Homocarnosinosis: A historical update and findings in the SPG11 gene.

OBJECTIVES: A family with homocarnosinosis was reported in the literature in 1976. Three affected siblings had spastic paraplegia, retinitis pigmentosa, mental retardation, and cerebrospinal fluid (CSF) homocarnosine concentrations 20 times higher than in controls. Based on the clinical findings and new genetic techniques, we have been able to establish a precise genetic diagnosis. METHOD: The medical records were re-evaluated, and genetic analyses were performed post-mortem in this original family. SNP array-based whole genome homozygosity mapping and Sanger sequencing of the SPG11 gene were performed. Seven additional Norwegian SPG11 patients and their disease-causing variants and clinical findings were evaluated. Homocarnosine levels in CSF were measured in four of these seven patients. RESULTS: A homozygous pathogenic splice-site variant in the SPG11 gene, c.2316 + 1G>A, was found. The clinical findings in the original family correlate with the heterogeneous SPG11 phenotype. The same variant was found in seven other Norwegian SPG11 patients, unrelated to the original family, either as homozygous or compound heterozygous constellation. Normal homocarnosine levels were found in the CSF of all unrelated SPG11 patients. CONCLUSIONS: A re-evaluation of the clinical symptoms and findings in the original family correlates with the SPG11 phenotype. The increased levels of homocarnosine do not seem to be a biomarker for SPG11 in our patients. Homocarnosinosis is still a biochemical aberration with unknown clinical significance.

Mutation analysis in Norwegian families with hereditary hemorrhagic telangiectasia: founder mutations in ACVRL1.

Hereditary hemorrhagic telangiectasia (HHT, Osler-Weber-Rendu disease) is an autosomal dominant inherited disease defined by the presence of epistaxis and mucocutaneous telangiectasias and arteriovenous malformations (AVMs) in internal organs. In most families (~85%), HHT is caused by mutations in the ENG (HHT1) or the ACVRL1 (HHT2) genes. Here, we report the results of genetic testing of 113 Norwegian families with suspected or definite HHT. Variants in ENG and ACVRL1 were found in 105 families (42 ENG, 63 ACVRL1), including six novel variants of uncertain pathogenic significance. Mutation types were similar to previous reports with more missense variants in ACVRL1 and more nonsense, frameshift and splice-site mutations in ENG. Thirty-two variants were novel in this study. The preponderance of ACVRL1 mutations was due to founder mutations, specifically, c.830C>A (p.Thr277Lys), which was found in 24 families from the same geographical area of Norway. We discuss the importance of founder mutations and present a thorough evaluation of missense and splice-site variants.

Vaccine-associated varicella and rubella infections in severe combined immunodeficiency with isolated CD4 lymphocytopenia and mutations in IL7R detected by tandem whole exome sequencing and chromosomal microarray.

In areas without newborn screening for severe combined immunodeficiency (SCID), disease-defining infections may lead to diagnosis, and in some cases, may not be identified prior to the first year of life. We describe a female infant who presented with disseminated vaccine-acquired varicella (VZV) and vaccine-acquired rubella infections at 13 months of age. Immunological evaluations demonstrated neutropenia, isolated CD4 lymphocytopenia, the presence of CD8(+) T cells, poor lymphocyte proliferation, hypergammaglobulinaemia and poor specific antibody production to VZV infection and routine immunizations. A combination of whole exome sequencing and custom-designed chromosomal microarray with exon coverage of primary immunodeficiency genes detected compound heterozygous mutations (one single nucleotide variant and one intragenic copy number variant involving one exon) within the IL7R gene. Mosaicism for wild-type allele (20-30%) was detected in pretransplant blood and buccal DNA and maternal engraftment (5-10%) demonstrated in pretransplant blood DNA. This may be responsible for the patient's unusual immunological phenotype compared to classical interleukin (IL)-7Rα deficiency. Disseminated VZV was controlled with anti-viral and immune-based therapy, and umbilical cord blood stem cell transplantation was successful. Retrospectively performed T cell receptor excision circle (TREC) analyses completed on neonatal Guthrie cards identified absent TREC. This case emphasizes the danger of live viral vaccination in severe combined immunodeficiency (SCID) patients and the importance of newborn screening to identify patients prior to high-risk exposures. It also illustrates the value of aggressive pathogen identification and treatment, the influence newborn screening can have on morbidity and mortality and the significant impact of newer genomic diagnostic tools in identifying the underlying genetic aetiology for SCID patients.

