Whole exome sequencing identifies multiple diagnoses in congenital glaucoma with systemic anomalies.

The genetic basis of congenital glaucoma with systemic anomalies is largely unknown. Whole exome sequencing (WES) in 10 probands with congenital glaucoma and variable systemic anomalies identified pathogenic or likely pathogenic variants in three probands; in two of these, a combination of two Mendelian disorders was found to completely explain the patients' features whereas in the third case only the ocular findings could be explained by the genetic diagnosis. The molecular diagnosis for glaucoma included two cases with compound heterozygous or homozygous pathogenic alleles in CYP1B1 and one family with a dominant pathogenic variant in FOXC1; the second genetic diagnosis for the additional systemic features included compound heterozygous mutations in NPHS1 in one family and a heterozygous 18q23 deletion in another pedigree. These findings show the power of WES in the analysis of complex conditions and emphasize the importance of CYP1B1 screening in patients with congenital glaucoma regardless of the presence/absence of other systemic anomalies.

Mitotic errors in somatic cells cause trisomy 21 in about 4.5% of cases and are not associated with advanced maternal age.

The study of DNA polymorphisms has permitted the determination of the parental and meiotic origin of the supernumerary chromosome 21 in families with free trisomy 21. Chromosomal segregation errors in somatic cells during mitosis were recognized after analysis of DNA markers in the pericentromeric region and (in order to identify recombination events) along the long arm of chromosome 21. Mitotic errors accounted for about 4.5% (11 of 238) of free trisomy 21 cases examined. The mean maternal age of mitotic errors was 28.5 years and there was no association with advanced maternal age. There was no preference in the parental origin of the duplicated chromosome 21. The 43 maternal meiosis II errors in this study had a mean maternal age of 34.1 years-the highest mean maternal age of all categories of chromosomal segregation errors.

Small nuclear ribonucleoprotein polypeptide N (SNRPN), an expressed gene in the Prader-Willi syndrome critical region.

Prader-Willi syndrome (PWS) is associated with paternally derived chromosomal deletions in region 15q11-13 or with maternal disomy for chromosome 15. Therefore, loss of the expressed paternal alleles of maternally imprinted genes must be responsible for the PWS phenotype. We have mapped the gene encoding the small nuclear RNA associated polypeptide SmN (SNRPN) to human chromosome 15q12 and a processed pseudogene SNRPNP1 to chromosome region 6pter-p21. Furthermore, SNRPN was mapped to the minimal deletion interval that is critical for PWS. The fact that the mouse Snrpn gene is maternally imprinted in brain suggests that loss of the paternally derived SNRPN allele may be involved in the PWS phenotype.

A gene for Holt-Oram syndrome maps to the distal long arm of chromosome 12.

Holt-Oram syndrome (HOS) is an autosomal dominant condition of unknown origin characterized by congenital septal heart defects with associated malformations of the upper limbs (radial ray). Here, we report on the mapping of a gene causing HOS to the distal long arm of chromosome 12 (12q21-qter) by linkage analysis in nine informative families (Zmax = 6.81 at theta = 0 at the D12S354 locus). Also, multipoint linkage analysis places the HOS gene within the genetic interval between D12S84 and D12S79 (multipoint lod-score in log base 10 = 8.10). The mapping of a gene for HOS is, to our knowledge, the first chromosomal localization of a gene responsible for congenital septal heart defect in human. The characterization of the HOS gene will hopefully shed light on the molecular mechanisms that govern heart septation in the early stages of embryogenesis.

A common mutation in the fibroblast growth factor receptor 1 gene in Pfeiffer syndrome.

Pfeiffer syndrome (PS) is one of the classic autosomal dominant craniosynostosis syndromes with craniofacial anomalies and characteristic broad thumbs and big toes. We have previously mapped one of the genes for PS to the centromeric region of chromosome 8 by linkage analysis. Here we present evidence that mutations in the fibroblast growth factor receptor-1 (FGFR1) gene, which maps to 8p, cause one form of familial Pfeiffer syndrome. A C to G transversion in exon 5, predicting a proline to arginine substitution in the putative extracellular domain, was identified in all affected members of five unrelated PS families but not in any unaffected individuals. FGFR1 therefore becomes the third fibroblast growth factor receptor to be associated with an autosomal dominant skeletal disorder.

Physical mapping of the holoprosencephaly critical region on chromosome 7q36.

Holoprosencephaly (HPE) is a developmental field defect involving the brain and face. Cytogenetic deletions in patients with HPE have localized one of the HPE genes to chromosomal region 7q36. We have characterized the 7q deletions in thirteen HPE patients. The result is the construction of a high resolution physical map of 7q32-qter. As a first step towards cloning an HPE gene crucial for normal brain development, we have defined the HPE minimal critical region in 7q36 between D7S292 and D7S392.

