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Papers by Andrea Zatkova

Disruption of exonic splicing enhancer elements is the principal cause of exon skipping associated with seven nonsense or missense alleles of NF1

Human Mutation, 2004

Nonsense, missense, and even silent mutation-associated exon skipping is recognized in an increas... more Nonsense, missense, and even silent mutation-associated exon skipping is recognized in an increasing number of genes as a novel form of splicing mutation. The analysis of individual mutations of this kind can shed light on basic pre-mRNA splicing mechanisms. Using cDNA-based mutation detection analysis, we have identified one missense and six nonsense mutations that lead to different extents of exon-lacking transcripts in neurofibromatosis type 1 (NF1) patients. We confirmed mutation-associated exon skipping in a heterologous hybrid minigene context. There is evidence that the disruption of functional exonic splicing enhancer (ESE) sequences is frequently the mechanism underlying mutation-associated exon skipping. Therefore, we examined the wild-type and mutant NF1 sequences with two available ESE-prediction programs. Either or both programs predicted the disruption of ESE motifs in six out of the seven analyzed mutations. To ascertain the function of the predicted ESEs, we quantitatively measured their ability to rescue splicing of an enhancer-dependent exon, and found that all seven mutant ESEs had reduced splicing enhancement activity compared to the wild-type sequences. Our results suggest that the wild-type sequences function as ESE elements, whose disruption is responsible for the mutation-associated exon skipping observed in the NF1 patients. Further, this study illustrates the utility of ESE-prediction programs for delineating candidate sequences that may serve as ESE elements. However, until more refined prediction algorithms have been developed, experimental data, preferably from patient tissues, remain indispensable to assess the clinical significance, particularly of missense and silent mutations, and to understand the structure-function relationship in the corresponding protein.

Mutation Screening of theHGDGene Identifies a Novel Alkaptonuria Mutation with Significant Founder Effect and High Prevalence

Annals of Human Genetics, Feb 12, 2014

Alkaptonuria (AKU) is an autosomal recessive disorder; caused by the mutations in the homogentisa... more Alkaptonuria (AKU) is an autosomal recessive disorder; caused by the mutations in the homogentisate 1, 2-dioxygenase (HGD) gene located on Chromosome 3q13.33. AKU is a rare disorder with an incidence of 1: 250,000 to 1: 1,000,000, but Slovakia and the Dominican Republic have a relatively higher incidence of 1: 19,000. Our study focused on studying the frequency of AKU and identification of HGD gene mutations in nomads. HGD gene sequencing was used to identify the mutations in alkaptonurics. For the past four years, from subjects suspected to be clinically affected, we found 16 positive cases among a randomly selected cohort of 41 Indian nomads (Narikuravar) settled in the specific area of Tamil Nadu, India. HGD gene mutation analysis showed that 11 of these patients carry the same homozygous splicing mutation c.87 + 1G > A; in five cases, this mutation was found to be heterozygous, while the second AKU-causing mutation was not identified in these patients. This result indicates that the founder effect and high degree of consanguineous marriages have contributed to AKU among nomads. Eleven positive samples were homozygous for a novel mutation c.87 + 1G > A, that abolishes an intron 2 donor splice site and most likely causes skipping of exon 2. The prevalence of AKU observed earlier seems to be highly increased in people of nomadic origin.

Bratislavské lekárske listy, Feb 27, 2023

BACKGROUND: Familial combined hypolipidaemia is a condition characterised by very low concentrati... more BACKGROUND: Familial combined hypolipidaemia is a condition characterised by very low concentrations of circulating very-low-density lipoprotein (VLDL), low-density lipoprotein cholesterol (LDL), and highdensity lipoprotein cholesterol (HDL). It is thought that low LDL/combined hypolipidaemia can protect from cardiovascular disease (CVD), but this is not what we found in a case we present. OBJECTIVE: We report on a 57-years-old male patient with combined hypolipidaemia who presented with premature peripheral vascular disease. We investigated also his two sons, 32-and 27-years-old, who manifested a tendency to low lipid levels. METHODS AND RESULTS: We used Illumina exome analysis in all three individuals and in all of them we could exclude the major effect of the variants within the genes most frequently mutated in hypolipidaemia, including recently reported LIPC gene variant. Instead, in all three individuals we identifi ed a novel ABCA1 variant, possibly responsible for the decreased HDL levels. The proband and one of his sons also share the splicing APOC3 variant rs138326449, known to be associated with decreased TG levels. CONCLUSION: The heterogeneous nature and the risk of atherosclerosis in combined hypolipidaemia seems to be variable, based on an interplay between low HDL and LDL levels, and it depends on the combination of variants that cause it (Tab. 2, Ref. 38).

RMD Open

ObjectivesOchronotic spondyloarthropathy represents one of the main clinical manifestations of al... more ObjectivesOchronotic spondyloarthropathy represents one of the main clinical manifestations of alkaptonuria (AKU); however, prospective data and description of the effect of nitisinone treatment are lacking.MethodsPatients with AKU aged 25 years or older were randomly assigned to receive either oral nitisinone 10 mg/day (N=69) or no treatment (N=69). Spine radiographs were recorded yearly at baseline, 12, 24, 36 and 48 months, and the images were scored for the presence of intervertebral space narrowing, soft tissue calcifications, vacuum phenomena, osteophytes/hyperostosis and spinal fusion in the cervical, thoracic and lumbosacral segment at each of the time points.ResultsAt baseline, narrowing of the intervertebral spaces, the presence of osteophytes/hyperostosis and calcifications were the three most frequent radiographic features in AKU. The rate of progression of the five main features during the 4 years, ranked from the highest to lowest was as follows: intervertebral spaces ...