Microdeletions including FMR1 in three female patients with intellectual disability - further delineation of the phenotype and expression studies.

Fragile X syndrome (FXS) is one of the most common causes of intellectual disability/developmental delay (ID/DD), especially in males. It is caused most often by CGG trinucleotide repeat expansions, and less frequently by point mutations and partial or full deletions of the FMR1 gene. The wide clinical spectrum of affected females partly depends on their X-inactivation status. Only few female ID/DD patients with microdeletions including FMR1 have been reported. We describe 3 female patients with 3.5-, 4.2- and 9.2-Mb de novo microdeletions in Xq27.3-q28 containing FMR1. X-inactivation was random in all patients, yet they presented with ID/DD as well as speech delay, macrocephaly and other features attributable to FXS. No signs of autism were present. Here, we further delineate the clinical spectrum of female patients with microdeletions. FMR1 expression studies gave no evidence for an absolute threshold below which signs of FXS present. Since FMR1 expression is known to be highly variable between unrelated females, and since FMR1 mRNA levels have been suggested to be more similar among family members, we further explored the possibility of an intrafamilial effect. Interestingly, FMR1 mRNA levels in all 3 patients were significantly lower than in their respective mothers, which was shown to be specific for patients with microdeletions containing FMR1.

X-linked congenital ptosis and associated intellectual disability, short stature, microcephaly, cleft palate, digital and genital abnormalities define novel Xq25q26 duplication syndrome.

Submicroscopic duplications along the long arm of the X-chromosome with known phenotypic consequences are relatively rare events. The clinical features resulting from such duplications are various, though they often include intellectual disability, microcephaly, short stature, hypotonia, hypogonadism and feeding difficulties. Female carriers are often phenotypically normal or show a similar but milder phenotype, as in most cases the X-chromosome harbouring the duplication is subject to inactivation. Xq28, which includes MECP2 is the major locus for submicroscopic X-chromosome duplications, whereas duplications in Xq25 and Xq26 have been reported in only a few cases. Using genome-wide array platforms we identified overlapping interstitial Xq25q26 duplications ranging from 0.2 to 4.76 Mb in eight unrelated families with in total five affected males and seven affected females. All affected males shared a common phenotype with intrauterine- and postnatal growth retardation and feeding difficulties in childhood. Three had microcephaly and two out of five suffered from epilepsy. In addition, three males had a distinct facial appearance with congenital bilateral ptosis and large protruding ears and two of them showed a cleft palate. The affected females had various clinical symptoms similar to that of the males with congenital bilateral ptosis in three families as most remarkable feature. Comparison of the gene content of the individual duplications with the respective phenotypes suggested three critical regions with candidate genes (AIFM1, RAB33A, GPC3 and IGSF1) for the common phenotypes, including candidate loci for congenital bilateral ptosis, small head circumference, short stature, genital and digital defects.

A translocation between Xq21.33 and 22q13.33 causes an intragenic SHANK3 deletion in a woman with Phelan-McDermid syndrome and hypergonadotropic hypogonadism.

Chromosome 22q13 monosomy has been described as a contiguous gene syndrome. Localized in the critical region, SHANK3 is likely to play a key role in the expression of the clinical phenotype. SHANK3 mutations have also been reported in autistic patients without a syndromic phenotype. We report on a 20-year-old woman with mental retardation carrying a de novo translocation between chromosome Xq21.33 and 22q13.33, associated with a duplication on Xq21.33 and deletion on 22q13.33. As a child her development was characterized by disturbed social interaction, stereotypic hand movements and ritualistic behavior and she was considered at one time to have autistic features. All these traits match the 22q13 deletion syndrome (Phelan-McDermid syndrome, OMIM 606232), likely due to the deletion overlapping the last two exons of the SHANK3 gene. Our patient harbors the smallest and most distal SHANK3 deletion described to date, yet resulting in the full spectrum of the Phelan-McDermid syndrome. In addition, she has hypergonadotropic hypogonadism with low estrogen level, high FSH level, and irregular menstruation. Intriguingly, chromosome translocations affecting the chromosome band Xq21 can result in premature ovarian failure.

15q11.2 microdeletion - seven new patients with delayed development and/or behavioural problems.

