Telomere length and mtDNA copy number in human cystathionine ß-synthase deficiency.

Telomere shortening and mitochondrial DNA (mtDNA) copy number are associated with human disease and a reduced life span. Cystathionine ß-synthase (CBS) is a housekeeping enzyme that catalyzes the first step in metabolic conversion of homocysteine (Hcy) to cysteine. Mutations in the CBS gene cause CBS deficiency, a rare recessive metabolic disease, manifested by severe hyperhomocysteinemia (HHcy) and thromboembolism, which ultimately reduces the life span. However, it was not known whether telomere shortening or mtDNA is involved in the pathology of human CBS deficiency. We quantified leukocyte telomere length (TL), mtDNA copy number, and plasma Hcy levels in CBS-/- patients (n = 23) and in sex- and age-matched unaffected CBS+/+ control individuals (n = 28) 0.08-57 years old. We found that TL was significantly increased in severely HHcy CBS-/- female patients but unaffected in severely HHcy CBS-/- male patients, relative to the corresponding CBS+/+ controls who had normal plasma Hcy levels. In multiple regression analysis TL was associated with CBS genotype in women but not in men. MtDNA copy number was not significantly affected by the CBS-/- genotype. Taken together, these findings identify the CBS gene as a new locus in human DNA that affects TL in women and illustrate a concept that a housekeeping metabolic gene can be involved in telomere biology. Our findings suggest that neither telomere shortening nor reduced mtDNA copy number contribute to the reduced life span in CBS-/- patients.

Serum Proteome Alterations in Human Cystathionine ß-Synthase Deficiency and Ischemic Stroke Subtypes.

Ischemic stroke induces brain injury via thrombotic or embolic mechanisms involving large or small vessels. Cystathionine ß-synthase deficiency (CBS), an inborn error of metabolism, is associated with vascular thromboembolism, the major cause of morbidity and mortality in affected patients. Because thromboembolism involves the brain vasculature in these patients, we hypothesize that CBS deficiency and ischemic stroke have similar molecular phenotypes. We used label-free mass spectrometry for quantification of changes in serum proteomes in CBS-deficient patients (n = 10) and gender/age-matched unaffected controls (n = 14), as well as in patients with cardioembolic (n = 17), large-vessel (n = 26), or lacunar (n = 25) ischemic stroke subtype. In CBS-deficient patients, 40 differentially expressed serum proteins were identified, of which 18 were associated with elevated homocysteine (Hcy) and 22 were Hcy-independent. We also identified Hcy-independent differentially expressed serum proteins in ischemic stroke patients, some of which were unique to a specific subtype: 10 of 32 for cardioembolic vs. large-vessel, six of 33 for cardioembolic vs. lacunar, and six of 23 for large-vessel vs. lacunar. There were significant overlaps between proteins affected by CBS deficiency and ischemic stroke, particularly the cardioembolic subtype, similar to protein overlaps between ischemic stroke subtypes. Top molecular pathways affected by CBS deficiency and ischemic stroke subtypes included acute phase response signaling and coagulation system. Similar molecular networks centering on NFκB were affected by CBS deficiency and stroke subtypes. These findings suggest common mechanisms involved in the pathologies of CBS deficiency and ischemic stroke subtypes.

Evidence for a relatively high proportion of DM2 mutations in a large group of Polish patients.

INTRODUCTION: Myotonic dystrophies (DMs) type 1 (DM1) and type 2 (DM2) are autosomal dominant, multisystem disorders, considered the most common dystrophies in adults. DM1 and DM2 are caused by dynamic mutations in the DMPK and CNBP genes, respectively. METHODS: Molecular analyses were performed by PCR and the modified RP-PCR in patients, in their at-risk relatives and prenatal cases. RESULTS: The analysis of Polish controls revealed the range of 5-31 CTG repeats for DM1 and 110-228 bp alleles for DM2. Among 318 confirmed probands - 196 (62%) were DM1 and 122 (38%) - DM2. Within DM1families, 10 subjects carried a low expanded CTG tract (< 100 repeats), which resulted in a full mutation in subsequent generations. Two related individuals had unstable alleles-188 bp and 196 bp without common interruptions. CONCLUSION: The relative frequencies of DM1/DM2 among Polish patients were 68% and 32%, respectively, with a relatively high proportion of DM2 mutations (1.6:1).

