Promoter polymorphisms in two overlapping 6p25 genes implicate mitochondrial proteins in cognitive deficit in schizophrenia.

In a previous study, we detected a 6p25-p24 region linked to schizophrenia in families with high composite cognitive deficit (CD) scores, a quantitative trait integrating multiple cognitive measures. Association mapping of a 10 Mb interval identified a 260 kb region with a cluster of single-nucleotide polymorphisms (SNPs) significantly associated with CD scores and memory performance. The region contains two colocalising genes, LYRM4 and FARS2, both encoding mitochondrial proteins. The two tagging SNPs with strongest evidence of association were located around the overlapping putative promoters, with rs2224391 predicted to alter a transcription factor binding site (TFBS). Sequencing the promoter region identified 22 SNPs, many predicted to affect TFBSs, in a tight linkage disequilibrium block. Luciferase reporter assays confirmed promoter activity in the predicted promoter region, and demonstrated marked downregulation of expression in the LYRM4 direction under the haplotype comprising the minor alleles of promoter SNPs, which however is not driven by rs2224391. Experimental evidence from LYRM4 expression in lymphoblasts, gel-shift assays and modelling of DNA breathing dynamics pointed to two adjacent promoter SNPs, rs7752203-rs4141761, as the functional variants affecting expression. Their C-G alleles were associated with higher transcriptional activity and preferential binding of nuclear proteins, whereas the G-A combination had opposite effects and was associated with poor memory and high CD scores. LYRM4 is a eukaryote-specific component of the mitochondrial biogenesis of Fe-S clusters, essential cofactors in multiple processes, including oxidative phosphorylation. LYRM4 downregulation may be one of the mechanisms involved in inefficient oxidative phosphorylation and oxidative stress, increasingly recognised as contributors to schizophrenia pathogenesis.

Genetic heterogeneity and minor CYP1B1 involvement in the molecular basis of primary congenital glaucoma in Gypsies.

Primary congenital glaucoma (PCG) is a genetically heterogeneous disorder of autosomal recessive inheritance, with mutations in the cytochrome P450 1B1 (CYP1B1) gene detected in an average of approximately 50% of cases worldwide. The Roma/Gypsies are considered to be a rare example of a single founder CYP1B1 mutation, E387K (identified in the Slovak Roma), accounting for 100% of disease alleles. Contrary to this concept, unusual genetic heterogeneity was revealed in this study of 21 Gypsy PCG patients from Bulgaria and 715 controls from the general Gypsy population. In our small sample of affected subjects, we identified five different CYP1B1 mutations - four known (E229K, R368H, E387K and R390C) and one novel and potentially pathogenic (F445I), which together accounted for approximately 30% of disease alleles. E387K was rare in both the patient and the control group, indicating that its high frequency in the Slovak Roma is the product of local founder effect not representative of the overall molecular pattern of PCG in the Gypsy population. Data on other Mendelian disorders and on the population genetics of the Gypsies suggest that a true founder mutation is likely to exist and has remained undetected. Our analysis of another candidate gene, MYOC, and the GLC3B and GLC3C loci did not provide support for their involvement. The molecular basis of PCG in the Gypsies is thus unresolved, and diagnostic analyses should be extended beyond the E387K mutation.

A novel Gypsy founder mutation, p.Arg1109X in the CMT4C gene, causes variable peripheral neuropathy phenotypes.

BACKGROUND: Linkage, haplotype and sequencing analysis in a large Spanish Gypsy kindred with multiple members affected by autosomal recessive peripheral neuropathy led to the identification of a novel mutation, p.Arg1109X, in the CMT4C gene. The screening of further unrelated patients, and of a panel of ethnically matched controls, showed that p.Arg1109X is an ancestral mutation which occurs in Gypsy populations across Europe and is the most common cause of autosomal recessive Charcot-Marie-Tooth disease in Spanish Gypsies. OBJECTIVE: To report the identification of a novel Gypsy founder mutation causing autosomal recessive CMT4C disease in a sample of homozygous affected individuals. RESULTS: The mutation was associated with a surprisingly broad spectrum of neuropathy phenotypes, with variation in the age at onset, rate of progression, severity of muscle and sensory involvement, the presence of scoliosis, and cranial nerve involvement. CONCLUSIONS: Ascertainment and further studies of CMT4C patients in this population will provide a unique opportunity for characterising the full range of clinical manifestations of the disease in a genetically homogeneous sample.

Genetic identity of Marinesco-Sjögren/myoglobinuria and CCFDN syndromes.