Endocrine Regulations

Alkaptonuria (AKU, OMIM, No. 203500) is a rare, slow-progressing, irreversible, multisystemic dis... more Alkaptonuria (AKU, OMIM, No. 203500) is a rare, slow-progressing, irreversible, multisystemic disease resulting from a deficiency of the homogentisate 1,2-dioxygenase enzyme, which leads to the accumulation of homogentisic acid (HGA) and subsequent deposition as pigment in connective tissues called ochronosis. As a result, severe arthropathy of large joints and spondyloarthropathy with frequent fractures, ligament ruptures, and osteoporosis develops in AKU patients. Since 2020, the first-time treatment with nitisinone has become available in the European Union. Nitisinone significantly reduces HGA production and arrests ochronosis in AKU patients. However, blocking of the tyrosine metabolic pathway by the drug leads to tyrosine plasma and tissue concentrations increase. The nitisinone-induced hypertyrosinemia can lead to the development of corneal keratopathy, and once it develops, the treatment needs to be interrupted. A decrease in overall protein intake reduces the risk of the ke...

Analysis of the SMN and NAIP Genes in Slovak Spinal Muscular Atrophy Patients

Human Heredity, 2000

We identified homozygous absence of exon 7 of the telomeric copy of the survival motor neuron gen... more We identified homozygous absence of exon 7 of the telomeric copy of the survival motor neuron gene (telSMN) in 88.4% (38/43) of spinal muscular atrophy (SMA) patients from Slovakia. Additional deletions within the neuronal apoptosis inhibitory protein (NAIP) gene were found in 38.5% of type I, 12.5% of type II and never in type III SMA patients. Neither the SMN nor the NAIP gene was deleted in 81 healthy relatives and 25 controls tested. In one family, pseudodominant inheritance was identified. Both the type III SMA father and type II SMA son carried the homozygous deletion of the telSMN gene. One SMA I patient showed an SMN hybrid gene, probably created by intrachromosomal deletion. In two haploidentical type II SMA sibs, the telSMN exon 7 was absent on one chromosome, while the other carried an A→G transition 96 bp upstream of exon 7 of the telSMN gene, a potential disease-causing mutation in these patients.

Metabolites, Oct 19, 2022

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

Molecularly confirmed pontocerebellar hypoplasia in a large family from Slovakia with four severely affected children

Bratislavské lekárske listy, 2022

Breakpoints characterisation of the genomic deletions identified by MLPA in alkaptonuria patients

Until recently, mainly DNA sequencing has been used to identify variants within the gene coding f... more Until recently, mainly DNA sequencing has been used to identify variants within the gene coding for homogentisate dioxygenase (HGD, 3q13.33) that cause alkaptonuria (AKU), an autosomal recessive inborn error of metabolism of tyrosine. In order to identify possible larger genomic deletions we have developed a novel Multiplex Ligation-dependent Probe Amplification (MLPA) assay specific for this gene (HGD-MLPA) and tested it successfully in healthy controls and in patients carrying two known previously identified HGD deletions. Subsequently, we analysed 22 AKU patients in whom only one or none classical HGD variant was found by sequencing. Using HGD-MLPA and sequencing, we identified four larger deletions encompassing from 1 to 4 exons of this gene and we defined their exact breakpoints: deletion of exons 1-4 (c.1-8460_282 + 6727del), deletion of exons 5 and 6 (c.283-9199_434 + 1688del), deletion of exon 11 (c.775-1915_879 + 1293del), and deletion of exon 13 (c.1007-1709_1188 + 1121del). We suggest including MLPA in the DNA diagnostic protocols for AKU in cases where DNA sequencing does not lead to identification of both HGD variants.

The Israel Medical Association journal : IMAJ, 2018

ments caused by alkaptonuria. The diagnosis was made by the typical black bone appearance seen at... more ments caused by alkaptonuria. The diagnosis was made by the typical black bone appearance seen at surgery.

Lakshminarayan R Ranganath, FRCP(Edin), FRCPath1, Anna M Milan PhD, FRCPath1, Andrew T Hughes MPh... more Lakshminarayan R Ranganath, FRCP(Edin), FRCPath1, Anna M Milan PhD, FRCPath1, Andrew T Hughes MPhil1, John J Dutton FIBMS1, Richard Fitzgerald MRCP2, Michael C Briggs FRCS3, Helen Bygott BSc1, Eftychia E Psarelli MSc4, Trevor F Cox PhD4, James A Gallagher PhD5, Jonathan C Jarvis PhD6, Christa van Kan7, Anthony K Hall MBBS, BSc, AKC8, Dinny Laan MSc7, Birgitta Olsson MSc9, Johan Szamosi MSc9, Mattias Rudebeck MSc, BMedSc9, Torbjörn Kullenberg MD9, Arvid Cronlund MSc9, Lennart Svensson PhD9, Carin Junestrand DDS9, Hana Ayoob BA10, Oliver G Timmis BA10, Nicolas Sireau PhD10, Kim-Hanh Le Quan Sang11, Federica Genovese PhD12, Daniela Braconi PhD13, Annalisa Santucci PhD13, Martina Nemethova MSc14, Andrea Zatkova PhD14, Judith McCaffrey MSc, Peter Christensen PhD, Gordon Ross PhD, Richard Imrich MD, PhD17, Jozef Rovensky17.

