Incidental finding of alpha-methylacyl-CoA racemase deficiency in a patient with oculocutaneous albinism type 4.

Genome-wide studies may lead to the discovery of genetic variants of potential clinical importance beyond the aims of the study. We performed single nucleotide polymorphism array analysis in a boy with oculocutaneous albinism to identify copy-neutral regions of homozygosity harboring genes involved in melanin biosynthesis. An unanticipated homozygous deletion of chromosome 5p13.3 was discovered, encompassing not only the OCA gene SLC45A2, but also four additional genes. This led to an unexpected presymptomatic diagnosis of alpha-methylacyl-CoA racemase deficiency in the same patient.

Complete FXN deletion in a patient with Friedreich's ataxia.

AIMS: Most patients (98%) with Friedreich's ataxia (FRDA) are homozygous for the GAA repeat expansion in FXN. Only a few compound heterozygous patients with an expanded repeat on one allele and a point mutation or an intragenic FXN deletion on the other allele are described. In a minority of the patients only a heterozygous pattern of the repeat expansion can be detected. Using array analysis after GAA repeat expansion testing, we identified a FRDA patient who is compound heterozygous for an expanded GAA repeat and a complete FXN deletion. Since not only repeat expansions and point mutations, but also large rearrangements can be the underlying cause of FRDA, a quantitative test should also be performed in case a patient shows only one allele with an expanded GAA repeat in FXN.

Multiplex ligation-depending probe amplification is not suitable for detection of low-grade mosaicism.

'Apparent non-penetrance' occurs in several genetic disorders, including tuberous sclerosis complex and neurofibromatosis type 1: clinically unaffected parents may have multiple affected offspring. Germ line or somatic mosaicism in one of the parents of the index patient is the probable cause and results in an enhanced recurrence risk. Therefore, it is of great importance to use the most sensitive technology for testing DNA of the parents of the index patient for the presence/absence of the familial mutation. To detect large rearrangements multiplex ligation-depending probe amplification (MLPA) is often used. Here we show that MLPA is less sensitive in detecting low-grade somatic mosaicism than fluorescence in situ hybridization (FISH) or a mutation-specific PCR test. Therefore, we recommend FISH (if possible) or PCR analysis for the analysis of parental DNA.

Loss of nuclear activity of the FBXO7 protein in patients with parkinsonian-pyramidal syndrome (PARK15).

Mutations in the F-box only protein 7 gene (FBXO7) cause PARK15, an autosomal recessive neurodegenerative disease presenting with severe levodopa-responsive parkinsonism and pyramidal disturbances. Understanding the PARK15 pathogenesis might thus provide clues on the mechanisms of maintenance of brain dopaminergic neurons, the same which are lost in Parkinson's disease. The protein(s) encoded by FBXO7 remain very poorly characterized. Here, we show that two protein isoforms are expressed from the FBXO7 gene in normal human cells. The isoform 1 is more abundant, particularly in primary skin fibroblasts. Both isoforms are undetectable in cell lines from the PARK15 patient of an Italian family; the isoform 1 is undetectable and the isoform 2 is severely decreased in the patients from a Dutch PARK15 family. In human cell lines and mouse primary neurons, the endogenous or over-expressed, wild type FBXO7 isoform 1 displays mostly a diffuse nuclear localization. An intact N-terminus is needed for the nuclear FBXO7 localization, as N-terminal modification by PARK15-linked missense mutation, or N-terminus tag leads to cytoplasmic mislocalization. Furthermore, the N-terminus of wild type FBXO7 (but not of mutant FBXO7) is able to confer nuclear localization to profilin (a cytoplasmic protein). Our data also suggest that overexpressed mutant FBXO7 proteins (T22M, R378G and R498X) have decreased stability compared to their wild type counterpart. In human brain, FBXO7 immunoreactivity was highest in the nuclei of neurons throughout the cerebral cortex, intermediate in the globus pallidum and the substantia nigra, and lowest in the hippocampus and cerebellum. In conclusion, the common cellular abnormality found in the PARK15 patients from the Dutch and Italian families is the depletion of the FBXO7 isoform 1, which normally localizes in the cell nucleus. The activity of FBXO7 in the nucleus appears therefore crucial for the maintenance of brain neurons and the pathogenesis of PARK15.

The use of comparative genomic hybridization and fluorescent in situ hybridization in postmortem pathology investigation of congenital malformations.