OBJECTIVE AND BACKGROUND: To describe three Gypsy families with Marinesco-Sjögren syndrome (MSS), demyelinating neuropathy, and recurrent episodes of myoglobinuria in five of the six affected subjects. Because these families originated from the same genetically isolated founder population as did patients with congenital cataracts facial dysmorphism neuropathy (CCFDN) syndrome, and because the two syndromes have clinical manifestations in common, we hypothesized that the two related, albeit distinct, syndromes may represent clinical variants of a single genetic disorder. METHODS: Clinical studies were conducted and linkage and haplotype analyses were performed for the three families. A total of 16 individuals, including the 6 with MSS and 10 unaffected relatives, were genotyped for six polymorphic microsatellite markers from the CCFDN region on 18qter. RESULTS: Linkage analysis of markers in the 18qter region, where we previously had located the CCFDN gene, produced a lod score of 3.55, demonstrating colocalization of the gene responsible for MSS with demyelinating neuropathy and myoglobinuria with the CCFDN gene. Moreover, the patients with MSS shared the conserved marker haplotype found in CCFDN chromosomes. CONCLUSIONS: These data suggest that Marinesco-Sjögren syndrome with peripheral neuropathy and myoglobinuria, and congenital cataracts facial dysmorphism neuropathy syndrome are genetically identical and are caused by a single founder mutation.

Angiotensin-converting enzyme activity and the ACE Alu polymorphism in autosomal dominant polycystic kidney disease.

BACKGROUND: Previous studies concerning Alu I/D polymorphism in the ACE gene and ADPKD severity have used the Alu genotypes as a representative of the true biological variable, namely ACE activity. However, wide individual and ethnic differences in the proportion of variance in ACE activity explained by the I/D genotype may have confounded these studies. This investigation examines the association between ADPKD severity and ACE in terms of plasma enzyme activity and I/D genotypes in individuals from three different countries. METHODS: Blood samples were collected from 307 ADPKD patients (116 Australian, 124 Bulgarian and 67 Polish) for determination of ACE activity levels and I/D genotypes. Chronic renal failure (CRF) was present in 117 patients and end-stage renal failure (ESRF) in 68 patients. RESULTS: ACE activity was related to the I/D genotype, showing a dosage effect of the D allele (P=0.006). The proportion of variance due to the Alu polymorphism was 14%. No difference in ACE activity and I/D genotype distribution was found between patients with CRF versus normal renal function (P=0.494; P=0.576) or between those with ESRF versus those without ESRF (P=0.872; P=0.825). No effect of the I/D genotype on age at development and progression to renal failure (CRF; ESRF) was detected in the overall group, and in subgroups based on ethnic origin, linkage status and sex. CONCLUSION: ACE is not likely to play a role as a determinant of ADPKD phenotype severity.

Origins and divergence of the Roma (gypsies).

The identification of a growing number of novel Mendelian disorders and private mutations in the Roma (Gypsies) points to their unique genetic heritage. Linguistic evidence suggests that they are of diverse Indian origins. Their social structure within Europe resembles that of the jatis of India, where the endogamous group, often defined by profession, is the primary unit. Genetic studies have reported dramatic differences in the frequencies of mutations and neutral polymorphisms in different Romani populations. However, these studies have not resolved ambiguities regarding the origins and relatedness of Romani populations. In this study, we examine the genetic structure of 14 well-defined Romani populations. Y-chromosome and mtDNA markers of different mutability were analyzed in a total of 275 individuals. Asian Y-chromosome haplogroup VI-68, defined by a mutation at the M82 locus, was present in all 14 populations and accounted for 44.8% of Romani Y chromosomes. Asian mtDNA-haplogroup M was also identified in all Romani populations and accounted for 26.5% of female lineages in the sample. Limited diversity within these two haplogroups, measured by the variation at eight short-tandem-repeat loci for the Y chromosome, and sequencing of the HVS1 for the mtDNA are consistent with a small group of founders splitting from a single ethnic population in the Indian subcontinent. Principal-components analysis and analysis of molecular variance indicate that genetic structure in extant endogamous Romani populations has been shaped by genetic drift and differential admixture and correlates with the migrational history of the Roma in Europe. By contrast, social organization and professional group divisions appear to be the product of a more recent restitution of the caste system of India.

Hereditary motor and sensory neuropathy-russe: new autosomal recessive neuropathy in Balkan Gypsies.