European Journal of Human Genetics, 2015

Alkaptonuria (AKU) is an autosomal recessive disorder caused by mutations in homogentisate-1,2-di... more Alkaptonuria (AKU) is an autosomal recessive disorder caused by mutations in homogentisate-1,2-dioxygenase (HGD) gene leading to the deficiency of HGD enzyme activity. The DevelopAKUre project is underway to test nitisinone as a specific treatment to counteract this derangement of the phenylalanine-tyrosine catabolic pathway. We analysed DNA of 40 AKU patients enrolled for SONIA1, the first study in DevelopAKUre, and of 59 other AKU patients sent to our laboratory for molecular diagnostics. We identified 12 novel DNA variants: one was identified in patients from Brazil (c.557T4A), Slovakia (c.500C4T) and France (c.440T4C), three in patients from India (c.469+6T4C, c.650-85A4G, c.158G4A), and six in patients from Italy (c.742A4G, c.614G4A, c.1057A4C, c.752G4A, c.119A4C, c.926G4T). Thus, the total number of potential AKU-causing variants found in 380 patients reported in the HGD mutation database is now 129. Using mCSM and DUET, computational approaches based on the protein 3D structure, the novel missense variants are predicted to affect the activity of the enzyme by three mechanisms: decrease of stability of individual protomers, disruption of protomer-protomer interactions or modification of residues in the region of the active site. We also present an overview of AKU in Italy, where so far about 60 AKU cases are known and DNA analysis has been reported for 34 of them. In this rather small group, 26 different HGD variants affecting function were described, indicating rather high heterogeneity. Twelve of these variants seem to be specific for Italy.

Molecular Genetics of Alkaptonuria

eLS, 2013

Alkaptonuria (AKU), the first defined human genetic disease with a recessive trait, is caused by ... more Alkaptonuria (AKU), the first defined human genetic disease with a recessive trait, is caused by mutations within the homogentisate 1,2-dioxygenase (HGD) gene (3q13.33). This prototypic inborn error of metabolism is characterised by typical bluish-black pigmentation in connective tissue ochronosis and severe form of osteoarthritis caused by the deposition of ochronotic pigment in the joints. AKU belongs to a group of rare diseases (1:250 000–1:1 000 000), however, several ethnities were reported, where an increased incidence of AKU was observed (Slovakia, Dominican Republic, Jordan and India). Mutation analysis was so far performed in approximately 350 out of more than 650 worldwide reported AKU patients. Rather high heterogeneity was observed with 122 AKU-causing mutations that are listed together with HGD polymorphisms in the global HGD mutation database (http://hgddatabase.cvtisr.sk/). Because HGD enzyme functions as hexamer, dimer of trimers, genotype/phenotype correlations are difficult to perform in this rare disease. Key Concepts: Alkaptonuria (AKU) is a prototypic inborn error in the metabolism of phenylalanine and tyrosine, characterised by the inability to metabolise homogentisic acid (HGA). The raised HGA levels in plasma and extracellular fluid lead to ochronosis, the deposition of polymers of HGA as pigment (ochronotic pigment) in connective tissues including cartilage, heart valves and sclera. Ochronosis leads to painful destruction of large weight-bearing joints as well as fusion of the vertebrae, scoliosis and tendon and ligament ruptures. AKU is caused by homozygous or compound heterozygous mutations in the homogentisate-1,2-dioxygenase gene (HGD) mapping to the chromosome 3q13.33. AKU belongs to a group of rare diseases (1:250 000–1:1 000 000), however, several ethnities were reported, where an increased incidence of AKU was observed (Slovakia, Dominican Republic, Jordan and India). In approximately 350 patients reported worldwide so far 122 different HGD mutations have been reported. It was also shown that AKU is caused also by the apparently partial loss-of-function mutations, however, the heterozygous carriers of AKU are healthy. HGD haplotype analysis helps to identify the origin of individual AKU-causing mutations in different countries. The triketone herbicide nitisinone or Orfadin inhibits the 4-hydroxyphenylpyruvate dioxygenase enzyme, which produces HGA, thus, it can decrease HGA and should therefore potentially be able to modify AKU. It has been shown that AKU is a novel type II AA amyloidosis, which opens new important perspectives for its therapy, since the control of the underlying inflammatory disorder can result in regression of the disease. Research on ochronosis in this monogenic disease can help to elucidate the molecular pathogenesis of the more common varieties of osteoarthritis, particularly the biochemical and structural changes at its initial stages. Keywords: AKU; alkaptonuria; HGD mutations; HGD mutation database; homogentisate 1,2-dioxygenase

A patient with de novo AML M1 and t(16;21) with karyotype evolution

Leukemia Research, 2007

Journal of Inherited Metabolic Disease, 2011

Alkaptonuria (AKU) is an autosomal recessive disorder caused by a deficiency of homogentisate 1,2... more Alkaptonuria (AKU) is an autosomal recessive disorder caused by a deficiency of homogentisate 1,2 dioxygenase (HGD) and characterized by homogentisic aciduria, ochronosis, and ochronotic arthritis. The defect is caused by mutations in the HGD gene, which maps to the human chromosome 3q21-q23. AKU shows a very low prevalence (1:100,000-250,000) in most ethnic groups, but there are countries such as Slovakia and the Dominican Republic in which the incidence of this disorder rises to as much as 1:19,000. In this work, we summarize the genetic aspects of AKU in general and the distribution of all known disease-causing mutations reported so far. We focus on special features of AKU in Slovakia, which is one of the countries with an increased incidence of this rare metabolic disorder.