Simultaneous Determination of Methionine and Homocysteine by on-column derivatization with o-phtaldialdehyde.

A fast and simple HPLC-based assay has been developed for the simultaneous determination of homocysteine (Hcy) and methionine (Met) in plasma and urine samples, utilizing as small volume of sample as 10µL. The assay uses on-column derivatization with o-phthaldialdehyde. The separation of Hcy and Met was achieved in 14min on a reversed phase C-18 column, followed by fluorescence detection (excitation at 348nm and emission at 438nm for Met; excitation at 370nm and emission at 480nm for Hcy). Linearity of the detector response was observed in the range of 2-60 µmol L-1 for Met and 2-40 µmol L-1 for Hcy. The method was successfully applied for Met and Hcy quantification in human and mouse plasma and urine samples from cystathionine ß-synthase deficient and unaffected individuals.

Molecular spectrum of the SPAST, ATL1 and REEP1 gene mutations associated with the most common hereditary spastic paraplegias in a group of Polish patients.

Hereditary spastic paraplegias (HSPs) consist of a heterogeneous group of genetically determined neurodegenerative disorders. Progressive lower extremity weakness and spasticity are the prominent features of HSPs resulting from retrograde axonal degeneration of the corticospinal tracts. Three genetic types, SPG3 (ATL1), SPG4 (SPAST) and SPG31 (REEP1), appear predominantly and may account for up to 50% of autosomal dominant hereditary spastic paraplegias (AD-HSPs). Here, we present the results of genetic testing of the three mentioned SPG genetic types in a group of 216 unrelated Polish patients affected with spastic paraplegia. Molecular evaluation was performed by multiplex ligation-dependent probe amplification (MLPA) and DNA sequencing. Nineteen novel mutations: 13 in SPAST, 4 in ATL1 and 2 in REEP1, were identified among overall 50 different mutations detected in 57 families. Genetic analysis resulted in the identification of molecular defects in 54% of familial and 8.4% of isolated cases. Our research expanded the causative mutations spectrum of the three most common genetic forms of HSPs found in a large cohort of probands originating from the Central Europe.

Identification of N-homocysteinylation sites in plasma proteins.

A protocol for the identification of N-homocysteinylation sites in plasma proteins is described. Human plasma or purified fibrinogen is subjected to trypsin digestion and analysis of N-Hcy-peptides by liquid chromatography/mass spectroscopy (LC/MS). Human fibrinogen is isolated from the plasma by the glycine precipitation method. Identification of N-Hcy-Lys-peptides in tryptic digests of in vivo-derived samples is facilitated by the use of N-Hcy-albumin and N-Hcy-fibrinogen synthesized in vitro from commercially available human proteins. This protocol allows identification of N-homocysteinylation sites at Lys4, Lys12, Lys137, and Lys525 in albumin directly in trypsin-digested human serum samples. N-Hcy-Lys562, N-Hcy-Lys344, and N-Hcy-Lys385 were identified in human fibrinogen from patients with cystathionine ß-synthase deficiency. The protocol can be completed in 4 days.

beta-Carotene deficiency in cholestatic liver disease of childhood is caused by beta-carotene malabsorption.

