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Pelizaeus-Merzbacher disease (PMD) is a rare Mendelian disorder characterised by central nervous system hypomyelination. PMD typically manifests in infancy or early childhood and is caused by mutations in proteolipid protein-1 (PLP1). However, variants in several other genes including gap junction protein gamma 2 (GJC2) can also cause a similar phenotype and are referred to PMD-like disease (PMLD). Whole-exome sequencing in two siblings presenting with clinical symptoms of PMD revealed a homozygous variant in the arginyl-tRNA synthetase (RARS) gene: NM_002887.3: c.[5A>G] p.(Asp2Gly). Subsequent screening of a PMD cohort without a genetic diagnosis identified an unrelated individual with novel compound heterozygous variants including a missense variant c.[1367C>T] p.(Ser456Leu) and a de novo deletion c.[1846_1847delTA] p.(Tyr616Leufs*6). Protein levels of RARS and the multi-tRNA synthetase complex into which it assembles were found to be significantly reduced by 80 and 90% by western blotting and Blue native-PAGE respectively using patient fibroblast extracts. As RARS is involved in protein synthesis whereby it attaches arginine to its cognate tRNA, patient cells were studied to determine their ability to proliferate with limiting amounts of this essential amino acid. Patient fibroblasts cultured in medium with limited arginine at 30 °C and 40 °C, showed a significant decrease in fibroblast proliferation (P<0.001) compared to control cells, suggestive of inefficiency of protein synthesis in the patient cells. Our functional studies provide further evidence that RARS is a PMD-causing gene.

X‐linked adrenoleukodystrophy (X‐ALD) is caused by mutations in the ABCD1 gene, which encodes a peroxisomal ABC half‐transporter (ALDP) involved in the import of very long‐chain fatty acids (VLCFA) into the peroxisome. The disease is characterized by a striking and unpredictable variation in phenotypic expression. Phenotypes include the rapidly progressive childhood cerebral form (CCALD), the milder adult form, adrenomyeloneuropathy (AMN), and variants without neurologic involvement. There is no apparent correlation between genotype and phenotype. In males, unambiguous diagnosis can be achieved by demonstration of elevated levels of VLCFA in plasma. In 15 to 20% of obligate heterozygotes, however, test results are false–negative. Therefore, mutation analysis is the only reliable method for the identification of heterozygotes. Since most X‐ALD kindreds have a unique mutation, a great number of mutations have been identified in the ABCD1 gene in the last seven years. In order to catalog and facilitate the analysis of these mutations, we have established a mutation database for X‐ALD ( http://www.x‐ald.nl). In this review we report a detailed analysis of all 406 X‐ALD mutations currently included in the database. Also, we present 47 novel mutations. In addition, we review the various X‐ALD phenotypes, the different diagnostic tools, and the need for extended family screening for the identification of new patients. Hum Mutat 18:499–515, 2001. © 2001 Wiley‐Liss, Inc.

Identifying genes and variants contributing to rare disease phenotypes and Mendelian conditions informs biology and medicine, yet potential phenotypic consequences for variation of >75% of the ~20,000 annotated genes in the human genome are lacking. Technical advances to assess rare variation genome-wide, particularly exome sequencing (ES), enabled establishment in the United States of the National Institutes of Health (NIH)-supported Centers for Mendelian Genomics (CMGs) and have facilitated collaborative studies resulting in novel “disease gene” discoveries. Pedigree-based genomic studies and rare variant analyses in families with suspected Mendelian conditions have led to the elucidation of hundreds of novel disease genes and highlighted the impact of de novo mutational events, somatic variation underlying nononcologic traits, incompletely penetrant alleles, phenotypes with high locus heterogeneity, and multilocus pathogenic variation. Herein, we highlight CMG collaborative discoveries that have contributed to understanding both rare and common diseases and discuss opportunities for future discovery in single-locus Mendelian disorder genomics. Phenotypic annotation of all human genes; development of bioinformatic tools and analytic methods; exploration of non-Mendelian modes of inheritance including reduced penetrance, multilocus variation, and oligogenic inheritance; construction of allelic series at a locus; enhanced data sharing worldwide; and integration with clinical genomics are explored. Realizing the full contribution of rare disease research to functional annotation of the human genome, and further illuminating human biology and health, will lay the foundation for the Precision Medicine Initiative.