A novel peripheral neuropathy of autosomal recessive inheritance has been identified in Balkan Gypsies and termed hereditary motor and sensory neuropathy-Russe (HMSN-R). We investigated 21 affected individuals from 10 families. Distal lower limb weakness began between the ages of 8 and 16 years, upper limb involvement beginning between 10 and 43 years, with an average of 22 years. This progressive disorder led to severe weakness of the lower limbs, generalized in the oldest subject (aged 57 years), and marked distal upper limb weakness. Prominent distal sensory loss involved all modalities, resulting in neuropathic joint degeneration in two instances. All patients showed foot deformity, and most showed hand deformity. Motor nerve conduction velocity was moderately reduced in the upper limbs but unobtainable in the legs. Sensory nerve action potentials were absent. There was loss of larger myelinated nerve fibers and profuse regenerative activity in the sural nerve. HMSN-R is a new form of autosomal recessive inherited HMSN caused by a single founder mutation in a 1 Mb interval on chromosome 10q.

Patterns of inter- and intra-group genetic diversity in the Vlax Roma as revealed by Y chromosome and mitochondrial DNA lineages.

Previous genetic studies, supported by linguistic and historical data, suggest that the European Roma, comprising a large number of socially divergent endogamous groups, may be a complex conglomerate of founder populations. The boundaries and characteristics of such founder populations and their relationship to the currently existing social stratification of the Roma have not been investigated. This study is an attempt to address the issues of common vs independent origins and the history of population fissioning in three Romani groups that are well defined and strictly endogamous relative to each other. According to linguistic classifications, these groups belong to the Vlax Roma, who account for a large proportion of the European Romani population. The analysis of mtDNA sequence variation has shown that a large proportion of maternal lineages are common to the three groups. The study of a set of Y chromosome markers of different mutability has revealed that over 70% of males belong to a single lineage that appears unique to the Roma and presents with closely related microsatellite haplotypes and MSY1 codes. The study unambiguously points to the common origins of the three Vlax groups and the recent nature of the population fissions, and provides preliminary evidence of limited genetic diversity in this young founder population.

Novel mutations in the GALK1 gene in patients with galactokinase deficiency.

Galactokinase deficiency is an inborn error of galactose metabolism whose major clinical manifestation is the development of cataracts during the first months of life. Only 20 mutations have been reported to date and understanding of the functionally important domains of the galactokinase protein is still limited. Here we report four novel mutations in GALK1 that were identified in two unrelated patients with galactokinase deficiency. Three of these were amino acid substitutions: 1569C-->T in exon 2 (R68C); 7093C-->T in exon 6 (T288M) and 7538G-->C in exon 8 (A384P). In addition, a single base-pair deletion was found in exon 5 (2833delC), predicted to result in a shift of the reading frame and a premature termination codon at position 263. Some differences with the GALK1 sequence deposited in Genbank are also reported.

Hereditary motor and sensory neuropathy-Lom (HMSNL) in a Spanish family: clinical, electrophysiological, pathological and genetic studies.

The clinical, electrophysiological, pathological and genetic findings are described in the first Spanish family diagnosed with hereditary motor and sensory neuropathy type Lom (HMSNL) initially identified by Kalaydjeva et al. in 1996. The three affected patients belong to a non-consanguineous family with Gypsy background that were followed up over 10 years. Serial clinical and neurophysiological examinations and genetic analysis were undertaken in every patient. Sural nerve biopsy was performed in the oldest patient. The clinical features are similar to those previously described in HMSNL and all of them showed abnormal brain auditory evoked potentials. The oldest brother developed sensorineural deafness at the age of 20. Conduction velocities were unobtainable in all patients and nerves tested except for the median nerve in the youngest child in whom conduction was severely slowed. Neuropathological examination revealed a severely depleted nerve with very few surviving myelinated fibers which possessed thin myelin sheaths. Schwann cell processes were arranged in circular configurations without typical onion bulb configuration. Genetic analysis showed that the maternal chromosome inherited by all three affected siblings displayed a very unusual haplotype. Our patients show the characteristic clinical, electrophysiological and pathological findings described in HMSNL and represent the first reported Spanish family affected from the disease. The genetic findings in this family have contributed to refine the HMSNL critical linkage region.

Hereditary motor and sensory neuropathy--Lom (HMSNL): refined genetic mapping in Romani (Gypsy) families from several European countries.

Hereditary motor and sensory neuropathy type Lom, initially identified in Roma (Gypsy) families from Bulgaria, has been mapped to 8q24. Further refined mapping of the region has been undertaken on DNA from patients diagnosed across Europe. The refined map consists of 25 microsatellite markers over approximately 3 cM. In this collaborative study we have identified a number of historical recombinations resulting from the spread of the hereditary motor and sensory neuropathy type Lom gene through Europe with the migration and isolation of Gypsy groups. Recombination mapping and the minimal region of homozygosity reduced the original 3 cM hereditary motor and sensory neuropathy type Lom region to a critical interval of about 200 kb.

