Clinician's guide to genes associated with Rett-like phenotypes-Investigation of a Danish cohort and review of the literature.

The differential diagnostics in Rett syndrome has evolved with the development of next generation sequencing-based techniques and many patients have been diagnosed with other syndromes or variants in newly described genes where the associated phenotype(s) is yet to be fully explored. The term Rett-like refers to phenotypes with distinct overlapping features of Rett syndrome where the clinical criteria are not completely fulfilled. In this study we have combined a review of Rett-like disorders with data from a Danish cohort of 35 patients with Rett-like phenotypes emphasizing the diagnostic overlap with Pitt-Hopkins syndrome, Cornelia de Lange syndrome with SMC1A variants, and epileptic encephalopathies, for example, due to STXBP1 variants. We also found a patient with a pathogenic variant in KCNB1, which has not been previously linked to a Rett-like phenotype. This study underlines the clinical and genetic heterogeneity of a Rett syndrome spectrum, and provides an overview of the Rett syndrome-related genes described to date, and hence serves as a guide for diagnosing patients with Rett-like phenotypes.

The MECP2 variant c.925C>T (p.Arg309Trp) causes intellectual disability in both males and females without classic features of Rett syndrome.

Missense MECP2 variants can have various phenotypic effects ranging from a normal phenotype to typical Rett syndrome (RTT). In females, the phenotype can also be influenced by the X-inactivation pattern. In this study, we present detailed clinical descriptions of six patients with a rare base-pair substitution affecting Arg309 at the C-terminal end of the transcriptional repression domain (TRD). All patients have intellectual disability and present with some RTT features, but they do not fulfill the clinical criteria for typical or atypical RTT. Most of the patients also have mild facial dysmorphism. Intriguingly, the mother of an affected male patient is an asymptomatic carrier of this variant. It is therefore likely that the p.(Arg309Trp) variation does not necessarily lead to male lethality, and it results in a wide range of clinical features in females, probably influenced by different X-inactivation patterns in target tissues.

Cornelia de Lange syndrome.

Cornelia de Lange syndrome (CdLS; MIM #122470, 300590, 610759, 614701, 300882) is a rare and clinically variable disorder that affects multiple organs. It is characterized by intellectual disability (mild to severe), distinctive facial features, prenatal and postnatal growth retardation, and hirsutism. Congenital anomalies include malformations of the upper limbs, gastrointestinal malformation/rotation, pyloric stenosis, diaphragmatic hernia, heart defects and genitourinary malformations. Gastroesophageal reflux disease is present in almost all patients. In addition to classic forms, milder phenotypes have been reported. To date five genes [NIPBL (Nipped-B-like protein), SMC1A (structural maintenance of chromosomes 1A), SMC3 (structural maintenance of chromosomes 3), RAD21 (human homolog of Schizosaccharomyces pombe radiation sensitive mutant 21) and HDAC8 (histone deacetylase 8)] have been associated with CdLS and mutations of these genes comprise the underlying defect in 70% of the patients. Here, we will provide a brief review of the clinical features of CdLS, summarize the known underlying genetic defects, prenatal and postnatal diagnosis possibilities, and genetic counseling.

Detection of triploidy at 11-14 weeks' gestation: a cohort study of 198 000 pregnant women.