Recent advances in next-generation sequencing strategies have led to the discovery of many novel disease genes. We describe here a non-consanguineous family with two affected boys presenting with early onset of severe axonal neuropathy, optic atrophy, intellectual disability, auditory neuropathy and chronic respiratory and gut disturbances. Whole-exome sequencing (WES) was performed on all family members and we identified compound heterozygous variants (c.[760C>A];[1528G>C];p.[(Gln254Lys);(Ala510Pro)] in the polyribonucleotide nucleotidyltransferase 1 (PNPT1) gene in both affected individuals. PNPT1 encodes the polynucleotide phosphorylase (PNPase) protein, which is involved in the transport of small RNAs into the mitochondria. These RNAs are involved in the mitochondrial translation machinery, responsible for the synthesis of mitochondrially encoded subunits of the oxidative phosphorylation (OXPHOS) complexes. Both PNPT1 variants are within highly conserved regions and predicted to be damaging. These variants resulted in quaternary defects in the PNPase protein and a clear reduction in protein and mRNA expression of PNPT1 in patient fibroblasts compared with control cells. Protein analysis of the OXPHOS complexes showed a significant reduction in complex I (CI), complex III (CIII) and complex IV (CIV). Enzyme activity of CI and CIV was clearly reduced in patient fibroblasts compared with controls along with a 33% reduction in total mitochondrial protein synthesis. In vitro rescue experiments, using exogenous expression of wild-type PNPT1 in patient fibroblasts, ameliorated the deficiencies in the OXPHOS complex protein expression, supporting the likely pathogenicity of these variants and the importance of WES in efficiently identifying rare genetic disease genes.

We have developed a simple and rapid nonradioactive method for detecting genetic variation and have applied it to the diagnosis of sickle cell anemia and β-thalassemia. The procedure involves the selective amplification of a segment of the human β-globin gene with oligonucleotide primers and a thermostable DNA polymerase, followed by hybridization of the amplified DNA with allele-specific oligonucleotide probes covalently labeled with horseradish peroxidase. The hybridized probes were detected with a simple colorimetric assay. We demonstrated the usefulness of this method in a retrospective analysis of two pregnancies at risk for β-thalassemia and one at risk for sickle cell anemia, as well as in an analysis of nine DNA samples simulating three family sets. (N Engl J Med 1988; 319:537–41.) SICKLE cell anemia and β-thalassemia are inherited hématologie disorders that are widespread in regions of the world where malaria was once endemic. 1 Both these autosomal recessive diseases are caused by mutations in the β-globin gene, a gene encoding a major protein component of hemoglobin A. These mutations generally involve the replacement, insertion, or deletion of one to four nucleotide bases from the DNA sequence of the normal (ßA) gene. 2 , 3 Sickle cell anemia, which is found primarily in African populations, is caused by homozygosity for a unique DNA base-pair substitution (βS) in the sixth codon of the gene. 4 β-Thalassemia . . .

We have previously linked aging, carcinogenesis, and de novo methylation within the promoter of the estrogen receptor (ER) gene in human colon. We now examine the dynamics of this process for the imprinted gene for insulin-like growth factor II (IGF2). In young individuals, the P2-4 promoters of IGF2 are methylated exclusively on the silenced maternal allele. During aging, this promoter methylation becomes more extensive and involves the originally unmethylated allele. Most adult human tumors, including colon, breast, lung, and leukemias, exhibit increased methylation at the P2-4 IGF2 promoters, suggesting further spreading during the neoplastic process. In tumors, this methylation is associated with diminished or absent IGF2 expression from the methylated P3 promoter but maintained expression from P1, an upstream promoter that is not contained within the IGF2 CpG island. Our results demonstrate a remarkable evolution of methylation patterns in the imprinted promoter of the IGF2 gene during aging and carcinogenesis, and provide further evidence for a potential link between aberrant methylation and diseases of aging.

DETECTION of sickle hemoglobin in the human fetus was first accomplished nearly 10 years ago. 1 , 2 This marked the beginning of a technology for prenatal diagnosis of the hemoglobinopathies. When methods for acquisition of fetal blood and for analysis of globin-chain synthesis were developed, the prenatal diagnosis of sickle-cell anemia and the thalassemia syndromes became a practical reality. 3 , 4 Nearly 2000 fetuses at risk for these disorders have now been studied worldwide. 5 However, a fetal loss of about 5 per cent due to these invasive procedures has provided the impetus for the development of diagnostic approaches that use fetal DNA rather than . . .

