Identification of SLC7A7, encoding y+LAT-1, as the lysinuric protein intolerance gene.

Lysinuric protein intolerance (LPI; OMIM 222700) is a rare, recessive disorder with a worldwide distribution, but with a high prevalence in the Finnish population; symptoms include failure to thrive, growth retardation, muscle hypotonia and hepatosplenomegaly. A defect in the plasma membrane transport of dibasic amino acids has been demonstrated at the baso-lateral membrane of epithelial cells in small intestine and in renal tubules and in plasma membrane of cultured skin fibroblasts from LPI patients. The gene causing LPI has been assigned by linkage analysis to 14q11-13. Here we report mutations in SLC7A7 cDNA (encoding y+L amino acid transporter-1, y+LAT-1), which expresses dibasic amino-acid transport activity and is located in the LPI region, in 31 Finnish LPI patients and 1 Spanish patient. The Finnish patients are homozygous for a founder missense mutation leading to a premature stop codon. The Spanish patient is a compound heterozygote with a missense mutation in one allele and a frameshift mutation in the other. The frameshift mutation generates a premature stop codon, eliminating the last one-third of the protein. The missense mutation abolishes y+LAT-1 amino-acid transport activity when co-expressed with the heavy chain of the cell-surface antigen 4F2 (4F2hc, also known as CD98) in Xenopus laevis oocytes. Our data establish that mutations in SLC7A7 cause LPI.

Population structure in contemporary Sweden--a Y-chromosomal and mitochondrial DNA analysis.

A population sample representing the current Swedish population was analysed for maternally and paternally inherited markers with the aim of characterizing genetic variation and population structure. The sample set of 820 females and 883 males were extracted and amplified from Guthrie cards of all the children born in Sweden during one week in 2003. 14 Y-chromosomal and 34 mitochondrial DNA SNPs were genotyped. The haplogroup frequencies of the counties closest to Finland, Norway, Denmark and the Saami region in the north exhibited similarities to the neighbouring populations, resulting from the formation of the Swedish nation during the past millennium. Moreover, the recent immigration waves of the 20th century are visible in haplogroup frequencies, and have led to increased diversity and divergence of the major cities. Signs of genetic drift can be detected in several counties in northern as well as in southern Sweden. With the exception of the most drifted subpopulations, the population structure in Sweden appears mostly clinal. In conclusion, our study yielded valuable information of the structure of the Swedish population, and demonstrated the usefulness of biobanks as a source of population genetic research. Our sampling strategy, nonselective on the current population rather than stratified according to ancestry, is informative for capturing the contemporary variation in the increasingly panmictic populations of the world.

Nutrient intake in lysinuric protein intolerance.

Lysinuric protein intolerance (LPI) is a rare autosomal recessive disorder characterized by defective transport of cationic amino acids. Poor intestinal absorption and increased renal loss of arginine, ornithine and lysine lead to low plasma concentrations of these amino acids and, subsequently, to impaired urea cycle function. The patients therefore have decreased nitrogen tolerance, which may lead to hyperammonaemia after ingestion of normal amounts of dietary protein. As a protective mechanism, most patients develop strong aversion to protein-rich foods early in life. Oral supplementation with citrulline, which is absorbed normally and metabolized to arginine and ornithine, improves protein tolerance to some extent, as do sodium benzoate and sodium phenylbutyrate also used by some patients. Despite effective prevention of hyperammonaemia, the patients still consume a very restricted diet, which may be deficient in energy, essential amino acids and some vitamins and minerals. To investigate the potential nutritional problems of patients with lysinuric protein intolerance, 77 three- to four-day food records of 28 Finnish LPI patients aged 1.5-61 years were analysed. The data suggest that the patients are clearly at risk for many nutritional deficiencies, which may contribute to their symptoms. Their diet is highly deficient in calcium, vitamin D, iron and zinc. Individualized nutritional supplementation accompanied by regular monitoring of dietary intake is therefore an essential part of the treatment of LPI.

Classification of European mtDNAs from an analysis of three European populations.

