36). Sequential addition of fat mass (FM) increased r(2) (r(2)((inc)remental)=0.08, r(2)=0.44, P<0.05) as did the subsequent addition of a block of fat distribution variables (extramyocellular fat, abdominal visceral fat, and sum of skinfolds; r(2)(inc)=0.11, r(2)=0.55, P<0.05). Sequential addition of a block of hormone variables (serum IGF-I and log((10)) leptin concentrations; r(2)(inc)=0.04, P>0.05) did not reliably improve r(2) beyond the physical characteristic and adiposity variables. In a second model, differences in sex and pubertal maturation were again held constant (r(2)=0.25), but body size differences were accounted for using percentage fat data. Sequential addition of percentage body fat (r(2)((inc)remental)=0.11, r(2)=0.36, P<0.05), then a block of fat distribution variables (percentage extramyocellular fat, percentage abdominal visceral fat, and percentage abdominal subcutaneous fat; r(2)(inc)=0.08, r(2)=0.44, P=0.058), and then a block of serum IGF-I and log((10)) leptin concentrations (r(2)(inc)=0.07, r(2)=0.51, P<0.05) increased r(2). Mean nocturnal GH release was not related to HOMA (r=-0.04, P=0.75) and therefore was not included in the hierarchical regression models. CONCLUSION: Increases in insulin resistance at puberty were most related to FM. Accumulation of fat in the abdominal visceral, subcutaneous and muscular compartments may increase insulin resistance at puberty beyond that due to total body fat. Serum concentrations of leptin and IGF-I may further modulate HOMA beyond the effects of adiposity and fat distribution. However, the results are limited by the cross-sectional design and the use of HOMA rather than a criterion measure of insulin resistance.

Systemic deficiency of the first component of the pyruvate dehydrogenase complex.

An infant with lactic acidosis and developmental delay had neuropathological changes consistent with Leigh's necrotizing encephalomyelopathy. Total pyruvate dehydrogenase complex (PDC) activity was low relative to controls in lymphocytes (0.2 versus 1.9 +/- 0.6 SD nmol/min/mg protein) and cultured skin fibroblasts (0.9 versus 2.7 +/- 1.0). Liver, muscle, heart, and kidney mitochondria oxidized several substrates normally, but did not oxidize pyruvate. PDC activity was absent in these mitochondria (0.1 versus 9.8 +/- 4.2 in liver and 0.7 versus 75 +/- 26 in muscle) and was very low in all tissue homogenates. Activity of the first component was low in liver mitochondria, whereas activities of the second and third components were normal. Western blot analysis of tissue proteins showed normal amounts of second and third component of PDC but undetectable to trace amounts of both alpha and beta subunits of the first component of PDC in liver, brain, kidney, heart, and skin fibroblasts. Thus, profound systemic deficiency of PDC was due to lack of both subunit proteins of the first component of PDC.

Molecular characterization of pyruvate carboxylase deficiency in two consanguineous families.

Pyruvate carboxylase (PC) is a biotinylated mitochondrial enzyme that catalyzes the conversion of pyruvate to oxaloacetate. Children with inborn errors of PC metabolism have lactic acidosis, hypoglycemia, and mental retardation. The variable severity of the clinical phenotype is dependent on both genetic and environmental factors. Two consanguineous families with moderate forms of PC deficiency were characterized at the biochemical and molecular levels. In both families, the probands were found to have low PC activity (range, 2-25% of control) in blood lymphocytes and skin fibroblasts associated with either diminished or normal protein levels. In the first case, sequencing of patient-specific PC cDNA demonstrated a T to C substitution at nucleotide 434, which causes a valine to alanine change at amino acid residue 145. Direct sequencing of the parents showed that they are heterozygous for this mutation. In the second family, a brother and sister had mental retardation and episodes of severe lactic/ketoacidosis in early childhood. In these cases, a C to T substitution at nucleotide 1351 results in a cysteine for arginine substitution at amino acid residue 451; the parents were also found to be heterozygous for this mutation. In both families, no other mutations were found, and both substitutions occurred in relatively conserved amino acid residues. These mutations, located in the biotin carboxylase domain, provide a unique opportunity to analyze how natural occurring mutations affect PC function.