OBJECTIVES: To assess the detection rate of triploidy at first-trimester screening for trisomy 21 and evaluate outcome in triploid pregnancies. METHODS: From 2008 to 2011, 198 427 women with singleton pregnancies underwent first-trimester screening between 11 + 2 and 14 + 0 weeks' gestation. Screening parameters included nuchal translucency, maternal serum free ß-human chorionic gonadotropin (ß-hCG) and pregnancy-associated plasma protein-A (PAPP-A). In all triploid fetuses, these parameters were re-evaluated. Karyotypes were established by invasive testing (chorionic villus sampling or amniocentesis) or postabortem and obtained from the Danish Cytogenetic Central Register and the Danish Fetal Medicine Database. RESULTS: A total of 30 triploid fetuses underwent first-trimester screening. Twenty-five were diagnosed as a result of abnormal first-trimester scan findings, a detection rate of 83.3%. Twenty-three fetuses were identified due to a high risk for trisomy 13, 18 or 21 and two fetuses due to structural abnormalities. The incidence of triploidy at first-trimester screening was 1:6614. A smaller crown-rump length than that estimated by date of last menstrual period was found in 95% of the fetuses with data available for evaluation. Eight fetuses had a larger biparietal diameter than expected for gestational age. Fetuses with a 69,XXX karyotype had significantly lower multiples of the median values for ß-hCG and PAPP-A than did 69,XXY fetuses (P = 0.045 and P = 0.02 forß-hCG and PAPP-A, respectively). No infants with triploidy were born in the study period. Among the triploid gestations detected on first-trimester screening, 20 (80.0%) women chose termination of pregnancy, four (16.0%) had spontaneous miscarriage and one (4.0%) was stillborn. CONCLUSION: First-trimester screening for trisomy 21 also provides a high detection rate for triploidy.

Exclusion of SNRPN as a major determinant of Prader-Willi syndrome by a translocation breakpoint.

The predominant genetic defects in Prader-Willi syndrome (PWS) are 15q11-q13 deletions of paternal origin and maternal chromosome 15 uniparental disomy (UPD). In contrast, maternal deletions and paternal chromosome 15 UPD are associated with a different neurogenetic disorder, Angelman syndrome (AS). In both disorders, these mutations are associated with parent-of-origin specific methylation at several 15q11-q13 loci. The critical PWS region has been narrowed to a approximately 320-kb region between D15S63 and D15S174, encoding several imprinted transcripts, including PAR5, IPW, PAR1 (refs 7,8) and SNRPN, which has so far been considered a strong candidate for the PWS gene. A few PWS-associated microdeletions involving a putative imprinting centre (IC) proximal to SNRPN have also been observed. We have mapped the breakpoint of a balanced translocation (9;15)pat associated with most of the PWS features between SNRPN and IPWIPAR1. Methylation and expression studies indicate that the paternal SNRPN allele is unaffected by the translocation, while IPW and PAR1 are unexpressed. This focuses the attention on genes distal to the breakpoint as the main candidate for PWS genes, and is consistent with a cis action of the putative IC, and suggests that further studies of translocational disruption of the imprinted region may establish genotype-phenotype relationships in this presumptive contiguous gene syndrome.

Breast cancer after bilateral risk-reducing mastectomy.

This study aims to evaluate the incidence of breast cancer after risk-reducing mastectomy (RRM) in healthy BRCA mutation carriers. This study is a long-term follow-up of 307 BRCA mutation carriers of whom 96 chose RRM. None of the study participants had a previous history of breast or ovarian cancer nor had they undergone RRM or risk-reducing bilateral salpingo-oophorectomy (BSO) prior to the time of BRCA testing. The annual incidence of post-mastectomy breast cancer was 0.8% compared with 1.7% in the non-operated group. Implications of these findings in relation to genetic counseling and future management are discussed.

Risk-reducing mastectomy and salpingo-oophorectomy in unaffected BRCA mutation carriers: uptake and timing.

Once female carriers of a BRCA mutation are identified they have to make decisions on risk management. The aim of this study is to outline the uptake of risk-reducing surgery in the Danish population of BRCA mutation positive women and to search for factors affecting this decision. We analysed data from 306 healthy BRCA carriers with no personal history of ovarian or breast cancer. We found a 10-year uptake of 75% for risk-reducing salpingo-oophorectomy and 50% for risk-reducing mastectomy by time to event analysis. Age and childbirth influenced this decision. The uptake rate has not changed significantly over the last decade. Risk-reducing surgeries are widely acceptable among Danish BRCA mutation positive women and the uptake of prophylactic mastectomy is higher than in most other countries.