Mitochondrial DNA (mtDNA) sequence variation was examined in Finns, Swedes and Tuscans by PCR amplification and restriction analysis. About 99% of the mtDNAs were subsumed within 10 mtDNA haplogroups (H, I, J, K, M, T, U, V, W, and X) suggesting that the identified haplogroups could encompass virtually all European mtDNAs. Because both hypervariable segments of the mtDNA control region were previously sequenced in the Tuscan samples, the mtDNA haplogroups and control region sequences could be compared. Using a combination of haplogroup-specific restriction site changes and control region nucleotide substitutions, the distribution of the haplogroups was surveyed through the published restriction site polymorphism and control region sequence data of Caucasoids. This supported the conclusion that most haplogroups observed in Europe are Caucasoid-specific, and that at least some of them occur at varying frequencies in different Caucasoid populations. The classification of almost all European mtDNA variation in a number of well defined haplogroups could provide additional insights about the origin and relationships of Caucasoid populations and the process of human colonization of Europe, and is valuable for the definition of the role played by mtDNA backgrounds in the expression of pathological mtDNA mutations.

mtDNA haplotype analysis in Finnish families with leber hereditary optic neuroretinopathy.

The mitochondrial DNA (mtDNA) sequence variation of 24 Finnish Leber hereditary optic neuroretinopathy (LHON) probands was characterized by sequencing and restriction endonuclease analyses. All LHON-associated substitutions and Caucasoid haplogroup-specific mutations were screened in the families. Analysis of the mtDNAs revealed that the Finnish LHON families have two unique features: an absence of the ND6/14484 mutation and a high number of families (10/24) without the primary mutations ND1/3460 and ND4/11778. Furthermore, the LHON families showed considerable mtDNA heterogeneity: among 24 families 22 haplotypes were detected. Overall, the haplogrouping of LHON families was similar to other European populations. However, the frequency of ND4/11778-positive families in haplogroup J was high, which may indicate that background mutations in this haplogroup together with the ND4/11778 primary mutation promote the penetrance of LHON.

Mitochondrial DNA and Y chromosome-specific polymorphisms in the Seminole Tribe of Florida.

Mitochondrial DNA (mtDNA) sequence variation was examined in 37 Seminoles from Florida by polymerase chain reaction amplification and high resolution restriction endonuclease analysis. The Y chromosome TaqI restriction fragment length polymorphisms detected by the probes 49a, 49f, and 12f2 were examined in the 26 males of this group. Analysis of the mtDNA revealed that all four Native American haplogroups (A, B, C and D) were present in the Seminoles encompassing about 95% of the Seminole mtDNAs. No European mtDNAs were found among the Seminoles, but two mtDNAs (about 5%) were members of the African-specific haplogroup L1, thus indicating that a limited number of African women were incorporated in the Seminole tribe. Analysis of Y chromosome haplotypes supports the hypothesis that haplotypes 18 and 63 are the most likely founding Native American Y chromosome haplotypes from Asia. However, 11% of the Seminole Y chromosomes represented haplotypes generally attributed to Europeans, though none harbored standard African haplotypes. These findings support historical evidence that the Seminole tribe has integrated individuals of European and African ancestry, but suggests that the sex ratio of nonnatives from different continents may have varied.

Characterization of lignin peroxidase-encoding genes from lignin-degrading basidiomycetes.

Two closely linked lignin peroxidase (LPO)-encoding genes (lpo) from Phanerochaete chrysosporium were isolated. Nucleotide sequence studies indicated that the two genes are separated by 1.3 kb of flanking DNA and transcribed in opposite directions. Cloned P. chrysosporium lpo gene probes have been shown to hybridize to multiple sequences present in the DNAs of the white-rot fungi, Bjerkandera adusta, Coriolus versicolor and Fomes lignosus, but no hybridization was detected with DNA from Pleurotus ostreatus. Thus, lpo gene families appear to be common in a number of lignin-degrading basidiomycetes, some of which have not yet been shown to produce LPO proteins.

Electron transfer properties of NADH:ubiquinone reductase in the ND1/3460 and the ND4/11778 mutations of the Leber hereditary optic neuroretinopathy (LHON).