BACKGROUND: : Depletion of beta-carotene (b-c) has not been extensively studied in children with chronic cholestatic liver disease. PATIENTS AND METHODS: : We assessed b-c serum concentration in 53 children with cholestatic liver disease: 19 patients operated on for biliary atresia, 12 with Alagille syndrome, and 22 with progressive familial intrahepatic cholestasis. To test b-c absorption, 6 children with chronic cholestasis received a load of 10 mg b-c/kg body weight. RESULTS: : We found decreased b-c concentrations in 45 patients. The absorption of b-c was not detectable in 5 of 6 children studied. CONCLUSIONS: : b-c depletion is a common problem of chronic cholestatic liver disease in childhood that can be attributed to disturbed intestinal absorption.

Clinical course of homozygous familial hypercholesterolemia during childhood: report on 4 unrelated patients with homozygous or compound heterozygous mutations in the LDLR gene.

Natural history of the disease in 4 unrelated Polish children with homozygous familial hypercholesterolemia (FH) is described. Their phenotypic homozygosity was established by identification of known LDLR gene mutations on both alleles, respectively: p.G592E & p.G592E in Patient 1; p.G592E & p.C667Y in Patient 2; p.S177L & p.R350X in Patient 3; and p.G592E & deletion in the promoter region, exons 1 and 2 in Patient 4. Heterozygosity of the mutations was revealed in all patients' mothers and fathers (obligatory heterozygotes) and in 1 out of 4 siblings studied. FH was diagnosed at the age of 4 months to 9 years by cholesterol screening among family members of patients with early cardiovascular disease episodes. At the time of FH detection, the children were asymptomatic. Only in 2, some skin eruptions were found. Antihyperlipidemic therapy was started, including a lipid-lowering diet, cholestyramine, and HMG-CoA inhibitors if necessary. No cardiovascular symptoms appeared during the observation up to the age of 18, 20, 19, and 17 years, respectively. An increase in external carotid artery diameter was found in a patient at the age of 9 years, and LDL-apheresis was introduced in his therapy. We conclude that the analysis of LDLR gene mutations in the studied FH children made it possible to identify 4 presymptomatic FH homozygotes and to introduce early appropriate treatment. Multicenter analysis of such persons would finally determine if the early lipid-lowering procedures can significantly reduce the risk of premature cardiovascular disease in homozygous FH.

The cystathionine beta-synthase (CBS) mutation c.1224-2A>C in Central Europe: Vitamin B6 nonresponsiveness and a common ancestral haplotype.

In homocystinuria due to cystathionine beta-synthase (CBS) deficiency, vitamin B6 response has been linked to distinct mutations and ruled out for others. The splice site mutation c.1224-2A>C leading to the deletion of exon 12 is predominantly found in patients from Central Europe, where it has been found on in average 14% of mutant alleles. In this study we analyzed the clinical picture in 17 CBS deficient carriers of c.1224-2A>C. Homozygotes for c.1224-2A>C did not respond to vitamin B6, while in compound heterozygotes the response to vitamin B6 depended on the mutation on the second allele. Maximum likelihood analysis revealed one common haplotype of the c.1224-2A>C alleles. Additionally, we report the four novel CBS mutations c.451G>A (p.Gly151?), c.740_769del (p.Lys247_Gly256del), c.862G>C (p.Ala288Pro) and c.1135C>T (p.Arg379Trp). In summary, the data of this study suggest that the CBS c.1224-2A>C allele confers vitamin B6 nonresponsiveness and that this mutant allele came from a common ancestor.

Identification and functional analysis of two novel mutations in the CBS gene in Polish patients with homocystinuria.

Homocystinuria due to cystathionine beta-synthase (CBS) deficiency is an inherited disorder of homocysteine transsulfuration, which manifests by neurological, vascular and connective tissue involvement. So far, 130 pathogenic mutations have been recognized in the CBS gene. We examined 10 independent alleles in Polish patients suffering from CBS deficiency, and we detected four already described mutations (c.1224-2A>C, c.684C>A, c.833T>C, and c.442G>A) and two novel mutations (c.429C>G and c.1039+1G>T). The pathogenicity of the novel mutations was demonstrated by expression in E.coli. This is the first published communication on mutations leading to CBS deficiency in Poland.