15q11.2 microdeletion has been suggested as a new microdeletion syndrome and several patients have been described in the literature. We report seven new patients belonging to six families, age 9-24 years old, with a 350 kb 15q11.2 deletion of the four highly conserved genes (TUBGCP5, NIPA1, NIPA2 and CYFIP1) earlier reported. All our patients had some degree of learning difficulties, delayed development and/or behavioural problems. Common dysmorphic features and congenital malformations were not characteristics of our patients. The deletion was inherited from a mildly affected parent in all cases tested (5/6 families available for testing both parents). These seven new cases confirm some of the features earlier reported to be associated with 15q11.2 deletion, and help to further delineate the phenotype associated with 15q11.2 deletion.

Transcriptional response to ionizing radiation in human radiation sensitive cell lines.

BACKGROUND AND PURPOSE: Radiation is a common treatment of cancer, but some patients show severe side effects when exposed to small doses of radiation. The aim of this study was to explore the underlying cause of radiation sensitivity in a group of radiation sensitive patients. MATERIALS AND METHODS: Lymphoblastoid cell lines from 5 normal individuals, 4 Ataxia Telangiectasia (AT), and 12 non-AT radiation sensitive (RS) patients were irradiated. RNA was isolated before and after radiation and hybridized to 15k cDNA microarrays and gene expression was recorded. RESULTS AND CONCLUSION: The RS cell lines showed an expression phenotype different from both the AT and normal cell lines. Six of the RS cell lines had a distinct expression profile before radiation. This implies that the RS patients are a heterogeneous group, but that six of the patients may have a common cause of radiation sensitivity.

The genetic algorithm applied to haplotype data at the LDL receptor locus.

Conventional statistical methods based upon single restriction fragment length polymorphisms often prove inadequate in studies of genetic variation. Cladistic analysis has been suggested as an alternative, but requires basic assumptions that usually cannot be met. We wanted to test whether it could be a workable approach to apply the genetic algorithm, an artificial intelligence method, to haplotype data. The genetic algorithm creates in-computer artificial 'individuals', all having 'genes' coding for solutions to a problem. The individuals are allowed to compete and 'mate', individuals with genes coding for better solutions mating more often. Genes coding for good solutions survive through generations of the genetic algorithm. At the end of the run, the best solutions can be extracted. We applied the genetic algorithm to data consisting of cholesterol values and haplotypes made up of seven restriction sites at the LDL receptor locus. The persons included were 114 FH (familial hypercholesterolemia) patients and 61 normals. The genetic algorithm found the restriction sites 1 (Sph1 in intron 6), 2 (StuI in exon 8), and 7 (ApaLI site in the 3' flanking region) were associated with high cholesterol levels. As a validity check we used runs of the genetic algorithm applied to 'artificial patients', i.e. artificially generated haplotypes linked to artificially generated cholesterol values. This demonstrated the genetic algorithm consistently found the appropriate haplotype. We conclude that the genetic algorithm may be a useful tool for studying genetic variation.

Mutant transcripts of the LDL receptor gene: mRNA structure and quantity.

mRNA of the low-density lipoprotein receptor (LDLR) gene from 22 heterozygous familial hypercholesterolemic subjects possessing different mutations in this gene was analyzed by Northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR) in order to detect abnormally spliced transcripts. These analyses revealed abnormally spliced transcripts for the two splice-site mutations 1359-1G-->A and 1705 + 1G-->T. The abnormally spliced transcript for mutation 1359-1G-->A was caused by activation of a cryptic acceptor splice site in exon 10. As a result, seven nucleotides of exon 10 were deleted. For mutation 1705 + 1G-->T, two mutant transcripts were observed. In the first transcript, exon 10 was spliced to exon 13, and in the second transcript intron 11 was retained. The relative amount of mutant transcripts from 14 of the 22 subjects was determined by use of an RT-PCR-based method. Quantitation of the relative amounts of mutant transcripts for five missense mutations resulted in a mean value (+/-SD) of 52.8% (+/-4.55). In comparison, quantitation of the relative amounts of mutant transcripts for five nonsense mutations resulted in a mean value of 31.8% (+/-6.91). This value was significantly lower than the value of 54.2% (+/-2.38) obtained for nine healthy subjects (P < 0.0001). The relative amount of mutant transcripts for the 1705 + 1G-->T mutation was 36%. Thus, transcripts from alleles containing premature stop codons are present in reduced amounts, whereas transcripts from alleles containing missense mutations are present in normal amounts. These findings underscore the importance of determining how mutations affect mRNA structure and quantity in order to understand how mutations cause disease.