Linkage of Rieger syndrome to the region of the epidermal growth factor gene on chromosome 4.

Rieger syndrome is an autosomal dominant disorder of morphogenesis in which previous cytogenetic arrangements have suggested chromosome 4 as a candidate chromosome. Using a group of highly polymorphic short tandem repeat polymorphisms (STRP), including a new tetranucleotide repeat for epidermal growth factor (EGF), significant linkage of Rieger syndrome to 4q markers has been identified. Tight linkage to EGF supports its role as a candidate gene, although a recombinant in an unaffected individual has been identified. This study demonstrates the utility of using polymorphic STRP markers when only a limited number of small families are available for study.

Mutations in human TBX3 alter limb, apocrine and genital development in ulnar-mammary syndrome.

Ulnar-mammary syndrome is a rare pleiotropic disorder affecting limb, apocrine gland, tooth and genital development. We demonstrate that mutations in human TBX3, a member of the T-box gene family, cause ulnar-mammary syndrome in two families. Each mutation (a single nucleotide deletion and a splice-site mutation) is predicted to cause haploinsufficiency of TBX3, implying that critical levels of this transcription factor are required for morphogenesis of several organs. Limb abnormalities of ulnar-mammary syndrome involve posterior elements. Mutations in TBX5, a related and linked gene, cause anterior limb abnormalities in Holt-Oram syndrome. We suggest that during the evolution of TBX3 and TBX5 from a common ancestral gene, each has acquired specific yet complementary roles in patterning the mammalian upper limb.

Epigenetic mutations of the imprinted IGF2-H19 domain in Silver-Russell syndrome (SRS): results from a large cohort of patients with SRS and SRS-like phenotypes.

BACKGROUND: Silver-Russell syndrome (SRS) is a clinically and genetically heterogeneous condition characterised by severe intrauterine and postnatal growth retardation. Loss of DNA methylation at the telomeric imprinting control region 1 (ICR1) on 11p15 is an important cause of SRS. METHODS: We studied the methylation pattern at the H19-IGF2 locus in 201 patients with suspected SRS. In an attempt to categorise the patients into different subgroups, we developed a simple clinical scoring system with respect to readily and unambiguously assessable clinical features. In a second step, the relationship between clinical score and epigenetic status was analysed. RESULTS AND CONCLUSIONS: The scoring system emerged as a powerful tool for identifying those patients with both a definite SRS phenotype and carrying an epimutation at 11p15. 53% of the 201 patients initially enrolled fulfilled the criteria for SRS and about 40% of them exhibited an epimutation at the H19-IGF2 locus. Methylation defects were restricted to patients who fulfilled the diagnostic criteria for SRS. Patients carrying epimutations had a more severe phenotype than either the SRS patients with mUPD7 or the idiopathic SRS patients. The majority of patients with methylation abnormalities showed hypomethylation at both the H19 and IGF2 genes. However, we also identified SRS patients where hypomethylation was restricted to either the H19 or the IGF2 gene. Interestingly, we detected epimutations in siblings of normal parents, most likely reflecting germ cell mosaicism in the fathers. In one family, we identified an epimutation in an affected father and his likewise affected daughter.

Persistent low thymic activity and non-cardiac mortality in children with chromosome 22q11.2 microdeletion and partial DiGeorge syndrome.

A subgroup of patients with 22q11.2 microdeletion and partial DiGeorge syndrome (pDGS) appears to be susceptible to non-cardiac mortality (NCM) despite sufficient overall CD4(+) T cells. To detect these patients, 20 newborns with 22q11.2 microdeletion and congenital heart disease were followed prospectively for 6 years. Besides detailed clinical assessment, longitudinal monitoring of naive CD4(+) and cytotoxic CD3(+)CD8(+) T cells (CTL) was performed. To monitor thymic activity, we analysed naive platelet endothelial cell adhesion molecule-1 (CD31(+)) expressing CD45RA(+)RO(-)CD4(+) cells containing high numbers of T cell receptor excision circle (T(REC))-bearing lymphocytes and compared them with normal values of healthy children (n = 75). Comparing two age periods, low overall CD4(+) and naive CD4(+) T cell numbers were observed in 65%/75%, respectively, of patients in period A (< 1 year) declining to 22%/50%, respectively, of patients in period B (> 1/< 7 years). The percentage of patients with low CTLs (< P10) remained robust until school age (period A: 60%; period B: 50%). Low numbers of CTLs were associated with abnormally low naive CD45RA(+)RO(-)CD4(+) T cells. A high-risk (HR) group (n = 11) and a standard-risk (SR) (n = 9) group were identified. HR patients were characterized by low numbers of both naive CD4(+) and CTLs and were prone to lethal infectious and lymphoproliferative complications (NCM: four of 11; cardiac mortality: one of 11) while SR patients were not (NCM: none of nine; cardiac mortality: two of nine). Naive CD31(+)CD45RA(+)RO(-)CD4(+), naive CD45RA(+)RO(-)CD4(+) T cells as well as T(RECs)/10(6) mononuclear cells were abnormally low in HR and normal in SR patients. Longitudinal monitoring of naive CD4(+) and cytotoxic T cells may help to discriminate pDGS patients at increased risk for NCM.