Chromosomal abnormalities are an important cause of multiple congenital anomalies (MCA). However, conventional cytogenetic analysis using culture is unsuccessful in 10% to 40% of the cases. The purpose of this study was to examine if retrospective chromosomal analysis was possible on paraffin-embedded autopsy material with new techniques, including comparative genomic hybridization (CGH) and fluorescent in situ hybridization (FISH). We investigated 92 patients, including 71 patients with MCA, 17 patients with an isolated congenital anomaly, and 4 normal controls, by conventional CGH analysis and/or FISH. The karyotype was known in 52 cases, of which 26 patients were normal and 26 had chromosomal anomalies. Comparative genomic hybridization or FISH confirmed all but 2 cases, which were not interpretable. In 40 patients the karyotype was unknown but could be analyzed successfully in 36 cases (90%) by CGH. In this series, we found 1 additional chromosomal aberration, 45,X (Turner syndrome). Furthermore, we examined the postmortem material of 12 patients by FISH, confirming a known abnormal karyotype in 9 patients, an abnormal karyotype found by CGH in 1 case, and confirming DiGeorge syndrome (22q11 deletion) in twins. Comparative genomic hybridization and FISH are reliable techniques with which to perform retrospective genetic analysis on paraffin-embedded autopsy material.

Interstitial 11q deletion derived from a maternal ins(4;11)(p14;q24.2q25): a patient report and review.

We present a family with multiple cytogenetic abnormalities, identified through a girl with several dysmorphic features and cardiac problems, suspected for Jacobsen syndrome. Cytogenetic analysis showed a 46,XX,del(11)(qter) karyotype, which was confirmed by fluorescence in situ hybridization (FISH). Cytogenetic investigation of the parents showed a chromosome aberration in both: the father had a t(11;12)(p13;q22) translocation and the mother was carrier of an ins(4;11)(p14;q24q25). FISH analysis with an 11q-subtelomeric probe from the second-generation telomere clone set and BACs from 11q24-q25 suggested a complex maternal rearrangement. However, subsequent array analysis showed a single interstitial deletion in the proband, derived from the maternal insertion. The aberrant karyotypes in both parents implicated an increased risk of unbalanced fetal chromosome composition, thus high risk for a child with multiple congenital abnormalities. Therefore, during the next pregnancy, the couple opted for prenatal diagnosis by means of amniocentesis. An interphase FISH strategy for uncultured amniotic fluid cells predicted two possible unbalanced fetal chromosome constitutions. Karyotyping of cultured amniotic cells confirmed one of the predicted unbalanced cytogenetic options, demonstrating the value of a fast interphase strategy for parents who both are carriers of a chromosomal abnormality. In addition, we present an overview of patients with Jacobsen syndrome and an interstitial 11q deletion reported thus far in literature.

Comparing two diagnostic laboratory tests for Williams syndrome: fluorescent in situ hybridization versus multiplex ligation-dependent probe amplification.

Most people with Williams syndrome (WS) have a heterozygous 1.55 Mb deletion on chromosome 7q11.23. For diagnostic purposes, fluorescence in situ hybridisation (FISH) with commercial FISH probes is commonly used to detect this deletion. We investigated whether multiplex ligation-dependent probe amplification (MLPA) is a reliable alternative for FISH. The MLPA kit (SALSA P029) contains probes for eight genes in the WS critical region: FKBP6, FZD9, TBL2, STX1A, ELN, LIMK1, RFC2, and CYLN2. The experimental FISH assay that was used consists of four probes covering the WS critical region. A total number of 63 patients was tested; in 53 patients, a deletion was detected both with FISH and MLPA(P029), in 10 patients both techniques failed to demonstrate a deletion. In only one patient, a deletion was detected which was not previously detected by two commercial FISH probes. This patient appeared to carry a small, atypical deletion. We conclude that MLPA is a reliable technique to detect WS. Compared with FISH, MLPA is less time consuming and has the possibility to detect also smaller, atypical deletions and duplications in the WS critical region.

A t(4;6)(q12;p23) translocation disrupts a membrane-associated O-acetyl transferase gene (MBOAT1) in a patient with a novel brachydactyly-syndactyly syndrome.

Here, we report a patient with a novel brachydactyly-syndactyly syndrome and a de novo translocation 46,XY,t(4;6)(q12;p23). We mapped the breakpoint and identified genes in the breakpoint region. One of the genes on chromosome 6, the membrane-associated O-acetyl transferase gene 1 (MBOAT1), was disrupted by the breakpoint. This gene consists of 13 exons and encodes a protein of 495 amino acids. MBOAT1 is predicted to be a transmembrane protein and belongs to the superfamily of membrane-bound O-acyltransferases. These proteins transfer organic compounds, usually fatty acids, onto hydroxyl groups of membrane-embedded targets. Identification of the transferred acyl group and the target may reveal the signaling pathways altered in this novel brachydactyly-syndactyly syndrome.

Clinical and genetic analysis of patients with Saethre-Chotzen syndrome.

