Outcomes of individuals with profound and partial biotinidase deficiency ascertained by newborn screening in Michigan over 25 years.

PURPOSE: Biotinidase deficiency, if untreated, usually results in neurological and cutaneous symptoms. Biotin supplementation markedly improves and likely prevents symptoms in those treated early. All states in the United States and many countries perform newborn screening for biotinidase deficiency. However, there are few studies about the outcomes of the individuals identified by newborn screening. METHODS: We report the outcomes of 142 children with biotinidase deficiency identified by newborn screening in Michigan over a 25-year period and followed in our clinic; 22 had profound deficiency and 120 had partial deficiency. RESULTS: Individuals with profound biotinidase and partial deficiency identified by newborn screening were started on biotin therapy soon after birth. With good compliance, these children appeared to have normal physical and cognitive development. Although some children exhibited mild clinical problems, these are unlikely attributable to the disorder. Biotin therapy appears to prevent the development of neurological and cutaneous problems in our population. CONCLUSION: Individuals with biotinidase deficiency ascertained by newborn screening and treated since birth appeared to exhibit normal physical and cognitive development. If an individual does develop symptoms, after compliance and dosage issues are excluded, then other causes must be considered.Genet Med 17 3, 205-209.

Extra alleles in FMR1 triple-primed PCR: artifact, aneuploidy, or somatic mosaicism?

Triple-primed PCR assays have become the preferred fragile X syndrome testing method. Using a commercially available assay, we detected a reproducible extra peak(s) in 0.5% of 13,161 clinical samples. The objectives of this study were to determine the cause of these extra peaks; to identify whether these peaks represent an assay specific artifact, an underlying chromosome aneuploidy, or somatic mosaicism; and to ascertain their clinical relevance. The presence of an extra allele(s) was confirmed by a laboratory-developed PCR, with sequencing of the FMR1 5' UTR or Southern blot for some samples. The laboratory-developed procedure detected the extra allele(s) in 57 of 64 samples. Thus, we confirmed an extra peak, typically of lower abundance, in approximately 0.4% of all samples. Of these samples, 5 were from males and 52 were from heterozygous or homozygous females. Six patients likely had X chromosome aneuploidies. In 82.3% of samples, the extra allele had fewer repeats than the predominant allele(s). Additional alleles detected by FMR1 triple-primed PCR are not an assay-specific artifact and are likely due to X chromosome aneuploidies or somatic repeat instability. Additional normal alleles likely have no clinical significance for fragile X syndrome carrier or affected status. Extra alleles in individuals with normal karyotypes probably represent FMR1 somatic variation.

Evaluation of the human fragile X mental retardation 1 polymerase chain reaction reagents to amplify the FMR1 gene: testing in a clinical diagnostic laboratory.

Fragile X syndrome (FXS) is caused by the absence of a functional fragile X mental retardation protein (FMRP). In most cases, the molecular mutation is an expansion and consequent methylation of the CGG trinucleotide repeat in the 5' end of the FMR1 gene. Polymerase chain reaction (PCR)-based assays that overcome the limitations of amplifying >100-150 CGG repeats have been designed. One such product, Human FMR1 PCR Reagents, can detect expanded mutation alleles without determining methylation status. We used this assay to amplify 70 clinical samples previously tested in three clinical laboratories, including 28 full mutation alleles, 17 premutation alleles, 6 gray zone alleles, and 21 normal samples (51 normal alleles including 5 homozygous females). The results were concordant with previously reported results. All full and premutation alleles were identifiable: repeat sizes are not assigned when the CGG repeat number is >200 and all full and premutation alleles were scored in the same category using this assay. All normal and gray zone alleles were within 0-1 repeat of their previously reported allele sizes. This method identified a mosaic premutation/full mutation pattern in 12/21 samples previously identified as full mutation only and in 5/7 samples previously reported as mosaic premutation/full mutation. These results demonstrate that this assay provides comparable results to the combination of PCR/Southern blot methodologies. Additional issues such as technologist time, reagent costs, turnaround times, and sample requirements are comparable to the PCR/Southern blotting assays currently utilized; however, methylation status cannot be determined using this assay. It is likely that PCR-only based assays will eventually replace previous methods for FXS and that Southern blotting or another methylation assay will only be utilized when determination of methylation status is necessary. This type of assay may also be utilized for other nucleotide expansion disorders.

An intergenerational contraction of a fully penetrant Huntington disease allele to a reduced penetrance allele: interpretation of results and significance for risk assessment and genetic counseling.

We report on a healthy 50-year-old woman who sought predictive testing due to a family history of Huntington disease (HD). Her 73-year-old mother had recently been confirmed to carry an HD allele of 42 CAG repeats, and started to show symptoms of HD at age 68. Clinically diagnosed HD is present in the maternal grandfather, maternal uncle, and three maternal cousins. Molecular analysis of the HD CAG repeat region identified an allele with 38 CAG repeats in the consultand, giving evidence of allele size contraction from the maternal 42 CAG repeat allele. Mitotic stability of the CAG repeat was demonstrated in DNA from a skin sample with the same allele size (38). In addition to sex of the parent and size of the repeat, recent data analysis of intergenerational stability of the CAG repeat size suggest a gender effect of the offspring on the likelihood of allele contraction or expansion. Discussion of these results with this patient presented challenges in providing appropriate risk assessment for developing the disease herself as well as the future risk to her offspring.