A new microduplication syndrome encompassing the region of the Miller-Dieker (17p13 deletion) syndrome.

BACKGROUND: The use of array comparative genome hybridisation (CGH) analyses for investigation of children with mental retardation has led to the identification of a growing number of new microdeletion and microduplication syndromes, some of which have become clinically well characterised and some that await further delineation. This report describes three children with de novo 17p13.1 duplications encompassing the PAFAH1B1 gene, who had similar phenotypic features, including mild to moderate developmental delay, hypotonia and facial dysmorphism, and compares them to the few previously reported cases with this duplication. METHODS: Multiplex ligation-dependent probe amplification (MLPA) or array-CGH was used to diagnose three developmentally delayed children with duplications of 17p13. The duplications were characterised further using Agilent array technology, revealing duplication sizes from 1.8 to 4.0 Mb, with a region of overlap corresponding to 1.8 Mb. Detailed clinical information was obtained from patient files and personal examinations. RESULTS: The developmental delay and similar clinical features in the three patients were most likely due to a common microduplication of 17p13. CONCLUSIONS: In contrast to patients with deletion of the region (Miller-Dieker syndrome) the patients reported here had mild to moderate retardation and displayed no lissencephaly or gross brain malformations. Further cases with similar duplications are expected to be diagnosed, and will contribute to the delineation of a potential new microduplication syndrome of 17p13.

Cancer in patients with ataxia-telangiectasia and in their relatives in the nordic countries.

BACKGROUND: Epidemiologic studies of the families of patients with ataxia-telangiectasia (A-T), a recessive genetic neurologic disorder caused by mutation of the ATM gene, suggest that heterozygous carriers of an ATM mutation are at increased risk of cancer. A population-based study of cancer incidence in A-T families with unbiased selection and tracing of relatives would confirm this hypothesis. METHODS: We conducted a study in the Nordic countries of 1218 blood relatives of 56 A-T patients from 50 families. The relatives were identified from population registries, and the occurrence of cancer was determined from cancer registry files in each country and compared with national incidence rates. All statistical tests were two-sided. RESULTS: Among the 56 patients with A-T, we observed six cases of cancer (four leukemias and two non-Hodgkin's lymphomas) compared with 0.16 expected, yielding a standardized incidence ratio (SIR) of 37 (95% confidence interval [CI] = 13 to 80). Among the 1218 relatives, 150 cancers were recorded, with 126 expected (SIR = 1.19; 95% CI = 1.01 to 1.40). Invasive breast cancer occurred in 21 female relatives of A-T patients (SIR = 1.54; 95% CI = 0.95 to 2.36), including five of the 50 mothers (all of whom are obligate ATM mutation carriers) (SIR = 7.1; 95% CI = 2.3 to 17). Relatives who were less likely to be carriers of a mutant ATM allele had no increase or only a modest, statistically nonsignificant increase in the risk of breast cancer. There was no evidence of increased risk for cancer at any other site. CONCLUSIONS: We confirmed the previously recognized high risk of lymphoma and leukemia in A-T patients. Our data are also consistent with an increased risk of breast cancer among blood relatives of A-T patients. The epidemiologic findings suggest, however, that, even if ATM mutations are responsible for some breast cancer cases, ATM is a relatively weak genetic risk factor for the disease.

Deletion of alpha-, beta-, and omega-interferon genes in malignant cells from children with acute lymphocytic leukemia.

Scanning densitometry and restriction fragment length polymorphism analysis were used to study the alpha-, beta-, gamma-, and omega-interferon (IFN) genes in malignant cells from 11 children with acute lymphocytic leukemia and in one cell line of T-cell origin. In the malignant cells of one patient there was a complete loss of alpha-, beta-, and omega-IFN genes, whereas in another patient one of the alleles of these genes had been deleted. Cytogenetic analysis revealed a deletion of the short arm of chromosome 9, i.e., the region containing the alpha-, beta-, and omega-IFN genes, in the latter patient. The normal cells of the patients with IFN gene deletions had two alleles of the alpha-, beta-, and omega-IFN genes. In cells from none of the patients could deletions or rearrangements of the gamma-IFN genes be detected. We conclude that in 2 of 11 patients with acute lymphocytic leukemia the malignant transformation is accompanied by loss of material on one or both chromosomes 9 and that the alpha-, beta-, and omega-IFN genes are included in these deletions.