We report the electron transfer properties of the NADH:ubiquinone oxidoreductase complex of the respiratory chain (Complex I) in mitochondria of cells derived from LHON patients with two different mutations in mitochondrial DNA (mtDNA). The mutations occur in the mtDNA genes coding for the ND1 and ND4 subunits of Complex I. The ND1/3460 mutation exhibits 80% reduction in rotenone-sensitive and ubiquinone-dependent electron transfer activity, whereas the proximal NADH dehydrogenase activity of the Complex is unaffected. This is in accordance with the proposal that the ND1 subunit interacts with rotenone and ubiquinone. In contrast, the ND4/11778 mutation had no effect on electron transfer activity of the Complex in inner mitochondrial membrane preparations; also Km for NADH and NADH dehydrogenase activity were unaffected. However, in isolated mitochondria with the ND4 mutation, the rate of oxidation of NAD-linked substrates, but not of succinate, was significantly decreased. This suggests that the ND4 subunit might be involved in specific aggregation of NADH-dependent dehydrogenases and Complex I, which may result in fast ('solid state') electron transfer from the former to the latter.

New methods for molecular diagnosis and demonstration of the (CCTG)n mutation in myotonic dystrophy type 2 (DM2).

Myotonic dystrophy types 1 and 2 are autosomal dominant, multisystemic disorders with many similarities in their clinical manifestations. Myotonic dystrophy type 1 is caused by a (CTG)n expansion in the 3' untranslated region of the DMPK gene in 19q13.3 and myotonic dystrophy type 2 by a (CCTG)n expansion in intron 1 of ZNF9 in 3q21.3. However, the clinical diagnosis of myotonic dystrophy type 2 is more complex than that of myotonic dystrophy type 1, and conventional molecular genetic methods used for diagnosing myotonic dystrophy type 1 are insufficient for myotonic dystrophy type 2. Herein we describe two in situ hybridization protocols for the myotonic dystrophy type 2 mutation detection. Chromogenic in situ hybridization was used to detect both the genomic expansion and the mutant transcripts in muscle biopsy sections. Chromogenic in situ hybridization can be used in routine myotonic dystrophy type 2 diagnostics. Fluorescence in situ hybridization on extended DNA fibers was used to directly visualize the myotonic dystrophy type 2 mutation and to estimate the repeat expansion sizes.

Mitochondrial DNA variation in an aboriginal Australian population: evidence for genetic isolation and regional differentiation.

The mitochondrial DNA (mt-DNA) variation of in the Walbiri tribe of the Northern Territories, Australia, was characterized by high resolution restriction fragment length polymorphism (HR-RFLP) analysis and control region sequencing. Surveying each mt-DNA for RFLPs with 14 different restriction enzymes detected 24 distinct haplotypes, whereas direct sequencing of the control region hypervariable segment I (HVS-I) of these mt-DNAs revealed 34 distinct sequences. Phylogenetic analysis of the RFLP haplotype and HVS-I sequence data depicted that the Walbiri have ten distinct haplotype groups (haplogroups), or mt-DNA lineages. The majority of the Walbiri RFLP haplotypes lacked polymorphisms common to Asian populations. In fact, most of the Walbiri haplogroups were unique to this population, although a few appeared to be subbranches of larger clusters of mt-DNAs that included other Aboriginal Australian and/or Papua New Guinea haplotypes. The similarity of these haplotypes suggested that Aboriginal Australian and Papua New Guinea populations may have once shared an ancient ancestral population(s), and then rapidly diverged from each other once geographically separated. Overall, the mt-DNA data corroborate the genetic uniqueness of Aboriginal Australian populations.

Migration waves to the Baltic Sea region.

In this study, the population history of the Baltic Sea region, known to be affected by a variety of migrations and genetic barriers, was analyzed using both mitochondrial DNA and Y-chromosomal data. Over 1200 samples from Finland, Sweden, Karelia, Estonia, Setoland, Latvia and Lithuania were genotyped for 18 Y-chromosomal biallelic polymorphisms and 9 STRs, in addition to analyzing 17 coding region polymorphisms and the HVS1 region from the mtDNA. It was shown that the populations surrounding the Baltic Sea are genetically similar, which suggests that it has been an important route not only for cultural transmission but also for population migration. However, many of the migrations affecting the area from Central Europe, the Volga-Ural region and from Slavic populations have had a quantitatively different impact on the populations, and, furthermore, the effects of genetic drift have increased the differences between populations especially in the north. The possible explanations for the high frequencies of several haplogroups with an origin in the Iberian refugia (H1, U5b, I1a) are also discussed.