Characterization of the supernumerary chromosome in cat eye syndrome.

Most individuals with cat eye syndrome (CES) have a supernumerary bisatellited chromosome which, on the basis of cytogenetic evidence, has been reported to originate from either chromosome 13 or 22. To resolve this question, a single-copy DNA probe, D22S9, was isolated and localized to 22q11 by in situ hybridization to metaphase chromosomes. The number of copies of this sequence was determined in CES patients by means of Southern blots and densitometry analysis of autoradiographs. In patients with the supernumerary chromosome, four copies were found, whereas in one patient with a duplication of part of chromosome 22, there were three copies. Therefore, the syndrome results from the presence of either three or four copies of DNA sequences from 22q11; there is no evidence that sequences from other chromosomes are involved. This work demonstrates how DNA sequence dosage analysis can be used to study genetic disorders that are not readily amenable to standard cytogenetic analysis.

Duplications in addition to terminal deletions are present in a proportion of ring chromosomes: clues to the mechanisms of formation.

BACKGROUND AND METHODS: Ring chromosomes are often associated with abnormal phenotypes because of loss of genomic material at one or both ends. In some cases no deletion has been detected and the abnormal phenotype has been attributed to mitotic ring instability. We investigated 33 different ring chromosomes in patients with phenotypic abnormalities by array based comparative genomic hybridisation (CGH) and fluorescence in situ hybridisation (FISH). RESULTS: In seven cases we found not only the expected terminal deletion but also a contiguous duplication. FISH analysis in some of these cases demonstrated that the duplication was inverted. Thus these ring chromosomes derived through a classical inv dup del rearrangement consisting of a deletion and an inverted duplication. DISCUSSION: Inv dup del rearrangements have been reported for several chromosomes, but hardly ever in ring chromosomes. Our findings highlight a new mechanism for the formation of some ring chromosomes and show that inv dup del rearrangements may be stabilised not only through telomere healing and telomere capture but also through circularisation. This type of mechanism must be kept in mind when evaluating possible genotype-phenotype correlations in ring chromosomes since in these cases: (1) the deletion may be larger or smaller than first estimated based on the size of the ring, with a different impact on the phenotype; and (2) the associated duplication will in general cause further phenotypic anomalies and might confuse the genotype-phenotype correlation. Moreover, these findings explain some phenotypic peculiarities which previously were attributed to a wide phenotypic variation or hidden mosaicism related to the instability of the ring.

Age and origin of major Smith-Lemli-Opitz syndrome (SLOS) mutations in European populations.

BACKGROUND: Smith-Lemli-Opitz syndrome (SLOS) (MIM 270 400) is an autosomal recessive multiple congenital anomalies/mental retardation syndrome caused by mutations in the Delta7-sterol reductase (DHCR7, E.C.1.3.1.21) gene. The prevalence of SLOS has been estimated to range between 1:15000 and 1:60000 in populations of European origin. METHODS AND RESULTS: We have analysed the frequency, origin, and age of DHCR7 mutations in European populations. In 263 SLOS patients 10 common alleles (c.964-1G>C, p.Trp151X, p.Thr93Met, p.Val326Leu, p.Arg352Trp, p.Arg404Cys, p.Phe302Leu, p.Leu157Pro, p.Gly410Ser, p.Arg445Gln) were found to constitute approximately 80% of disease-causing mutations. As reported before, the mutational spectra differed significantly between populations, and frequency peaks of common mutations were observed in North-West (c.964-1G>C), North-East (p.Trp151X, p.Val326Leu) and Southern Europe (p.Thr93Met). SLOS was virtually absent from Finland. The analysis of nearly 8000 alleles from 10 different European populations confirmed a geographical distribution of DHCR7 mutations as reported in previous studies. The common Null mutations in Northern Europe (combined ca. 1:70) occurred at a much higher frequency than expected from the reported prevalence of SLOS. In contrast the most common mutation in Mediterranean SLOS patients (p.Thr93Met) had a low population frequency. Haplotypes were constructed for SLOS chromosomes, and for wild-type chromosomes of African and European origins using eight cSNPs in the DHCR7 gene. The DHCR7 orthologue was sequenced in eight chimpanzees (Pan troglodytes) and three microsatellites were analysed in 50 of the SLOS families in order to estimate the age of the three major SLOS-causing mutations. CONCLUSIONS: The results indicate a time of first appearance of c.964-1G>C and p.Trp151X some 3000 years ago in North-West and North-East Europe, respectively. The p.Thr93Met mutations on the J haplotype has probably first arisen approximately 6000 years ago in the Eastern Mediterranean. Together, it appears that a combination of founder effects, recurrent mutations, and drift have shaped the present frequency distribution of DHCR7 mutations in Europe.