Chemotherapy-related - late occurring - Philadelphia chromosome in AML, ALL and CML. Similar events related to treatment with DNA topoisomerase II inhibitors?

Therapy with DNA topoisomerase II inhibitors has been shown to result in an increased risk of acute myeloid leukemia (AML), often presenting balanced translocations to chromosome bands 11q23 and 21q22. Also other balanced aberrations, more rarely observed in therapy-related AML (t-AML), such as t(15;17) and inv(16) have been associated with these drugs. Recently we observed a case of chronic myeloid leukemia (CML) with t(9;22) after therapy of a germ cell tumor with etoposide, cisplatin and bleomycin. Based on this case and a review of chemotherapy-related leukemias with t(9;22) from the literature, we suggest a causal relationship between therapy with DNA topoisomerase II inhibitors and development of various types of leukemia carrying the Philadelphia chromosome.

Interferon system defects in malignant T-cells.

Deletions of chromosome 9p21-22 occur in acute lymphocytic leukemia (ALL), melanoma and glioma. With some exceptions, these deletions include the alpha- and beta-interferon (IFN) genes. In this study, the frequency of alpha- and beta-IFN gene deletions was investigated in 17 T-cell lines, and losses of IFN genes were related to other aspects of the IFN system. Deletions of alpha-/beta-IFN genes were observed in 7/17 cell lines. In two cases the deletions were homozygous for both loci. In most cases aberrations of chromosome 9 were also apparent on cytogenetic analysis. An increased proportion (40% as compared to the expected 13%) of the remaining ten cell lines showed homozygosity for all five common polymorphic alpha-/beta-IFN markers, possibly implicating allelic deletion by loss of heterozygosity (LOH) in some of these clones. The cell lines showed a large variability in IFN production, IFN-alpha receptor number, susceptibility to IFN measured as induction of the enzyme 2',5' oligoadenylate synthetase and cell growth inhibition. No correlations between loss of IFN genes and IFN-producing capacity, or susceptibility to IFN, were found. Of the seven cell lines with a normal IFN-gene dosage and heterozygosity for the alpha- and beta-IFN genes, three had a deficiency in their IFN-producing capacity and one was also insensitive to growth inhibition by IFN. All IFN-producing cell lines predominantly produced beta-IFN.

Disease associated balanced chromosome rearrangements: a resource for large scale genotype-phenotype delineation in man.

Disease associated balanced chromosomal rearrangements (DBCRs), which truncate, delete, or otherwise inactivate specific genes, have been instrumental for positional cloning of many disease genes. A network of cytogenetic laboratories, Mendelian Cytogenetics Network (MCN), has been established to facilitate the identification and mapping of DBCRs. To get an estimate of the potential of this approach, we surveyed all cytogenetic archives in Denmark and southern Sweden, with a population of approximately 6.6 million. The nine laboratories have performed 71 739 postnatal cytogenetic tests. Excluding Robertsonian translocations and chromosome 9 inversions, we identified 216 DBCRs ( approximately 0.3%), including a minimum estimate of 114 de novo reciprocal translocations (0.16%) and eight de novo inversions (0.01%). Altogether, this is six times more frequent than in the general population, suggesting a causal relationship with the traits involved in most of these cases. Of the identified cases, only 25 (12%) have been published, including 12 cases with known syndromes and 13 cases with unspecified mental retardation/congenital malformations. The remaining DBCRs were associated with a plethora of traits including mental retardation, dysmorphic features, major congenital malformations, autism, and male and female infertility. Several of the unpublished DBCRs defined candidate breakpoints for nail-patella, Prader-Willi, and Schmidt syndromes, ataxia, and ulna aplasia. The implication of the survey is apparent when compared with MCN; altogether, the 292 participating laboratories have performed >2.5 million postnatal analyses, with an estimated approximately 7500 DBCRs stored in their archives, of which more than half might be causative mutations. In addition, an estimated 450-500 novel cases should be detected each year. Our data illustrate that DBCRs and MCN are resources for large scale establishment of phenotype-genotype relationships in man.