Leber hereditary optic neuropathy: clinical and molecular genetic findings.

Leber hereditary optic neuropathy (LHON) is a maternally inherited disease characterized by acute or subacute painless central visual loss usually in young adults, predominantly in males. Except for optic atrophy, LHON patients are usually otherwise healthy. Occasionally, LHON is associated with neurological, cardiac, and skeletal changes. The clinical course of LHON has several stages. Peripapillary microangiopathy is present from the beginning. Microangiopathy disappears as the disease progresses towards the end stages. Simultaneously, the retinal nerve fiber layer fades from view, first papillomacular nerve fiber bundles, and months later, the whole nerve fiber layer becomes atrophic. At the end stage the centrocecal scotoma is large and absolute. Loss of vision is usually permanent, but spontaneous recovery can occur. Despite a few attempts, no effective treatment to prevent or halt LHON has been found. Several mitochondrial DNA (mtDNA) mutations are associated with LHON, but the pathogenic processes leading to optic nerve atrophy are largely unknown. About 15% of the families are heteroplasmic, i.e., both mutant and wild type mtDNA coexist within an individual. The level of heteroplasmy between different tissues can vary markedly. mtDNA mutations are not sufficient to cause visual loss in LHON, since not all individuals harboring a pathogenic LHON mutation express the disease. There are additional genetic and/or environmental precipitating factors, but thus far they are unknown.

Tandem repeats and length variation in the mitochondrial DNA control region of Epirrita autumnata (Lepidoptera: Geometridae).

The organization of the mitochondrial DNA (mtDNA) control region (CR) of the autumnal moth, Epirrita autumnata, is described. The E. autumnata CR presents a distinct type of lepidopteran CR with domains of non-repetitive and repetitive sequences. The CRs show considerable length variation owing to a variable number of short approximately 29-bp sequence blocks that are repeated between 6 and 14 times in tandem. The organization of such a tandem array is unique among the insect CRs examined so far. Furthermore, the E. autumnata CR, which may reach 1075 bp in length, is considerably longer than previously reported lepidopteran CRs, which reach 311-499 bp in length. Like other lepidopteran CRs, the E. autumnata CR contains two long homopolymer runs that may be involved in mtDNA replication and (or) transcription.

The spectrum of mitochondrial DNA mutations in families with Leber hereditary optic neuroretinopathy.

The mitochondrial complex I genes were sequenced in seven Leber hereditary optic neuroretinopathy (LHON) families without the ND4/11,778 and ND1/3460 mutations. Four replacement mutations restricted only to LHON families were found, one in the ND1 gene at nt 4025, and three in the ND5 gene at nt 12,811, 13,637, and 13,967. The mutations did not change evolutionarily conserved amino acids suggesting that they are not primary LHON mutations in these families. They may be considered as secondary LHON mutations serving as exacerbating factors in an appropriate genetic background. A complex III mutation, cyt b/15,257, has been suggested to be one of the primary mutations causing LHON. Its presence was determined for 23 Finnish LHON families, and it was detected in two families harboring the ND4/11,778 mutation. Similarly, complex IV mutation COI/7444 was screened in Finnish LHON families, and it was found in one family carrying the ND1/3460 mutation.

Quantification of point mutations associated with Leber hereditary optic neuroretinopathy by solid-phase minisequencing.

About two-thirds of patients with Leber hereditary optic neuroretinopathy (LHON) harbor mutations in mitochondrial DNA at positions 11778 (ND4) or 3460 (ND1). Thus, the clinical diagnosis of LHON can often be confirmed with mutation analysis. Detection of pathogenic mutations and quantification of heteroplasmy has mainly relied on PCR and restriction site analysis and densitometric scanning. We applied the recently developed solid-phase minisequencing method, based on primer-guided nucleotide incorporation, to the simultaneous detection and quantitation of the ND4/11778 and ND1/3460 mutations. The method was highly sensitive, heteroplasmy as low as 1.5% being easily detected. Rapid, reproducible, and accurate results prove solid-phase minisequencing to be the method of choice for quantitative analysis of LHON mutations.