Thirty-Nine Novel Neurofibromatosis 1 ( NF1) Gene Mutations Identified in Slovak Patients

Annals of Human Genetics, 2013

We performed a complex analysis of the neurofibromatosis type 1 (NF1) gene in Slovakia based on d... more We performed a complex analysis of the neurofibromatosis type 1 (NF1) gene in Slovakia based on direct cDNA sequencing supplemented by multiple ligation dependent probe amplification (MLPA) analysis. All 108 patients had café-au-lait spots, 85% had axilary and/or inguinal freckling, 61% neurofibromas, 36% Lisch nodules of the iris and 31% optic pathway glioma, 5% suffered from typical skeletal disorders, and 51% of patients had family members with NF1. In 78 of the 86 (90.7%) index patients our analysis revealed the presence of NF1 mutations, 68 of which were small changes (87.2%), including 39 (50%) novel. Among the identified mutations the most prevalent were small deletions and insertions causing frameshift (42.3%), followed by nonsense (14.1%), missense (12.8%), and typical splicing (11.5%) mutations. Type 1 NF1 deletions and intragenic deletions/duplication were identified in five cases each (6.4%). Interestingly, in five other cases nontypical splicing variants were found, whose real effect on NF1 transcript would have remained undetected if using a DNA-based method alone, thus underlying the advantage of using the cDNA-based sequencing. We show that Slovak NF1 patients have a similar repertoire of NF1 germline mutations compared to other populations, with some prevalence of small deletions/insertions and a decreased proportion of nonsense mutations.

Acta Facultatis Pharmaceuticae Universitatis Comenianae, 2015

Alkaptonuria (AKU) is the first described inborn error of metabolism and a classical example of r... more Alkaptonuria (AKU) is the first described inborn error of metabolism and a classical example of rare autosomal recessive disease. AKU patients carry homozygous or compound heterozygous mutations of the gene coding for enzyme homogentisate dioxygenase (HGD) involved in metabolism of tyrosine. The metabolic block in AKU causes accumulation of homogentisic acid (HGA) that, with advancing age of the patient, leads to severe and painful ochronotic arthropathy.

Alkaptonuria (AKU) is characterised by a typical bluish-black pigmentation in connective tissue (... more Alkaptonuria (AKU) is characterised by a typical bluish-black pigmentation in connective tissue (ochronosis) that usually occurs after the age of 30 years. AKU is the first inborn error of metabolism to be understood as a recessive trait. It is caused by mutations within the gene located on the human chromosome 3q13.33, coding for the enzyme homogentisate 1,2-dioxygenase (HGD). About 650 AKU patients have been reported worldwide, and mutation analysis performed so far in about 270 cases shows a rather high heterogeneity, since 117 AKU-causing mutations have been found, also summarized in a novel HGD mutation database. Several ethnicities have been reported in which an increased incidence of AKU is observed, compared to its worldwide low prevalence (1 : 250 000 – 1 : 1 000 000). S t r e s z c z e n i e

Alkaptonuria (AKU) is a disorder of phenylalanine/tyrosine metabolism due to a defect in the enzy... more Alkaptonuria (AKU) is a disorder of phenylalanine/tyrosine metabolism due to a defect in the enzyme homogentisate 1,2-dioxygenase (HGD). This recessive disease is caused by mutations in the HGD gene. We report a 14-year-old girl who was referred after presenting black urine. Careful examination revealed ochronosis of the conjunctiva. There was no affection of the cardiac valves. Elevated excretion of homogentisic acid in urine was found. Sequence analysis of the HGD gene from genomic DNA revealed that the patient is a compound heterozygote with a previously described mutation (c.473C>T, p.Pro158Leu), and a novel one (c.821C>T, p.Pro274Leu). Her mother is heterozygous for the novel mutation, while the brother is heterozygous for the previously described mutation. In summary, we describe an alkaptonuric patient with ocular ochronosis and a novel HGD mutation, c.821C>T, p.Pro274Leu.

Alkaptonuria in Russia

European Journal of Human Genetics

Alkaptonuria is characterized by the accumulation of homogentisic acid (HGA), part of which is ex... more Alkaptonuria is characterized by the accumulation of homogentisic acid (HGA), part of which is excreted in the urine but the excess HGA forms a dark brown ochronotic pigment that deposits in the connective tissue (ochronosis), eventually leading to early-onset severe arthropathy. We analyzed a cohort of 48 Russian AKU families by sequencing all 14 exons (including flanking intronic sequences) of the homogentisate 1,2-dioxygenase gene (HGD) and Multiplex Ligation-dependent Probe Amplification (MLPA) analysis. Nine novel likely pathogenic HGD variants were identified, which have not been reported previously in any other country. Recently, Bychkov et al. [1] reported on the variant spectrum in another cohort of 49 Russian AKU patients. Here we summarize complete data from both cohorts that include 82 Russian AKU families. Taken together, 31 different HGD variants were found in these patients, of which 14 are novel and found only in Russia. The most common variant was c.481G>A (p.(Gly161Arg)), present in almost 54% of all AKU alleles.