Mutation and phenotypic spectrum in patients with cardio-facio-cutaneous and Costello syndrome.

Cardio-facio-cutaneous (CFC) and Costello syndrome (CS) are congenital disorders with a significant clinical overlap. The recent discovery of heterozygous mutations in genes encoding components of the RAS-RAF-MAPK pathway in both CFC and CS suggested a similar underlying pathogenesis of these two disorders. While CFC is heterogeneous with mutations in BRAF, MAP2K1, MAP2K2 and KRAS, HRAS alterations are almost exclusively associated with CS. We carried out a comprehensive mutation analysis in 51 CFC-affected patients and 31 individuals with CS. Twelve different BRAF alterations were found in twenty-four patients with CFC (47.0%), two MAP2K1 mutations in five (9.8%) and two MAP2K2 sequence variations in three CFC-affected individuals (5.9%), whereas three patients had a KRAS alteration (5.9%). We identified four different missense mutations of HRAS in twenty-eight cases with CS (90.3%), while KRAS mutations were detected in two infants with a phenotype meeting criteria for CS (6.5%). In 14 informative families, we traced the parental origin of HRAS alterations and demonstrated inheritance of the mutated allele exclusively from the father, further confirming a paternal bias in the parental origin of HRAS mutations in CS. Careful clinical evaluation of patients with BRAF and MAP2K1/2 alterations revealed the presence of slight phenotypic differences regarding craniofacial features in MAP2K1- and MAP2K2-mutation positive individuals, suggesting possible genotype-phenotype correlations.

Cryptic deletions are a common finding in "balanced" reciprocal and complex chromosome rearrangements: a study of 59 patients.

Using array comparative genome hybridisation (CGH) 41 de novo reciprocal translocations and 18 de novo complex chromosome rearrangements (CCRs) were screened. All cases had been interpreted as "balanced" by conventional cytogenetics. In all, 27 cases of reciprocal translocations were detected in patients with an abnormal phenotype, and after array CGH analysis, 11 were found to be unbalanced. Thus 40% (11 of 27) of patients with a "chromosomal phenotype" and an apparently balanced translocation were in fact unbalanced, and 18% (5 of 27) of the reciprocal translocations were instead complex rearrangements with >3 breakpoints. Fourteen fetuses with de novo, apparently balanced translocations, all but two with normal ultrasound findings, were also analysed and all were found to be normal using array CGH. Thirteen CCRs were detected in patients with abnormal phenotypes, two in women who had experienced repeated spontaneous abortions and three in fetuses. Sixteen patients were found to have unbalanced mutations, with up to 4 deletions. These results suggest that genome-wide array CGH may be advisable in all carriers of "balanced" CCRs. The parental origin of the deletions was investigated in 5 reciprocal translocations and 11 CCRs; all were found to be paternal. Using customized platforms in seven cases of CCRs, the deletion breakpoints were narrowed down to regions of a few hundred base pairs in length. No susceptibility motifs were associated with the imbalances. These results show that the phenotypic abnormalities of apparently balanced de novo CCRs are mainly due to cryptic deletions and that spermatogenesis is more prone to generate multiple chaotic chromosome imbalances and reciprocal translocations than oogenesis.

Initial maternal meiotic I error leading to the formation of a maternal i(2q) and a paternal i(2p) in a healthy male.