Phenotypic consequences of a deletion of exons 2 and 3 of the LDL receptor gene.

Screening for structural alterations of the low density lipoprotein (LDL) receptor gene by Southern blot analysis revealed an abnormal band pattern in one subject with a clinical diagnosis of homozygous familial hypercholesterolemia (FH). The molecular defect was further characterized by polymerase chain reaction and cDNA sequencing. These analyses identified a 4.8 kb in-frame deletion of exons 2 and 3, where exon 1 was spliced to exon 4. This deletion is expected to produce a receptor that has lost the two first cysteine-rich repeats of the ligand-binding domain. Previously published data of in vitro site-directed mutagenesis has shown that binding of LDL to such a receptor is reduced to 70% of normal. A mild phenotype in our FH homozygote is consistent with that observation. In contrast, heterozygotes carrying this deletion have a relatively more severe phenotype that is comparable to that of heterozygotes carrying a null-allele. A severe phenotype was also found in a compound heterozygote carrying this deletion. Possible mechanisms for this phenotypic variability are discussed.-Rødningen, O. K., S. Tonstad, J. D. Medh, D. A. Chappell, L. Ose, and T. P. Leren. Phenotypic consequences of a deletion of exons 2 and 3 of the LDL receptor gene.

Effects of a 9.6-kb deletion of the LDL receptor gene (FH Helsinki) on structure and levels of mRNA.

FH Helsinki is a deletion of the low-density lipoprotein receptor (LDLR) gene that deletes 9.6 kb from intron 15 to exon 18. Screening for mutant transcripts by Northern blot analysis from a patient heterozygous for FH Helsinki revealed two mutant transcripts. One was a transcript where the proximal part of intron 15 was retained in mRNA. The second was a transcript where exon 15 was spliced to nucleotide 4186 of exon 18. Thus, this transcript was generated using the normal donor splice site in intron 15, and a cryptic AG acceptor splice site in exon 18. Translation of the two mutant transcripts is predicted to give nonfunctional proteins, as both the membrane-spanning domain and the cytoplasmic domain of the receptor are deleted. Scanning of the autoradiograms showed that the amounts of each of the two mutant transcripts were approximately 10 times higher than that of the normal transcript in our heterozygous patient. The finding of higher levels of mutant transcripts was confirmed by an allele-specific transcript quantitation method, in which the amount of the two mutant transcripts together was approximately 5 times higher than the amount of the normal transcript. Deletion of destabilizing elements (AU-rich elements) by FH Helsinki are proposed to cause the increased levels of mutant transcripts.

Molecular genetics of familial hypercholesterolaemia in Norway.

OBJECTIVES: To characterize mutations in the low density lipoprotein (LDL) receptor gene causing familial hypercholesterolaemia (FH) amongst Norwegian patients. DESIGN: Molecular genetic analyses of the LDL receptor gene have been performed in patients with a clinical diagnosis of FH. SUBJECTS: A total of 742 probands have been studied. Of these, 476 had a diagnosis of definite FH. The rest had a diagnosis of possible FH. RESULTS: Twenty-three different mutations in the LDL receptor gene as well as the apolipoprotein B-3500 mutation have been found. Six of the mutations in the LDL receptor gene are novel mutations. A molecular genetic diagnosis was achieved in 295 of the probands with definite FH (62%) and in 317 probands total. Of the 317 probands, 3% carried the apolipoprotein B-3500 mutation. When family members were included, a total of 624 persons carried a mutation in the LDL receptor gene and 20 carried the apolipoprotein B-3500 mutation. CONCLUSIONS: Approximately 5% of Norwegian FH patients have been provided with a molecular genetic diagnosis. Our data suggest that molecular diagnosis of FH in Norway is feasible and should be implemented in clinical medicine.

[Application of gene technology in the diagnosis of familial hypercholesterolemia]. / Genteknologisk diagnostikk av familiaer hyperkolesterolemi.

Familial hypercholesterolaemia is an autosomal dominant disorder characterized by hypercholesterolaemia, xanthomas and premature coronary heart disease. Treatment of hypercholesterolemia is effective and consists of dietary changes and lipid lowering drugs. Only a minor proportion of familial hypercholesterolaemia patients are adequately treated, however. One explanation for this is assumed to be the relatively vague clinical diagnostic criteria applied. Because familial hypercholesterolaemia is caused by a mutation in the gene encoding the low density lipoprotein (LDL) receptor, mutation analysis of this gene could form the basis for specific diagnosis. 29 different mutations in the LDL receptor gene have been found to cause familial hypercholesterolaemia among Norwegian patients, and a total of 681 patients from 322 unrelated families have been provided with a molecular genetic diagnosis. We conclude that the use of molecular genetic analysis is feasible, and should be used clinically.