Disruption of exonic splicing enhancer elements is the principal cause of exon skipping associated with seven nonsense or missense alleles of NF1

Human Mutation, 2004

Nonsense, missense, and even silent mutation-associated exon skipping is recognized in an increas... more Nonsense, missense, and even silent mutation-associated exon skipping is recognized in an increasing number of genes as a novel form of splicing mutation. The analysis of individual mutations of this kind can shed light on basic pre-mRNA splicing mechanisms. Using cDNA-based mutation detection analysis, we have identified one missense and six nonsense mutations that lead to different extents of exon-lacking transcripts in neurofibromatosis type 1 (NF1) patients. We confirmed mutation-associated exon skipping in a heterologous hybrid minigene context. There is evidence that the disruption of functional exonic splicing enhancer (ESE) sequences is frequently the mechanism underlying mutation-associated exon skipping. Therefore, we examined the wild-type and mutant NF1 sequences with two available ESE-prediction programs. Either or both programs predicted the disruption of ESE motifs in six out of the seven analyzed mutations. To ascertain the function of the predicted ESEs, we quantitatively measured their ability to rescue splicing of an enhancer-dependent exon, and found that all seven mutant ESEs had reduced splicing enhancement activity compared to the wild-type sequences. Our results suggest that the wild-type sequences function as ESE elements, whose disruption is responsible for the mutation-associated exon skipping observed in the NF1 patients. Further, this study illustrates the utility of ESE-prediction programs for delineating candidate sequences that may serve as ESE elements. However, until more refined prediction algorithms have been developed, experimental data, preferably from patient tissues, remain indispensable to assess the clinical significance, particularly of missense and silent mutations, and to understand the structure-function relationship in the corresponding protein.

Mutation Screening of theHGDGene Identifies a Novel Alkaptonuria Mutation with Significant Founder Effect and High Prevalence

Annals of Human Genetics, Feb 12, 2014

Alkaptonuria (AKU) is an autosomal recessive disorder; caused by the mutations in the homogentisa... more Alkaptonuria (AKU) is an autosomal recessive disorder; caused by the mutations in the homogentisate 1, 2-dioxygenase (HGD) gene located on Chromosome 3q13.33. AKU is a rare disorder with an incidence of 1: 250,000 to 1: 1,000,000, but Slovakia and the Dominican Republic have a relatively higher incidence of 1: 19,000. Our study focused on studying the frequency of AKU and identification of HGD gene mutations in nomads. HGD gene sequencing was used to identify the mutations in alkaptonurics. For the past four years, from subjects suspected to be clinically affected, we found 16 positive cases among a randomly selected cohort of 41 Indian nomads (Narikuravar) settled in the specific area of Tamil Nadu, India. HGD gene mutation analysis showed that 11 of these patients carry the same homozygous splicing mutation c.87 + 1G > A; in five cases, this mutation was found to be heterozygous, while the second AKU-causing mutation was not identified in these patients. This result indicates that the founder effect and high degree of consanguineous marriages have contributed to AKU among nomads. Eleven positive samples were homozygous for a novel mutation c.87 + 1G > A, that abolishes an intron 2 donor splice site and most likely causes skipping of exon 2. The prevalence of AKU observed earlier seems to be highly increased in people of nomadic origin.

Bratislavské lekárske listy, Feb 27, 2023

BACKGROUND: Familial combined hypolipidaemia is a condition characterised by very low concentrati... more BACKGROUND: Familial combined hypolipidaemia is a condition characterised by very low concentrations of circulating very-low-density lipoprotein (VLDL), low-density lipoprotein cholesterol (LDL), and highdensity lipoprotein cholesterol (HDL). It is thought that low LDL/combined hypolipidaemia can protect from cardiovascular disease (CVD), but this is not what we found in a case we present. OBJECTIVE: We report on a 57-years-old male patient with combined hypolipidaemia who presented with premature peripheral vascular disease. We investigated also his two sons, 32-and 27-years-old, who manifested a tendency to low lipid levels. METHODS AND RESULTS: We used Illumina exome analysis in all three individuals and in all of them we could exclude the major effect of the variants within the genes most frequently mutated in hypolipidaemia, including recently reported LIPC gene variant. Instead, in all three individuals we identifi ed a novel ABCA1 variant, possibly responsible for the decreased HDL levels. The proband and one of his sons also share the splicing APOC3 variant rs138326449, known to be associated with decreased TG levels. CONCLUSION: The heterogeneous nature and the risk of atherosclerosis in combined hypolipidaemia seems to be variable, based on an interplay between low HDL and LDL levels, and it depends on the combination of variants that cause it (Tab. 2, Ref. 38).

RMD Open

ObjectivesOchronotic spondyloarthropathy represents one of the main clinical manifestations of al... more ObjectivesOchronotic spondyloarthropathy represents one of the main clinical manifestations of alkaptonuria (AKU); however, prospective data and description of the effect of nitisinone treatment are lacking.MethodsPatients with AKU aged 25 years or older were randomly assigned to receive either oral nitisinone 10 mg/day (N=69) or no treatment (N=69). Spine radiographs were recorded yearly at baseline, 12, 24, 36 and 48 months, and the images were scored for the presence of intervertebral space narrowing, soft tissue calcifications, vacuum phenomena, osteophytes/hyperostosis and spinal fusion in the cervical, thoracic and lumbosacral segment at each of the time points.ResultsAt baseline, narrowing of the intervertebral spaces, the presence of osteophytes/hyperostosis and calcifications were the three most frequent radiographic features in AKU. The rate of progression of the five main features during the 4 years, ranked from the highest to lowest was as follows: intervertebral spaces ...