Analysis of mitochondrial ND4 gene DNA sequence in Finnish families with Leber hereditary optic neuroretinopathy.

A mutation in the mitochondrial DNA at nt 11,778 has recently been found in Leber hereditary optic neuroretinopathy (LHON), a maternally inherited ocular disease. The mutation is located in the ND4 gene encoding subunit 4 of the respiratory chain enzyme NADH dehydrogenase. The mutation was subsequently not found in 9 of the 20 known Finnish families with LHON, implying that there are at least two different mutations associated with the disease. Using direct sequencing of PCR-amplified mtDNA, we have now sequenced the entire ND4 region in the families without the nt 11,778 mutation to find the other mutations. No new mutations in the ND4 region were found, suggesting that the putative mtDNA mutation in these families may be in the coding regions for other subunits of NADH dehydrogenase enzyme. The sequence of ND4 gene as found to be highly homogeneous.

A new mtDNA mutation associated with Leber hereditary optic neuroretinopathy.

A single base mutation at nucleotide position 3460 (nt 3460) in the ND1 gene in human mtDNA was found to be associated with Leber hereditary optic neuroretinopathy (LHON). The G-to-A mutation converts an alanine to a threonine at the 52d codon of the gene. The mutation also abolishes an AhaII restriction site and thus can be detected easily by RFLP analysis. The mutation was found in three independent Finnish LHON families but in none of the 60 controls. None of the families with the nt 3460 mutation in ND1 had the previously reported nt 11778 mutation in the ND4 gene. The G-to-A change at nt 3460 is the second mutation so far detected in LHON.

Ophthalmologic findings in Leber hereditary optic neuropathy, with special reference to mtDNA mutations.

BACKGROUND: Leber hereditary optic neuropathy (LHON) is associated with primary and secondary mutations in mitochondrial DNA. Clinical studies suggest that there is a wide spectrum of clinical expression. METHODS: Fifty-three affected and 131 unaffected maternal relatives from 21 pedigrees with LHON were studied neuro-ophthalmologically and followed over a period of 14 years. Mitochondrial DNA analysis was performed on their blood specimens. RESULTS: Thirty-two affected (60%) individuals from ten families harbored the 11778 mutation and ten individuals (19%) from three families harbored the 3460 mutation. No confirmed primary mutation was detected in 11 (21%) affected individuals from eight families. The visual outcome was better in families with the 3460 mutation than in those with the 11778 mutation. Secondary mutations did not affect the penetrance or the visual outcome. Fifteen patients had a favorable outcome; seven of whom had subclinical disease, two had slowly progressive LHON with a favorable visual outcome, and six had classic LHON with spontaneous recovery. In seven patients, the onset of the disease had been in childhood. These patients had a more favorable prognosis than the adults. Results of eye examinations of asymptomatic maternal relatives showed subclinically affected individuals. CONCLUSIONS: In addition to classic LHON, the disease can manifest itself in three different atypical forms: subclinical disease, slowly progressive LHON with a favorable visual outcome, and LHON with the classic acute stage but spontaneous visual recovery. The current study suggests that the ophthalmologic findings and outcome in LHON are independent of secondary mutations.

Pre-excitation syndrome in Leber's hereditary optic neuropathy.

Pre-excitation syndrome is common in families with Leber's hereditary optic neuropathy (LHON). 24 Finnish families with LHON were screened for the 11778 and the 3460 mitochondrial DNA mutations. 5 of 30 individuals with LHON and the 11778 mutation had the Wolff-Parkinson-White pre-excitation syndrome. None of 10 with the 3460 mutation or of 11 with "other" mutations had this syndrome. Overall, 5 of 51 LHON patients and 9 of 112 symptom-free maternal relatives had Wolff-Parkinson-White syndrome (9%). In paternal relatives, the frequency was 1.6%. Mitochondrial DNA causal for LHON may contribute to pre-excitation syndrome.