A novel locus for autosomal recessive peripheral neuropathy in the EGR2 region on 10q23.

During our studies of Romany (Gypsy) families with hereditary motor and sensory neuropathy-Lom, we have identified a large kindred with two independently segregating autosomal recessive neuropathies. The novel disorder, named "hereditary motor and sensory neuropathy-Russe" (HMSNR), presented as a severe disabling form of Charcot-Marie-Tooth disease with prominent sensory loss, moderately reduced motor nerve conduction velocity, and a high threshold for electrical nerve stimulation. A genome scan in two branches of the large kindred detected linkage to the 10q22-q23 region containing the early growth response 2 gene (EGR2), a transcription factor with a key role in peripheral nerve myelination. The results of sequence analysis and the detection of an intragenic polymorphism allowed us to exclude EGR2 as the HMSNR gene. Further analysis done using linkage and recombination mapping refined the position of the HMSNR gene to a small interval on 10q23.2, flanked by markers D10S581 and D10S1742, telomeric to EGR2. In this interval, a conserved seven-marker haplotype is shared by all disease chromosomes, suggesting a single founder mutation. The homozygosity region is contained in bacterial-artificial-chromosome contig 1570 of the Sanger Centre physical map and has an estimated physical size of approximately 500 kb.

N-myc downstream-regulated gene 1 is mutated in hereditary motor and sensory neuropathy-Lom.

Hereditary motor and sensory neuropathies, to which Charcot-Marie-Tooth (CMT) disease belongs, are a common cause of disability in adulthood. Growing awareness that axonal loss, rather than demyelination per se, is responsible for the neurological deficit in demyelinating CMT disease has focused research on the mechanisms of early development, cell differentiation, and cell-cell interactions in the peripheral nervous system. Autosomal recessive peripheral neuropathies are relatively rare but are clinically more severe than autosomal dominant forms of CMT, and understanding their molecular basis may provide a new perspective on these mechanisms. Here we report the identification of the gene responsible for hereditary motor and sensory neuropathy-Lom (HMSNL). HMSNL shows features of Schwann-cell dysfunction and a concomitant early axonal involvement, suggesting that impaired axon-glia interactions play a major role in its pathogenesis. The gene was previously mapped to 8q24.3, where conserved disease haplotypes suggested genetic homogeneity and a single founder mutation. We have reduced the HMSNL interval to 200 kb and have characterized it by means of large-scale genomic sequencing. Sequence analysis of two genes located in the critical region identified the founder HMSNL mutation: a premature-termination codon at position 148 of the N-myc downstream-regulated gene 1 (NDRG1). NDRG1 is ubiquitously expressed and has been proposed to play a role in growth arrest and cell differentiation, possibly as a signaling protein shuttling between the cytoplasm and the nucleus. We have studied expression in peripheral nerve and have detected particularly high levels in the Schwann cell. Taken together, these findings point to NDRG1 having a role in the peripheral nervous system, possibly in the Schwann-cell signaling necessary for axonal survival.

A founder mutation in the GK1 gene is responsible for galactokinase deficiency in Roma (Gypsies).

Galactokinase deficiency is an inborn error in the first step of galactose metabolism. Its major clinical manifestation is the development of cataracts in the first weeks of life. It has also been suggested that carriers of the deficiency are predisposed to presenile cataracts developing at age 20-50 years. Newborn screening data suggest that the gene frequency is very low worldwide but is higher among the Roma in Europe. Since the cloning of the galactokinase gene (GK1) in 1995, only two disease-causing mutations, both confined to single families, have been identified. Here we present the results of a study of six affected Romani families from Bulgaria, where index patients with galactokinase deficiency have been detected by the mass screening. Genetic linkage mapping placed the disease locus on 17q, and haplotype analysis revealed a small conserved region of homozygosity. Using radiation hybrid mapping, we have shown that GK1 is located in this region. The founder Romani mutation identified in this study is a single nucleotide substitution in GK1 resulting in the replacement of the conserved proline residue at amino acid position 28 with threonine (P28T). The P28T carrier rate in this endogamous population is approximately 5%, suggesting that the mutation may be an important cause of early childhood blindness in countries with a sizeable Roma minority.

Congenital cataracts facial dysmorphism neuropathy (CCFDN) syndrome: a novel developmental disorder in Gypsies maps to 18qter.