Endocrine Regulations

Alkaptonuria (AKU, OMIM, No. 203500) is a rare, slow-progressing, irreversible, multisystemic dis... more Alkaptonuria (AKU, OMIM, No. 203500) is a rare, slow-progressing, irreversible, multisystemic disease resulting from a deficiency of the homogentisate 1,2-dioxygenase enzyme, which leads to the accumulation of homogentisic acid (HGA) and subsequent deposition as pigment in connective tissues called ochronosis. As a result, severe arthropathy of large joints and spondyloarthropathy with frequent fractures, ligament ruptures, and osteoporosis develops in AKU patients. Since 2020, the first-time treatment with nitisinone has become available in the European Union. Nitisinone significantly reduces HGA production and arrests ochronosis in AKU patients. However, blocking of the tyrosine metabolic pathway by the drug leads to tyrosine plasma and tissue concentrations increase. The nitisinone-induced hypertyrosinemia can lead to the development of corneal keratopathy, and once it develops, the treatment needs to be interrupted. A decrease in overall protein intake reduces the risk of the ke...

Analysis of the SMN and NAIP Genes in Slovak Spinal Muscular Atrophy Patients

Human Heredity, 2000

We identified homozygous absence of exon 7 of the telomeric copy of the survival motor neuron gen... more We identified homozygous absence of exon 7 of the telomeric copy of the survival motor neuron gene (telSMN) in 88.4% (38/43) of spinal muscular atrophy (SMA) patients from Slovakia. Additional deletions within the neuronal apoptosis inhibitory protein (NAIP) gene were found in 38.5% of type I, 12.5% of type II and never in type III SMA patients. Neither the SMN nor the NAIP gene was deleted in 81 healthy relatives and 25 controls tested. In one family, pseudodominant inheritance was identified. Both the type III SMA father and type II SMA son carried the homozygous deletion of the telSMN gene. One SMA I patient showed an SMN hybrid gene, probably created by intrachromosomal deletion. In two haploidentical type II SMA sibs, the telSMN exon 7 was absent on one chromosome, while the other carried an A→G transition 96 bp upstream of exon 7 of the telSMN gene, a potential disease-causing mutation in these patients.

Metabolites, Oct 19, 2022

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

Molecularly confirmed pontocerebellar hypoplasia in a large family from Slovakia with four severely affected children

Bratislavské lekárske listy, 2022

Breakpoints characterisation of the genomic deletions identified by MLPA in alkaptonuria patients

Until recently, mainly DNA sequencing has been used to identify variants within the gene coding f... more Until recently, mainly DNA sequencing has been used to identify variants within the gene coding for homogentisate dioxygenase (HGD, 3q13.33) that cause alkaptonuria (AKU), an autosomal recessive inborn error of metabolism of tyrosine. In order to identify possible larger genomic deletions we have developed a novel Multiplex Ligation-dependent Probe Amplification (MLPA) assay specific for this gene (HGD-MLPA) and tested it successfully in healthy controls and in patients carrying two known previously identified HGD deletions. Subsequently, we analysed 22 AKU patients in whom only one or none classical HGD variant was found by sequencing. Using HGD-MLPA and sequencing, we identified four larger deletions encompassing from 1 to 4 exons of this gene and we defined their exact breakpoints: deletion of exons 1-4 (c.1-8460_282 + 6727del), deletion of exons 5 and 6 (c.283-9199_434 + 1688del), deletion of exon 11 (c.775-1915_879 + 1293del), and deletion of exon 13 (c.1007-1709_1188 + 1121del). We suggest including MLPA in the DNA diagnostic protocols for AKU in cases where DNA sequencing does not lead to identification of both HGD variants.

The Israel Medical Association journal : IMAJ, 2018

ments caused by alkaptonuria. The diagnosis was made by the typical black bone appearance seen at... more ments caused by alkaptonuria. The diagnosis was made by the typical black bone appearance seen at surgery.

Lakshminarayan R Ranganath, FRCP(Edin), FRCPath1, Anna M Milan PhD, FRCPath1, Andrew T Hughes MPh... more Lakshminarayan R Ranganath, FRCP(Edin), FRCPath1, Anna M Milan PhD, FRCPath1, Andrew T Hughes MPhil1, John J Dutton FIBMS1, Richard Fitzgerald MRCP2, Michael C Briggs FRCS3, Helen Bygott BSc1, Eftychia E Psarelli MSc4, Trevor F Cox PhD4, James A Gallagher PhD5, Jonathan C Jarvis PhD6, Christa van Kan7, Anthony K Hall MBBS, BSc, AKC8, Dinny Laan MSc7, Birgitta Olsson MSc9, Johan Szamosi MSc9, Mattias Rudebeck MSc, BMedSc9, Torbjörn Kullenberg MD9, Arvid Cronlund MSc9, Lennart Svensson PhD9, Carin Junestrand DDS9, Hana Ayoob BA10, Oliver G Timmis BA10, Nicolas Sireau PhD10, Kim-Hanh Le Quan Sang11, Federica Genovese PhD12, Daniela Braconi PhD13, Annalisa Santucci PhD13, Martina Nemethova MSc14, Andrea Zatkova PhD14, Judith McCaffrey MSc, Peter Christensen PhD, Gordon Ross PhD, Richard Imrich MD, PhD17, Jozef Rovensky17.