Mutations in SIRT2 deacetylase which regulate enzymatic activity but not its interaction with HDAC6 and tubulin.

Human SIRT2 is a cytoplasmic NAD-dependent deacetylase implicated in the mitotic regulation of microtubule dynamics by its association with the class II histone deacetylase 6 (HDAC6). We have previously reported that SIRT2 is multiply phosphorylated in a cell cycle dependent pattern. Here, we demonstrate that HDAC6 binds to both phosphorylated and unphosphorylated forms of SIRT2 and that tubulin binds only to the SIRT2-HDAC6 complex. Tubulin does not bind to either HDAC6 or SIRT2 individually. In addition, we show that replacement of specific serines with alanines in either isoform of SIRT2 regulates its enzymatic activity. We also found that overexpression of isoform2 was deleterious to cell survival. SIRT2 was found to be phosphorylated at serines 368 and 372, outside the conserved core domain of the Sir2 protein family. Double replacement of S368A and S372A reduced SIRT2 deacetylase activity by 44% compared to wildtype activity. Replacements of other serine, threonine, and tyrosine residues, which did not alter the phosphorylation pattern, had varying effects on SIRT2 deacetylase activity but no effect on tubulin/HDAC6 binding.

Expression profiling identifies three pathways altered in cellular immortalization: interferon, cell cycle, and cytoskeleton.

Abrogation of cellular senescence, resulting in immortalization, is a necessary step in the tumorigenic transformation of a cell. Four independent, spontaneously immortalized Li-Fraumeni syndrome (LFS) cell lines were used to analyze the gene expression changes that may have given these cell lines the growth advantage required to become immortal. A cellular senescence-like phenotype can be induced in immortal LFS cells by treating them with the DNA methyltransferase (DNMT) inhibitor 5-aza-deoxycytidine. We hypothesized, therefore, that genes epigenetically silenced by promoter methylation are potentially key regulators of senescence. We used microarrays to compare the epigenetic gene expression profiles of precrisis LFS cells with immortal LFS cells. Gene ontology analysis of the expression data revealed a statistically significant contribution of interferon pathway, cell cycle, and cytoskeletal genes in the process of immortalization. The identification of the genes and pathways regulating immortalization will lead to a better understanding of cellular immortalization and molecular targets in cancer and aging.

Diagnostic markers of ovarian cancer by high-throughput antigen cloning and detection on arrays.

A noninvasive screening test would significantly facilitate early detection of epithelial ovarian cancer. This study used a combination of high-throughput selection and array-based serologic detection of many antigens indicative of the presence of cancer, thereby using the immune system as a biosensor. This high-throughput selection involved biopanning of an ovarian cancer phage display library using serum immunoglobulins from an ovarian cancer patient as bait. Protein macroarrays containing 480 of these selected antigen clones revealed 65 clones that interacted with immunoglobulins in sera from 32 ovarian cancer patients but not with sera from 25 healthy women or 14 patients having other benign or malignant gynecologic diseases. Sequence analysis data of these 65 clones revealed 62 different antigens. Among the markers, we identified some known antigens, including RCAS1, signal recognition protein-19, AHNAK-related sequence, nuclear autoantogenic sperm protein, Nijmegen breakage syndrome 1 (Nibrin), ribosomal protein L4, Homo sapiens KIAA0419 gene product, eukaryotic initiation factor 5A, and casein kinase II, as well as many previously uncharacterized antigenic gene products. Using these 65 antigens on protein microarrays, we trained neural networks on two-color fluorescent detection of serum IgG binding and found an average sensitivity and specificity of 55% and 98%, respectively. In addition, the top 6 of the most specific clones resulted in an average sensitivity and specificity of 32% and 94%, respectively. This global approach to antigenic profiling, epitomics, has applications to cancer and autoimmune diseases for diagnostic and therapeutic studies. Further work with larger panels of antigens should provide a comprehensive set of markers with sufficient sensitivity and specificity suitable for clinical testing in high-risk populations.

Role for human SIRT2 NAD-dependent deacetylase activity in control of mitotic exit in the cell cycle.

Studies of yeast have shown that the SIR2 gene family is involved in chromatin structure, transcriptional silencing, DNA repair, and control of cellular life span. Our functional studies of human SIRT2, a homolog of the product of the yeast SIR2 gene, indicate that it plays a role in mitosis. The SIRT2 protein is a NAD-dependent deacetylase (NDAC), the abundance of which increases dramatically during mitosis and is multiply phosphorylated at the G(2)/M transition of the cell cycle. Cells stably overexpressing the wild-type SIRT2 but not missense mutants lacking NDAC activity show a marked prolongation of the mitotic phase of the cell cycle. Overexpression of the protein phosphatase CDC14B, but not its close homolog CDC14A, results in dephosphorylation of SIRT2 with a subsequent decrease in the abundance of SIRT2 protein. A CDC14B mutant defective in catalyzing dephosphorylation fails to change the phosphorylation status or abundance of SIRT2 protein. Addition of 26S proteasome inhibitors to human cells increases the abundance of SIRT2 protein, indicating that SIRT2 is targeted for degradation by the 26S proteasome. Our data suggest that human SIRT2 is part of a phosphorylation cascade in which SIRT2 is phosphorylated late in G(2), during M, and into the period of cytokinesis. CDC14B may provoke exit from mitosis coincident with the loss of SIRT2 via ubiquitination and subsequent degradation by the 26S proteasome.