Further delineation of the 22q13 deletion syndrome.

A chromosomal deletion syndrome associated with a 22q13 microdeletion has previously been reported in approximately 75 children. We report six cases from Denmark with a deletion of 22q13. One was cytogenetically visible by conventional karyotyping, one was diagnosed by high resolution karyotyping after the demonstration of low arylsulfatase A activity. Two were diagnosed by high resolution CGH analysis, one was diagnosed by multisubtelomeric FISH analysis and one was diagnosed serendipitously as lack of the control signal in a FISH analysis for 22q11 deletion. One of the cases was a mosaic with 16% of cells showing two signals. The phenotype of the children included: generalized developmental delay, compromised language development, hypotonia, normal or accelerated growth and minor facial dysmorphism. Other features were partial agenesis of the corpus callosum, bilateral ureteropelvic stricture, gastroesophageal reflux and hearing loss. One case had a different phenotype, and showed a deletion as well as a duplication. The extent of the deletion was studied by quantitative PCR analysis of a number of DNA markers in the 22q13 region. The deletions varied in size, extending from 4.0 to 9.0 Mb. The clinical phenotype seemed rather similar although some specific features might be attributable to differences in deletions.

Characterization of ATM mutations in 41 Nordic families with ataxia telangiectasia.

The Ataxia Telangiectasia Mutation (ATM) gene is mutated in the rare recessive syndrome Ataxia Telangiectasia (AT), which is characterized by cerebellar degeneration, immunodeficiency, and cancer predisposition. In this study, 41 AT families from Denmark, Finland, Norway, and Sweden were screened for ATM mutations. The protein truncation test (PTT), fragment length and heteroduplex analyses of large (0.8-1.2 kb) cDNA fragments were used. In total, 67 of 82 (82%) of the disease-causing alleles were characterized. Thirty-seven unique mutations were detected of which 25 have not previously been reported. The mutations had five different consequences for the ATM transcript: mutations affecting splicing (43%); frameshift mutations (32%); nonsense mutations (16%); small in-frame deletions (5%); and one double substitution (3%). In 28 of the probands mutations were found in both alleles, in 11 of the probands only one mutated allele was detected, and no mutations were detected in two Finnish probands. One-third of the probands (13) were homozygous, whereas the majority of the probands (26) were compound heterozygote with at least one identified allele. Ten alleles were found more than once; one Norwegian founder mutation constituted 57% of the Norwegian alleles. Several sequence variants were identified, none of them likely to be disease-causing. Some of them even involved partial skipping of exons, leading to subsequent truncation of the ATM protein.

Mutational analysis of PAX6: 16 novel mutations including 5 missense mutations with a mild aniridia phenotype.

Mutations in the developmental control gene PAX6 have been shown to be the genetic cause of aniridia, which is a severe panocular eye disease characterised by iris hypoplasia. The inheritance is autosomal dominant with high penetrance but variable expressivity. Here we describe a mutational analysis of 27 Danish patients using a dideoxy fingerprinting method, which identified PAX6 mutations in 18 individuals with aniridia. A thorough phenotype description was made for the 18 patients. A total of 19 mutations, of which 16 were novel, are described. Among these were five missense mutations which tended to be associated with a milder aniridia phenotype, and in fact one of them seemed to be non-penetrant. Four of the five missense mutations were located in the paired domain. We also describe a third alternative spliced PAX6 isoform in which two of the four missense mutations would be spliced out. Our observations support the concept of dosage effects of PAX6 mutations as well as presenting evidence for variable expressivity and gonadal mosaicism.