BACKGROUND: Saethre-Chotzen syndrome is a craniosynostosis syndrome further characterized by distinctive facial and limb abnormalities. It shows complete penetrance and variable expressivity and has been linked to the TWIST gene on chromosome 7p21; more than 80 different intragenic mutations and, recently, large deletions have been detected in Saethre-Chotzen patients. The aim of this study was to genetically and phenotypically characterize patients with a clinical diagnosis of Saethre-Chotzen syndrome. METHODS: Patients with a clinical diagnosis as well as those with a genetic diagnosis of Saethre-Chotzen syndrome (n = 34) were included in the study. RESULTS: The study showed that the important features of Saethre-Chotzen syndrome are brachycephaly (occurring in 74 percent of patients), a broad, depressed nasal bridge (65 percent), a high forehead (56 percent), ptosis (53 percent), and prominent auricular crura (56 percent). Furthermore, using different molecular techniques, pathogenic mutations in the TWIST gene were identified in 71 percent of patients. CONCLUSIONS: Patients with deletions of the TWIST gene did not differ from those with intragenic TWIST mutations in frequency or severity of craniofacial abnormalities. However, they did distinguish themselves by the presence of many additional anomalies and diseases and--most importantly--the high frequency of mental retardation, which was borderline significant. The authors conclude that when using stringent inclusion criteria for studies of Saethre-Chotzen syndrome, patients who have a pathogenic mutation of the TWIST gene should be excluded.

Selective chromosome analysis in couples with two or more miscarriages: case-control study.

OBJECTIVE: To identify additional factors, such as maternal age or factors related to previous reproductive outcome or family history, and the corresponding probability of carrying a chromosome abnormality in couples with two or more miscarriages. DESIGN: Nested case-control study. SETTING: Six centres for clinical genetics in the Netherlands. PARTICIPANTS: Couples referred for chromosome analysis after two or more miscarriages in 1992-2000; 279 carrier couples were marked as cases, and 428 non-carrier couples served as controls. MAIN OUTCOME MEASURES: Independent factors influencing the probability of carrier status and the corresponding probability of carrier status. RESULTS: Four factors influencing the probability of carrier status could be identified: maternal age at second miscarriage, a history of three or more miscarriages, a history of two or more miscarriages in a brother or sister of either partner, and a history of two or more miscarriages in the parents of either partner. The calculated probability of carrier status in couples referred for chromosome analysis after two or more miscarriages varied between 0.5% and 10.2%. CONCLUSIONS: The probability of carrier status in couples with two or more miscarriages is modified by additional factors. Selective chromosome analysis would result in a more appropriate referral policy, could decrease the annual number of chromosome analyses, and could therefore lower the costs.

Mutational analysis of the TSC1 and TSC2 genes in a diagnostic setting: genotype--phenotype correlations and comparison of diagnostic DNA techniques in Tuberous Sclerosis Complex.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterised by the development of hamartomas in multiple organs and tissues. TSC is caused by mutations in either the TSC1 or TSC2 gene. We searched for mutations in both genes in a cohort of 490 patients diagnosed with or suspected of having TSC using a combination of denaturing gradient gel electrophoresis, single-strand conformational polymorphism, direct sequencing, fluorescent in situ hybridisation and Southern blotting. We identified pathogenic mutations in 362 patients, a mutation detection rate of 74%. Of these 362 patients, 276 had a definite clinical diagnosis of TSC and in these patients 235 mutations were identified, a mutation detection rate of 85%. The ratio of TSC2:TSC1 mutations was 3.4:1. In our cohort, both TSC1 mutations and mutations in familial TSC2 cases were associated with phenotypes less severe than de novo TSC2 mutations. Interestingly, consistent with other studies, the phenotypes of the patients in which no mutation was identified were, overall, less severe than those of patients with either a known TSC1 or TSC2 mutation.

Isolated postaxial polydactyly type B with mosaicism of a submicroscopic unbalanced translocation leading to an extended phenotype in offspring.

Postaxial polydactyly (PAP) is characterized by the presence of one or more extra ulnar or fibular digits or parts of it. PAP type B presents frequently as a skin tag on the hand(s). It is usually an isolated malformation, but in 6.6% it is associated with other congenital abnormalities, mostly well recognizable syndromes. We present a male with PAP-B only and his daughter with an extended phenotype including mental retardation and minor dysmorphisms. Both share a cytogenetically balanced t(4;7)(p15.2;q35), present in mosaicism in the father. We found microdeletions associated with the breakpoints. The chromosomal regions described here have not been previously associated with the PAP-B phenotype. We present the first case of an individual with isolated PAP-B and a submicroscopic chromosome abnormality.

A novel case of infantile sacral teratoma and a constitutional t(12;15)(q13;q25) pat.

Cytogenetic analysis of peripheral lymphocytes of an infantile patient with a sacral teratoma revealed a constitutional translocation (12;15)(q13;q25) pat. The same translocation was found in four additional relatives. Loss of heterozygosity analysis of the patient's tumor material showed retention of both translocation-derived chromosomes. Since allelic loss in the 12q13 region has been observed in germ cell tumors, we hypothesize that disregulation of genes located at or near the 12q13 breakpoint may be related to the development of this sacral teratoma. As a first step towards the identification of these genes, a 12q13 genomic contig that spans the breakpoint has been constructed.