DNA polymorphisms are normal inherited variations in DNA that can often be used to document the inheritance of genes that produce disease. In this report we summarize our experience with prenatal diagnosis in 95 pregnancies in which the fetus was at risk for a hemoglobinopathy; the diagnosis was performed with use of DNA polymorphisms located so near the β-globin gene that they are inherited along with that gene. Of the 95 pregnancies, 57 involved fetuses at risk for sickle-cell anemia, 32 fetuses at risk for β-thalassemia, and 6 fetuses at risk for other β-chain hemoglobinopathies. Diagnosis was achieved solely by analysis of DNA polymorphisms in cells recovered by amniocentesis in 82 cases (86 per cent) and was completed by fetoscopy and fetal-blood study in an additional 6 cases (6 per cent). Prenatal diagnosis was proved correct in all 78 cases that have been available for confirmation to date. Our experience demonstrates that DNA polymorphisms can be useful for the prenatal diagnosis of genetic diseases in which the basic defect cannot be directly detected. (N Engl J Med 1983; 308:1054–8.) Sickle-cell anemia and β-thalassemia are autosomal recessive disorders in which couples at risk have a 25 per cent chance with each pregnancy of producing an affected child. Prenatal diagnosis of these hemoglobinopathies has been carried out by analysis of globin-chain synthesis in fetal blood samples since 1975. 1 , 2 These fetal blood samples were obtained by fetoscopy or placental aspiration — specialized techniques that are performed routinely at very few medical centers and that carry approximately a 3 per cent risk of fetal mortality. 2 In 1978 Kan and Dozy discovered a DNA polymorphism located extremely close to the β-globin gene on chromosome . . .

By using probes for ε -, ψ β1-, and β -globin genes, we found four additional polymorphic restriction sites that have frequencies >0.1 in persons of Mediterranean area origin, Asian Indians, and American Blacks. Three of these (HincII sites) and the two previously described polymorphic HindIII sites [one in intervening sequence (IVS) II of each γ -globin gene] are distributed over 32 kilobases (kb) of DNA located 5′to the δ -globin gene. This region of DNA comprises two-thirds of the β -globin gene cluster. Since each of these five polymorphic sites can be present (+) or absent (-), in theory there exist 32 possible combinations of sites (haplotypes). However, in Italians, Greeks, Indians, and Turks, 3 of the 32 haplotypes, (+ - - - -), (- + - + +), and (- + + - +), account for 92% of 89 βAchromosomes examined. The observed frequencies for these haplotypes are 0.64, 0.15, and 0.13 in the populations studied, in contrast to expected frequencies (based on the observed gene frequencies at each of the five sites) of 0.20, 0.006, and 0.005, respectively. In American Blacks, a fourth haplotype, (- - - - +), which is rare in non-Black populations, has a frequency of 0.37 in contrast to its expected frequency of 0.05. These results suggest a nonrandom association of DNA sequences over 32 kb 5′to the δ -globin gene in all populations studied. Two other polymorphic sites 3′to the δ gene (the newly discovered Ava II site in IVS II of the β -globin gene and the BamHI site 3′to it) are nonrandomly associated with each other but randomly distributed with respect to the above haplotypes. This suggests that randomization of sequences has occurred within 12 kb of DNA between these two nonrandomly associated sequence clusters. Nonrandom association of polymorphic restriction sites has practical consequences in that it limits the usefulness of these additional HincII sites for prenatal diagnosis of hemoglobinopathies by linkage analysis. These sites provide little additional information for detection of β -thalassemia, while the polymorphic Ava II site, which lies outside the nonrandomly associated sequences 5′to the δ gene, improves the test applicability from 52% to 70% of couples at risk.

In order to investigate the origin(s) of the mutation(s) leading to the βS-globin gene in North American populations of African ancestry, we analyzed DNA polymorphisms in the βS-globin gene cluster in a large number of both βA- and βS-globin gene-bearing chromosomes in U.S. and Jamaican Blacks. We found 16 different haplotypes of polymorphic sites associated with 170 βS-globin gene-bearing chromosomes. The three most common βShaplotypes, which account for 151/170 of the βS-globin gene-bearing chromosomes, are only rarely seen in the chromosomes bearing the βA-globin gene in these populations (6/47). Two observations suggest multiple origins or interallelic gene conversion, or both, of the βSmutation. First, the mutation is present in all three β -globin gene frameworks. Second, the βShaplotypes can be divided into four groups, each of which cannot be derived from any other by less than two crossing-over events. In summary, our observation of the βSmutation on 16 different haplotypes in African populations can be best explained by (i) a number of simple recombination events 5′to the βS-globin gene and (ii) up to four independent mutations and/or interallelic gene conversions.