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.

PRKACA mediates resistance to HER2-targeted therapy in breast cancer cells and restores anti-apoptotic signaling.

Targeting HER2 with antibodies or small molecule inhibitors in HER2-positive breast cancer leads to improved survival, but resistance is a common clinical problem. To uncover novel mechanisms of resistance to anti-HER2 therapy in breast cancer, we performed a kinase open reading frame screen to identify genes that rescue HER2-amplified breast cancer cells from HER2 inhibition or suppression. In addition to multiple members of the MAPK (mitogen-activated protein kinase) and PI3K (phosphoinositide 3-kinase) signaling pathways, we discovered that expression of the survival kinases PRKACA and PIM1 rescued cells from anti-HER2 therapy. Furthermore, we observed elevated PRKACA expression in trastuzumab-resistant breast cancer samples, indicating that this pathway is activated in breast cancers that are clinically resistant to trastuzumab-containing therapy. We found that neither PRKACA nor PIM1 restored MAPK or PI3K activation after lapatinib or trastuzumab treatment, but rather inactivated the pro-apoptotic protein BAD, the BCl-2-associated death promoter, thereby permitting survival signaling through BCL-XL. Pharmacological blockade of BCL-XL/BCL-2 partially abrogated the rescue effects conferred by PRKACA and PIM1, and sensitized cells to lapatinib treatment. These observations suggest that combined targeting of HER2 and the BCL-XL/BCL-2 anti-apoptotic pathway may increase responses to anti-HER2 therapy in breast cancer and decrease the emergence of resistant disease.

Mosaic supernumerary ring chromosome 1 in a three-generational family: 10-year follow-up report.

Additional small ring chromosome 1 is described with increasing rate of mosaicism in three generations. Ten years after the first examination, the mosaic rates in the patients were strikingly similar. An increase in the expression of phenotypic anomalies was also observed in the successive generations. FISH examinations following microdissection revealed signals which were positive for 1p13 and 1q21 indicating that the ring contained euchromatic segments on both ends. Additionally, array-CGH whole-genome analysis showed a single copy gain corresponding to band 1p12 to band 1q21-1 of chromosome 1 in the patients. The presence of euchromatic material from chromosome 1 in the ring suggests that the relationship between the cytogenetic findings and the clinical manifestation is likely causative. These unique observations might be explained by mitotic loss of the ring at early embryogenesis, and would indicate different mitotic vulnerability of certain chromosome abnormalities at early postzygotic stages versus later during development.

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.

The fate of children with microdeletion 22q11.2 syndrome and congenital heart defect: clinical course and cardiac outcome.

BACKGROUND: This study aimed to evaluate the cardiac outcome for children with microdeletion 22q11.2 and congenital heart defect (CHD). METHODS: A total of 49 consecutive children with 22q11.2 and CHD were retrospectively identified. The CHD consisted of tetralogy of Fallot and variances (n = 22), interrupted aortic arch (n = 10), ventricular septal defect (n = 8), truncus arteriosus (n = 6), and double aortic arch (n = 1). Extracardiac anomalies were present in 46 of 47 children. RESULTS: The median follow-up time was 8.5 years (range, 3 months to 23.5 years). Cardiac surgical repair was performed for 35 children, whereas 5 had palliative surgery, and 9 never underwent cardiac surgery. The median age at repair was 7.5 months (range, 2 days to 5 years). The mean hospital stay was 35 days (range, 7-204 days), and the intensive care unit stay was 15 days (range, 3-194 days). Significant postoperative complications occurred for 26 children (74%), and surgery for extracardiac malformations was required for 21 patients (43%). The overall mortality rate was 22% (11/49), with 1-year survival for 86% and 5-year survival for 80% of the patients. A total of 27 cardiac reinterventions were performed for 16 patients (46%) including 15 reoperations and 12 interventional catheterizations. Residual cardiac findings were present in 25 patients (71%) at the end of the follow-up period. CONCLUSIONS: Children with microdeletion 22q11.2 and CHD are at high risk for mortality and morbidity, as determined by both the severity of the cardiac lesions and the extracardiac anomalies associated with the microdeletion.

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.