We have identified a novel developmental disorder with complex phenotypic characteristics involving primarily the nervous system, which appears to be common in a specific Gypsy group in Bulgaria. We propose to refer to the syndrome as congenital cataracts facial dysmorphism neuropathy (CCFDN). We have assigned the disease locus to the telomeric region of chromosome 18q. Linkage disequilibrium and highly conserved haplotypes suggest genetic homogeneity and founder effect. CCFDN co-localises with an EST which shows high homology to a conserved Drosophila gene involved in the regulation of nervous system development in vertebrates.

Hereditary auditory, vestibular, motor, and sensory neuropathy in a Slovenian Roma (Gypsy) kindred.

Members of a Roma (Gypsy) family with hereditary motor and sensory peripheral neuropathy (HMSN) and concomitant auditory and vestibular cranial neuropathies were identified in Kocevje, Slovenia. The illness begins in childhood with a severe and progressive motor disability and the deafness is delayed until the second decade. There are no symptoms of vestibular dysfunction. The family structure is consistent with an autosomal recessive pattern of inheritance and the genetic locus for the disorder is linked to the same region of chromosome 8q24 as other Roma families with HMSN and deafness from Lom, Bulgaria (HMSN-Lom). The present study shows that the deafness is caused by a neuropathy of the auditory nerve with preserved measures of cochlear outer hair cell function (otoacoustic emissions and cochlear microphonics) but absent neural components of auditory brainstem potentials. The hearing loss affects speech comprehension out of proportion to the pure tone loss. Vestibular testing showed absence of caloric responses. Physiological and neuropathological studies of peripheral nerves were compatible with the nerve disorder contemporaneously affecting Schwann cells and axons resulting in both slowed nerve conduction and axonal loss. Genetic linkage studies suggest a refinement of the 8q24 critical region containing the HMSN-Lom locus that affects peripheral motor and sensory nerves as well as the cranial auditory and vestibular nerves.

Cystic fibrosis mutations and associated haplotypes in Bulgaria - a comparative population genetic study.

We present data on the population genetics of cystic fibrosis (CF) in Bulgaria, obtained by comprehensive mutation analysis and the construction of intragenic microsatellite haplotypes. The sample of 262 CF alleles analysed is representative of the patients diagnosed during the period of referral and of the three main ethnic groups in the country. deltaF508 accounted for 100% of Gypsy CF alleles, which thus differed significantly from both Bulgarians and ethnic Turks. In Bulgarian and Turkish CF patients, 92% of the mutant alleles were identified, yielding a total of 25 different mutations, of which only 7 occurred at frequencies higher than 1%. The findings were compared to other European populations and to the distribution of phenylketonuria mutations. Genetic distances and population trees demonstrated that in the south-eastern tip of Europe, the overall distribution of CF mutations and polymorphic haplotypes is very close to that of Mediterranean populations, with a high frequency of N1303K and G542X, a large number of rare mutations and a prevalence of the 23 31 13 haplotype in association with deltaF508. These findings are consistent with a main role for the Neolithic expansion in the shaping of the CF mutation spectrum in Bulgaria and southern Europe.

SSCP analysis: a blind sensitivity trial.

Studies of the sensitivity of SSCP analysis usually have been performed under conditions contrary to the rules of quality control trials and have produced widely different results. We have performed a blind trial of the sensitivity of SSCP analysis for the detection of mutations in fragments up to 500 bp in length under a fixed single set of electrophoretic conditions. The mutation detection rate was 84%. In addition, we have identified a second mutation in nine samples. All these mutations are polymorphisms, including a novel polymorphism 1248 + 52T/C first reported in the present work.

Gene mapping in Gypsies identifies a novel demyelinating neuropathy on chromosome 8q24.

Founder effect and linkage disequilibrium have been successfully exploited to map single gene disorders, and the study of isolated populations is emerging as a major approach to the investigation of genetically complex diseases. In the search for genetic isolates ranging from Pacific islands to Middle East deserts, the 10 million Gypsies resident in Europe have largely escaped the attention of geneticists. Because of their geographical ubiquity, lack of written history and the presumed social and cultural nature of their isolation, Gypsies are construed as not meeting the criteria for a well defined founder population. Gypsy society has a complex structure with subdivisions and stratifications that are incomprehensible to the surrounding populations. Marginalization by the health care systems in most countries results in a lack of information on causes of morbidity and mortality and little is known about hereditary disorders or the population genetic characteristics of Gypsies. This study is the first example of mapping a disease gene in endogamous Gypsy groups. Using lod score analysis and linkage disequilibrium, we have located a novel demyelinating neuropathy to a narrow interval on chromosome 8q24. We show that the disease, occurring in Gypsy groups of different identity and history of migrations, is caused by a single mutation whose origin predates the divergence of these groups.