Application of long polymerase chain reaction in the study of the LDL receptor gene.

In this report we have applied an improved method of the polymerase chain reaction (PCR) in order to detect structural aberrations and point mutations in the low density lipoprotein receptor (LDLR) gene. Except from intron 1, we were able to amplify the entire gene in two fragments of 16.1 and 20.0 kb, respectively. We were also able to detect a 9.6-kb deletion, known as FH Helsinki, as well as a restriction fragment length polymorphism in the 2.7-kb intron 6. We conclude that PCR can almost completely replace Southern blot analysis in molecular genetic studies of the LDLR gene.

Two novel missense mutations in the LDL receptor gene causing familial hypercholesterolemia.

We have employed analysis of single-strand conformation polymorphisms to identify mutations in the low density lipoprotein receptor gene causing familial hypercholesterolemia. Two familial hypercholesterolemia heterozygotes had abnormal single-strand conformation polymorphism patterns of exons 4 and 8. DNA sequencing revealed that the abnormal pattern of exon 4 was due to heterozygosity (G/T) at nucleotide 502. Nucleotide 502 is the first base of codon 147, and the G->T mutation (D147Y) changes this codon from AspGAC to TyrUAC. The abnormal pattern of exon 8 was due to heterozygosity (A/G) at nucleotide 1097. Nucleotide 1097 is the second base of codon 345, and the A->G mutation (Q345R) changes this codon from GlnCAG to ArgCGG. Based upon screening of 437 unrelated familial hypercholesterolemia heterozygotes, both D147Y and Q345R account for about 0.5% of the mutations causing familial hypercholesterolemia in Norway.

Two novel point mutations in the EGF precursor homology domain of the LDL receptor gene causing familial hypercholesterolemia.

Familial hypercholesterolemia is caused by mutations in the low density lipoprotein (LDL) receptor gene. Analysis of single-strand conformation polymorphisms of exons 10 and 11 of the LDL receptor gene from familial hypercholesterolemia heterozygotes indicated the presence of two mutations, which were characterized by DNA sequencing. One mutation (delta N466) was a 3-bp deletion in exon 10 that deletes Asn in codon 466. The other (intron 11 +1, G-->T) was a splice donor mutation at position +1 of intron 11.

Screening for known mutations in the LDL receptor gene causing familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is caused by defective low density lipoprotein (LDL) receptors and is characterized by hypercholesterolemia and premature coronary heart disease. Two strategies can be used to identify the mutation in the LDL receptor gene underlying FH. One strategy is to search for novel mutations by DNA sequencing with or without prior mutation screening. The other strategy is to screen for known mutations. In this study we employed the latter strategy to screen 75 unrelated, Norwegian FH subjects for 38 known mutations. Three of the 38 mutations were detected in our group of FH subjects. Two subjects had FH-Padova, one had FH-Cincinnati-2 and one had FH-Gujerat. When additional unrelated FH heterozygotes were screened for the three mutations, the gene frequencies were 1.3%, 1.0% and 3.0%, respectively. In addition to identifying known mutations we also detected a novel stop codon in codon 541 (S541X). We conclude that screening for known mutations in the LDL receptor gene should be used as a complementary strategy to screening for novel mutations in order to understand the molecular genetics of FH.

Identification of the apo B-3500 mutation in the Norwegian population.

Familial defective apolipoprotein B-100 (FDB) is caused by a mutation in codon 3500 of the apo B gene. It is inherited in a co-dominant fashion and is characterized by hypercholesterolaemia. Thus, FDB has similar features to familial hypercholesterolaemia (FH). In order to investigate whether some of the Norwegian subjects diagnosed as having FH actually have FDB, we have screened 208 Norwegian FH heterozygotes for the apo B-3500 mutation. One of the subjects possessed the mutation which was on a haplotype compatible with the mutation-bearing haplotype found in other populations. Although, hypercholesterolaemia segregated with haplotypes both at the apolipoprotein B and low density lipoprotein (LDL) receptor loci in the proband's family, LDL receptor analysis revealed that the proband was not doubly heterozygous for FDB and FH.