Clinical profiles of four patients with Rett syndrome carrying a novel exon 1 mutation or genomic rearrangement in the MECP2 gene.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the X-linked MECP2 gene encoding methyl CpG binding protein 2 (MeCP2). Recently, a new isoform of MeCP2 including exon 1 was identified. This new isoform is more abundantly expressed in brain than the isoform including exons 2-4. Very little is known about the phenotypes associated with mutations in exon 1 of MECP2 since only a limited number of RTT patients carrying such mutations have been identified so far. In this study, we screened a cohort of 20 girls with RTT for exon 1 mutations by sequencing and multiplex ligation-dependent probe amplification (MLPA). We identified one girl with a novel exon 1 mutation (c.30delCinsGA) by sequencing and three with genomic rearrangements by MLPA. Comparison of the phenotypes showed that the girls carrying a mutation or rearrangement encompassing exon 1 were more severely affected than the girls with rearrangements not affecting exon 1.

Survival with trisomy 18--data from Switzerland.

We collected records of 352 cases of trisomy 18 karyotyped between 1964 and May 2003 from the two major cytogenetic laboratories in Northeastern Switzerland. For more detailed information about the cases we contacted the referring physicians and/or the families of the patients. In this way we collected data about survival and malformations of 161 live births, 136 induced abortions and 29 stillborns or spontaneous abortions. In 26 cases of trisomy 18, only incomplete records were available. We observed that each year more cases of trisomy 18 were cytogenetically diagnosed in the two laboratories. Before 1984 almost no prenatal diagnoses were made; however, after this date the number of prenatal diagnoses increased and in the last 10 years, accounted for 75% of all cases. A decrease in the number of postnatally diagnosed cases was also observed over the same period of time. One third of the live-born children with trisomy 18 died during the first day of life. After 1 week, 1 month and 1 year of life the survival rates were 40, 22 and 6%, respectively. The median survival was 4 days, and only 1% of the children survived until their 10th birthday. Females were more likely to survive long term. In 63 cases autopsy reports were available for review. In 97% of these cases three or more malformations were found: 67% had VSD, 32% had horseshoe kidneys, 21% had esophageal atresia, 14% had omphalocele, 14% had facial clefts, and 11% had diaphragmatic hernias. In more than 50% genital hypoplasia was also described. We further analyzed survival of live-borns in relation to the length of gestation and to VSD and esophageal atresia.

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.

A case with de novo interstitial deletion of chromosome 7q21.1-q22.

A case with de novo interstitial deletion of chromosome 7q21.1-q22: A patient with multiple congenital anomalies was found to have a de novo proximal interstitial deletion of chromosome 7q21.1-q22. The patient was 10.5 years of age, and manifestations include growth retardation (below 3rd percentile), mental retardation, mild microcephaly, hypersensitivity to noise, mild spasticity, short palpebral fissures, alternant exotropia, compensated hypermetropic astigmatism, hypotelorism, hypoplastic labia majora and minora, clinodactyly of fingers 4 and 5. Molecular studies revealed that the deletion had a paternal origin, while chromosomes of both parents cytogenetically were shown to be normal. Molecular, and fluorescence in situ hybridization (FISH) analyses confirmed no deletion at the Williams-Beuren Syndrome region. Some of the heterogeneous clinical findings were consistent with previously reported cases of same chromosomal breakpoints.

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.

Parental origin of the two additional haploid sets of chromosomes in an embryo with tetraploidy.

We report on the molecular investigations performed on an embryo with tetraploidy, karyotype 92,XXXY. The embryo was spontaneously aborted after eight weeks of gestation. Molecular analyses were performed in order to determine the parental origin and mode of formation of the two additional haploid sets of chromosomes. Microsatellite markers mapping to pericentromeric chromosome regions were used. Our results show a maternal origin of one additional set of chromosomes most likely due to the incorporation of the polar body of meiosis I and a paternal origin of the second additional set of chromosomes most likely due to dispermy. The karyotype 92,XXXY is rather unusual, indeed the vast majority of cases with tetraploidy have the karyotypes 92,XXXX or 92,XXYY. To the best of our knowledge this is the first case with 92,XXXY for which molecular investigations have been performed.