European Journal of Human Genetics, 2015

Alkaptonuria (AKU) is an autosomal recessive disorder caused by mutations in homogentisate-1,2-di... more Alkaptonuria (AKU) is an autosomal recessive disorder caused by mutations in homogentisate-1,2-dioxygenase (HGD) gene leading to the deficiency of HGD enzyme activity. The DevelopAKUre project is underway to test nitisinone as a specific treatment to counteract this derangement of the phenylalanine-tyrosine catabolic pathway. We analysed DNA of 40 AKU patients enrolled for SONIA1, the first study in DevelopAKUre, and of 59 other AKU patients sent to our laboratory for molecular diagnostics. We identified 12 novel DNA variants: one was identified in patients from Brazil (c.557T4A), Slovakia (c.500C4T) and France (c.440T4C), three in patients from India (c.469+6T4C, c.650-85A4G, c.158G4A), and six in patients from Italy (c.742A4G, c.614G4A, c.1057A4C, c.752G4A, c.119A4C, c.926G4T). Thus, the total number of potential AKU-causing variants found in 380 patients reported in the HGD mutation database is now 129. Using mCSM and DUET, computational approaches based on the protein 3D structure, the novel missense variants are predicted to affect the activity of the enzyme by three mechanisms: decrease of stability of individual protomers, disruption of protomer-protomer interactions or modification of residues in the region of the active site. We also present an overview of AKU in Italy, where so far about 60 AKU cases are known and DNA analysis has been reported for 34 of them. In this rather small group, 26 different HGD variants affecting function were described, indicating rather high heterogeneity. Twelve of these variants seem to be specific for Italy.

Molecular Genetics of Alkaptonuria

eLS, 2013

Alkaptonuria (AKU), the first defined human genetic disease with a recessive trait, is caused by ... more Alkaptonuria (AKU), the first defined human genetic disease with a recessive trait, is caused by mutations within the homogentisate 1,2-dioxygenase (HGD) gene (3q13.33). This prototypic inborn error of metabolism is characterised by typical bluish-black pigmentation in connective tissue ochronosis and severe form of osteoarthritis caused by the deposition of ochronotic pigment in the joints. AKU belongs to a group of rare diseases (1:250 000–1:1 000 000), however, several ethnities were reported, where an increased incidence of AKU was observed (Slovakia, Dominican Republic, Jordan and India). Mutation analysis was so far performed in approximately 350 out of more than 650 worldwide reported AKU patients. Rather high heterogeneity was observed with 122 AKU-causing mutations that are listed together with HGD polymorphisms in the global HGD mutation database (http://hgddatabase.cvtisr.sk/). Because HGD enzyme functions as hexamer, dimer of trimers, genotype/phenotype correlations are difficult to perform in this rare disease. Key Concepts: Alkaptonuria (AKU) is a prototypic inborn error in the metabolism of phenylalanine and tyrosine, characterised by the inability to metabolise homogentisic acid (HGA). The raised HGA levels in plasma and extracellular fluid lead to ochronosis, the deposition of polymers of HGA as pigment (ochronotic pigment) in connective tissues including cartilage, heart valves and sclera. Ochronosis leads to painful destruction of large weight-bearing joints as well as fusion of the vertebrae, scoliosis and tendon and ligament ruptures. AKU is caused by homozygous or compound heterozygous mutations in the homogentisate-1,2-dioxygenase gene (HGD) mapping to the chromosome 3q13.33. AKU belongs to a group of rare diseases (1:250 000–1:1 000 000), however, several ethnities were reported, where an increased incidence of AKU was observed (Slovakia, Dominican Republic, Jordan and India). In approximately 350 patients reported worldwide so far 122 different HGD mutations have been reported. It was also shown that AKU is caused also by the apparently partial loss-of-function mutations, however, the heterozygous carriers of AKU are healthy. HGD haplotype analysis helps to identify the origin of individual AKU-causing mutations in different countries. The triketone herbicide nitisinone or Orfadin inhibits the 4-hydroxyphenylpyruvate dioxygenase enzyme, which produces HGA, thus, it can decrease HGA and should therefore potentially be able to modify AKU. It has been shown that AKU is a novel type II AA amyloidosis, which opens new important perspectives for its therapy, since the control of the underlying inflammatory disorder can result in regression of the disease. Research on ochronosis in this monogenic disease can help to elucidate the molecular pathogenesis of the more common varieties of osteoarthritis, particularly the biochemical and structural changes at its initial stages. Keywords: AKU; alkaptonuria; HGD mutations; HGD mutation database; homogentisate 1,2-dioxygenase

A patient with de novo AML M1 and t(16;21) with karyotype evolution

Leukemia Research, 2007

Journal of Inherited Metabolic Disease, 2011

Alkaptonuria (AKU) is an autosomal recessive disorder caused by a deficiency of homogentisate 1,2... more Alkaptonuria (AKU) is an autosomal recessive disorder caused by a deficiency of homogentisate 1,2 dioxygenase (HGD) and characterized by homogentisic aciduria, ochronosis, and ochronotic arthritis. The defect is caused by mutations in the HGD gene, which maps to the human chromosome 3q21-q23. AKU shows a very low prevalence (1:100,000-250,000) in most ethnic groups, but there are countries such as Slovakia and the Dominican Republic in which the incidence of this disorder rises to as much as 1:19,000. In this work, we summarize the genetic aspects of AKU in general and the distribution of all known disease-causing mutations reported so far. We focus on special features of AKU in Slovakia, which is one of the countries with an increased incidence of this rare metabolic disorder.