We report on the investigation of the parental origin and mode of formation of the two isochromosomes, i(2p) and i(2q), detected in a healthy adult male. Conventional cytogenetic analysis revealed the proband's lack of structurally normal chromosomes 2, these being replaced by an i(2p) and an i(2q). Investigation of the parental origin of the isochromosomes revealed a paternal origin of the i(2p) chromosome and a maternal origin of the i(2q) chromosome. Thus, the formation of both isochromosomes, or at least of the paternal i(2p), appears to have occurred postzygotically. Interestingly, whilst a paternal isodisomy was observed for the entire 2p, maternal heterodisomy was detected for two segments of 2q, separated by a segment showing isodisomy. The results are indicative of an initial error (non-disjunction or i(2q) formation) concerning the maternal chromosomes 2 during meiosis I, which likely favored the subsequent mitotic recombination event resulting in the presence of two isochromosomes. To the best of our knowledge this is the first case of an initial meiotic error, followed by postzygotic trisomy rescue through the formation of isochromosomes, resulting in a normal phenotype. A prenatal detection, by cytogenetic and molecular analysis, of such chromosome abnormality would have led to the incorrect conclusion of a most likely poor prognosis for the fetus.

European Cytogeneticists Association Register of Unbalanced Chromosome Aberrations (ECARUCA); an online database for rare chromosome abnormalities.

During recent years a considerable improvement in diagnostic techniques has enabled cytogeneticists to find more and smaller chromosomal aberrations. However, accurate clinical knowledge about rare chromosome disorders is frequently lacking, mostly due to a significant decline in publishable cases. On the other hand, there is an increasing demand from parents and physicians for reliable information. In order to improve the quality and the quantity of data available, we designed a new database named the European Cytogeneticists Association Register of Unbalanced Chromosome Aberrations (ECARUCA) at http://www.ecaruca.net. This Internet-database contains cytogenetic and clinical data of patients with rare chromosome abnormalities, including microscopically visible aberrations, as well as microdeletions and -duplications. Cases with certain breakpoints collected in the Zurich Cytogenetic Database were transferred to ECARUCA. The advantages of ECARUCA compared to existing sources are that ECARUCA is interactive, dynamic and has long-term possibilities to store cytogenetic, molecular and clinical data. Professionals can login to submit new cases and perform searches in the database through the Internet. Currently the database contains 1500 unique chromosomal aberrations from almost 4000 patients. A frequent submission of new data ensures the up-to-date quality of the collection. Individual parent accounts allow parents to inform the ECARUCA team about the follow-up of their child. The ECARUCA database provides health care workers with accurate information on clinical aspects of rare chromosome disorders. Additionally, detailed correlations between chromosome aberrations and their phenotypes are of invaluable help in localising genes for mental retardation and congenital anomalies.

Unbalanced 18q/21q translocation in a patient previously reported as monosomy 21.

We describe a patient in whom full monosomy 21 was initially assumed from routine GTG-banded karyotyping. Re-examination with chromosome painting demonstrated an unbalanced translocation between the long arms of chromosomes 18 and 21. Fluorescence in situ hybridisation (FISH) and microsatellite marker analysis revealed partial monosomy of chromosome 21 (pter-q21) and 18(q22-qter). The patient, 18 years old at the second examination, revealed multiple dysmorphic features, genital hypoplasia, dilated cerebral ventricles, muscular hypotonia and severe mental retardation. In not one out of all patients investigated postnatally in whom an initial examination had revealed monosomy 21, this could be confirmed by FISH; in all of them, re-examination detected an unbalanced rearrangement leading to only partial monosomy 21 plus partial monosomy of another chromosome to which the distal 21q segment was attached. Thus, it is still highly likely that full monosomy 21 is incompatible with intra-uterine survival.

Non-random asynchronous replication at 22q11.2 favours unequal meiotic crossovers leading to the human 22q11.2 deletion.

BACKGROUND: Analyses of the replication timing at 22q11.2 were prompted by our finding of a statistically significant bias in the origin of the regions flanking the deletion site in patients with 22q11.2 deletions, the proximal region being in the majority of cases of grandmaternal origin. We hypothesised that asynchronous replication may be involved in the formation of the 22q11.2 deletion, the most frequently occurring interstitial deletion in humans, by favouring the mispairing of low-copy repeats. METHODS: Replication timing during S phase at 22q11.2 was investigated by fluorescent in situ hybridisation on interphase nuclei. We report on the detection of non-random asynchronous replication at the human chromosome region 22q11.2, an autosomal locus believed not to contain imprinted genes. RESULTS: Asynchronous replication at 22q11.2 was observed without exception in all 20 tested individuals; these comprised individuals with structurally normal chromosomes 22 (10 cases), individuals with translocations involving the locus 22q11.2 (eight cases), and patients with a 22q11.2 deletion (two cases). The non-random nature of the asynchronous replication was observed in all individuals for whom the chromosomes 22 were distinguishable. The earlier replicating allele was found to be of paternal origin in all cases where the parental origin of the translocation or deletion was known.