High carrier frequency of the 35delG deafness mutation in European populations. Genetic Analysis Consortium of GJB2 35delG.

Congenital deafness accounts for about 1 in 1000 infants and approximately 80% of cases are inherited as an autosomal recessive trait. Recently, it has been demonstrated that connexin 26 (GJB2) gene is a major gene for congenital sensorineural deafness. A single mutation (named 35delG) was found in most recessive families and sporadic cases of congenital deafness, among Caucasoids, with relative frequencies ranging from 28% to 63%. We present here the analysis of the 35delG mutation in 3270 random controls from 17 European countries. We have detected a carrier frequency for 35delG of 1 in 35 in southern Europe and 1 in 79 in central and northern Europe. In addition, 35delG was detected in five out of 376 Jewish subjects of different origin, but was absent in other non-European populations. The study suggests either a single origin for 35delG somewhere in Europe or in the Middle East, and the possible presence of a carrier advantage together with a founder effect. The 35delG carrier frequency of 1 in 51 in the overall European population clearly indicates that this genetic alteration is a major mutation for autosomal recessive deafness in Caucasoids. This finding should facilitate diagnosis of congenital deafness and allow early treatment of the affected subjects.

Analysis of FMR1 (CGG)n alleles and FRAXA microsatellite haplotypes in the population of Greenland: implications for the population of the New World from Asia.

The fragile X syndrome is caused by the expansion of a polymorphic (CGG)n tract in the promoter region of the FMR1 gene. Apparently the incidence of fragile X syndrome is rare in the population of Greenland. In order to examine population-related factors involved in stability of the (CGG)n sequence, DNA samples obtained randomly from the Greenlandic population were analysed for size and AGG interspersion pattern of the FMR1 (CGG)n region and associated DXS548-FRAXAC1 haplotypes. In addition a large Greenland family with unstable transmission in the premutation range was analysed. The (CGG)n allele sizes in the Greenland population showed a narrow distribution similar to that reported for Asian populations. DNA sequencing of alleles with 36 CGG repeats revealed an AGG(CGG)6 insertion previously reported exclusively in Asian populations and a high frequency of alleles with a (CGG)10AGG(CGG)9AGG(CGG)9 or (CGG)9AGG(CGG)9AGG(CGG)6AGG(CGG)9 sequence pattern was found. Thus the data confirm the Asian origin of the Greenlandic (Eskimo) population and indicates that some (CGG)n alleles have remained stable for 15-30,000 years, since the population of the New World arrived from Asia via the Bering Strait.

Tetrasomy 18p de novo: parental origin and different mechanisms of formation.

We have used eight PCR-based DNA polymorphisms to determine the parental origin and mechanisms of formation in 9 patients with de novo nonmosaic tetrasomy 18p. The 9 patients, 4 girls and 5 boys, had clinical features characteristic of i(18p) syndrome. The supernumerary marker chromosome was identified by fluorescence in situ hybridization (FISH) analysis using centromeric probes and a flow-sorted 18p-specific library. The isochromosome was of maternal origin in all 9 cases. The formation of tetrasomy 18p cannot be explained by a single model. In 6 cases, meiosis II nondisjunction, followed by subsequent postzygotic misdivsion, and in 1 case postzygotic nondisjunction and postzygotic misdivision were the most likely mechanisms of formation. Alternative mechanisms are suggested in the remaining 2 cases.

Growth pattern in boys with fragile X.

The impression that a proportion of boys with fra(X) may show overgrowth in early infancy was investigated in a small series of 7 boys born after 1970. The fra(X) positive boys had been referred for mental retardation, or were identified in investigations of familial mental retardation. The mean birth weight was increased, and average linear growth was consistently above the mean for Danish boys with the greatest increase in the second year of life. In contrast, the weight measurements were on average below the mean until age 2 years. It is suggested that in fra(X) patients there is a disturbance of early infantile growth.