Thirty-Nine Novel Neurofibromatosis 1 ( NF1) Gene Mutations Identified in Slovak Patients

Annals of Human Genetics, 2013

We performed a complex analysis of the neurofibromatosis type 1 (NF1) gene in Slovakia based on d... more We performed a complex analysis of the neurofibromatosis type 1 (NF1) gene in Slovakia based on direct cDNA sequencing supplemented by multiple ligation dependent probe amplification (MLPA) analysis. All 108 patients had café-au-lait spots, 85% had axilary and/or inguinal freckling, 61% neurofibromas, 36% Lisch nodules of the iris and 31% optic pathway glioma, 5% suffered from typical skeletal disorders, and 51% of patients had family members with NF1. In 78 of the 86 (90.7%) index patients our analysis revealed the presence of NF1 mutations, 68 of which were small changes (87.2%), including 39 (50%) novel. Among the identified mutations the most prevalent were small deletions and insertions causing frameshift (42.3%), followed by nonsense (14.1%), missense (12.8%), and typical splicing (11.5%) mutations. Type 1 NF1 deletions and intragenic deletions/duplication were identified in five cases each (6.4%). Interestingly, in five other cases nontypical splicing variants were found, whose real effect on NF1 transcript would have remained undetected if using a DNA-based method alone, thus underlying the advantage of using the cDNA-based sequencing. We show that Slovak NF1 patients have a similar repertoire of NF1 germline mutations compared to other populations, with some prevalence of small deletions/insertions and a decreased proportion of nonsense mutations.

Acta Facultatis Pharmaceuticae Universitatis Comenianae, 2015

Alkaptonuria (AKU) is the first described inborn error of metabolism and a classical example of r... more Alkaptonuria (AKU) is the first described inborn error of metabolism and a classical example of rare autosomal recessive disease. AKU patients carry homozygous or compound heterozygous mutations of the gene coding for enzyme homogentisate dioxygenase (HGD) involved in metabolism of tyrosine. The metabolic block in AKU causes accumulation of homogentisic acid (HGA) that, with advancing age of the patient, leads to severe and painful ochronotic arthropathy.

Alkaptonuria (AKU) is characterised by a typical bluish-black pigmentation in connective tissue (... more Alkaptonuria (AKU) is characterised by a typical bluish-black pigmentation in connective tissue (ochronosis) that usually occurs after the age of 30 years. AKU is the first inborn error of metabolism to be understood as a recessive trait. It is caused by mutations within the gene located on the human chromosome 3q13.33, coding for the enzyme homogentisate 1,2-dioxygenase (HGD). About 650 AKU patients have been reported worldwide, and mutation analysis performed so far in about 270 cases shows a rather high heterogeneity, since 117 AKU-causing mutations have been found, also summarized in a novel HGD mutation database. Several ethnicities have been reported in which an increased incidence of AKU is observed, compared to its worldwide low prevalence (1 : 250 000 – 1 : 1 000 000). S t r e s z c z e n i e

Alkaptonuria (AKU) is a disorder of phenylalanine/tyrosine metabolism due to a defect in the enzy... more Alkaptonuria (AKU) is a disorder of phenylalanine/tyrosine metabolism due to a defect in the enzyme homogentisate 1,2-dioxygenase (HGD). This recessive disease is caused by mutations in the HGD gene. We report a 14-year-old girl who was referred after presenting black urine. Careful examination revealed ochronosis of the conjunctiva. There was no affection of the cardiac valves. Elevated excretion of homogentisic acid in urine was found. Sequence analysis of the HGD gene from genomic DNA revealed that the patient is a compound heterozygote with a previously described mutation (c.473C>T, p.Pro158Leu), and a novel one (c.821C>T, p.Pro274Leu). Her mother is heterozygous for the novel mutation, while the brother is heterozygous for the previously described mutation. In summary, we describe an alkaptonuric patient with ocular ochronosis and a novel HGD mutation, c.821C>T, p.Pro274Leu.

Alkaptonuria in Russia

European Journal of Human Genetics

Alkaptonuria is characterized by the accumulation of homogentisic acid (HGA), part of which is ex... more Alkaptonuria is characterized by the accumulation of homogentisic acid (HGA), part of which is excreted in the urine but the excess HGA forms a dark brown ochronotic pigment that deposits in the connective tissue (ochronosis), eventually leading to early-onset severe arthropathy. We analyzed a cohort of 48 Russian AKU families by sequencing all 14 exons (including flanking intronic sequences) of the homogentisate 1,2-dioxygenase gene (HGD) and Multiplex Ligation-dependent Probe Amplification (MLPA) analysis. Nine novel likely pathogenic HGD variants were identified, which have not been reported previously in any other country. Recently, Bychkov et al. [1] reported on the variant spectrum in another cohort of 49 Russian AKU patients. Here we summarize complete data from both cohorts that include 82 Russian AKU families. Taken together, 31 different HGD variants were found in these patients, of which 14 are novel and found only in Russia. The most common variant was c.481G>A (p.(Gly161Arg)), present in almost 54% of all AKU alleles.