Does non-invasive prenatal testing affect the livebirth prevalence of Down syndrome in the Netherlands? A population-based register study.

OBJECTIVE: To evaluate if non-invasive prenatal testing (NIPT) affects livebirth (LB) prevalence of Down syndrome (DS) in the Netherlands. METHOD: Data from clinical genetics laboratories and the Working Party on Prenatal Diagnosis and Therapy (2014-2018) and previous published data (1991-2013) were used to assess trends for DS LB prevalence and reduction percentage (the net decrease in DS LBs resulting from selective termination of pregnancies). Statistics Netherlands provided general population data. RESULTS: DS LB prevalence increased from 11.6/10,000 in 1991 to 15.9/10,000 in 2002 (regression coefficient 0.246 [95% CI: 0.105-0.388; p = 0.003]). After 2002, LB prevalence decreased to 11.3/10,000 in 2014 and further to 9.9/10,000 in 2018 (regression coefficient 0.234 (95% CI: -0.338 to -0.131; p < 0.001). The reduction percentage increased from 26% in 1991 to 55.2% in 2018 (regression coefficient 0.012 (95% CI: 0.010-0.013; p < 0.001)). There were no trend changes after introducing NIPT as second-tier (2014) and first-tier test (2017). CONCLUSIONS: Introducing NIPT did not change the decreasing trend in DS LB prevalence and increasing trend in reduction percentage. These trends may be caused by a broader development of more prenatal testing that had already started before introducing NIPT.

Perinatal follow-up of children born after preimplantation genetic diagnosis between 1995 and 2014.

PURPOSE: We aim to evaluate the safety of PGD. We focus on the congenital malformation rate and additionally report on adverse perinatal outcome. METHODS: We collated data from a large group of singletons and multiples born after PGD between 1995 and 2014. Data on congenital malformation rates in live born children and terminated pregnancies, misdiagnosis rate, birth parameters, perinatal mortality, and hospital admissions were prospectively collected by questionnaires. RESULTS: Four hundred thirty-nine pregnancies in 381 women resulted in 364 live born children. Nine children (2.5%) had major malformations. This percentage is consistent with other PGD cohorts and comparable to the prevalence reported by the European Surveillance of Congenital Anomalies (EUROCAT). We reported one misdiagnosis resulting in a spontaneous abortion of a fetus with an unbalanced chromosome pattern. 20% of the children were born premature (< 37 weeks) and less than 15% had a low birth weight. The incidence of hospital admissions is in line with prematurity and low birth weight rate. One child from a twin, one child from a triplet, and one singleton died at 23, 32, and 37 weeks of gestation respectively. CONCLUSIONS: We found no evidence that PGD treatment increases the risk on congenital malformations or adverse perinatal outcome. TRIAL REGISTRATION NUMBER: NCT 2 149485.

Can Characteristics of Reciprocal Translocations Predict the Chance of Transferable Embryos in PGD Cycles?

Translocation carriers have an increased risk of miscarriage or the birth of a child with congenital anomalies. Preimplantation genetic diagnosis (PGD) is performed in translocation carriers to select for balanced embryos and, thus, increase the chance of an ongoing pregnancy. However, a common experience is that reciprocal translocation carriers produce a high percentage of unbalanced embryos, which cannot be transferred. Therefore, the pregnancy rates in PGD in this patient group are low. In a cohort of 85 reciprocal translocation carriers undergoing PGD we have searched for cytogenetic characteristics of the translocations that can predict the percentage of balanced embryos. Using shape algorithms, the most likely segregation mode per translocation was determined. Shape algorithm, breakpoint location, and relative chromosome segment sizes proved not to be independent predictors of the percentage of balanced embryos. The ratio of the relative sizes of the translocated segments of both translocation chromosomes can give some insight into the chance of transferable embryos: Very asymmetrical translocations have a higher risk of unbalanced products (p = 0.048). Counseling of the couples on the pros and cons of all their reproductive options remains very important.

A new syndrome of microtia with unilateral renal agenesis and short stature.

We report on a 13-month-old girl of first cousin parents who presented with a combination of short stature, bilateral microtia, proportionate short stature, distinctive facial features (bitemporal narrowing, long philtrum), and agenesis of the left kidney and a small right kidney. Clinical findings did not match any previously described syndromes with the anomalies seen in the patient. We performed SNP array analysis to characterize the observation as a novel syndrome and this was normal. We propose that this represents a new syndrome, likely of autosomal recessive inheritance.

SNP array-based copy number and genotype analyses for preimplantation genetic diagnosis of human unbalanced translocations.

Preimplantation genetic diagnosis (PGD) for chromosomal rearrangements (CR) is mainly based on fluorescence in situ hybridisation (FISH). Application of this technique is limited by the number of available fluorochromes, the extensive preclinical work-up and technical and interpretative artefacts. We aimed to develop a universal, off-the-shelf protocol for PGD by combining single-nucleotide polymorphism (SNP) array-derived copy number (CN) determination and genotyping for detection of unbalanced translocations in cleavage-stage embryos. A total of 36 cleavage-stage embryos that were diagnosed as unbalanced by initial PGD FISH analysis were dissociated (n=146) and amplified by multiple displacement amplification (MDA). SNP CNs and genotypes were determined using SNP array. Epstein-Barr Virus-transformed cell lines with known CR were used for optimising the genomic smoothing (GS) length setting to increase signal to noise ratio. SNP CN analysis showed 23 embryos (64%) that were unbalanced in all blastomeres for the chromosomes involved in the translocation, 5 embryos (14%) that were normal or balanced in all blastomeres and 8 embryos (22%) that were mosaic. SNP genotyping, based on analysis of informative SNP loci with opposing homozygous parental genotypes, confirmed partial monosomies associated with inheritance of unbalanced translocation in surplus embryos. We have developed a universal MDA-SNP array technique for chromosome CN analysis in single blastomeres. SNP genotyping could confirm partial monosomies. This combination of techniques showed improved diagnostic specificity compared with FISH and may provide more reliable PGD analysis associated with higher embryo transfer rate.

MYT1L is a candidate gene for intellectual disability in patients with 2p25.3 (2pter) deletions.

A partial deletion of chromosome band 2p25.3 (2pter) is a rarely described cytogenetic aberration in patients with intellectual disability (ID). Using microarrays we identified deletions of 2p25.3, sized 0.37-3.13 Mb, in three adult siblings and three unrelated patients. All patients had ID, obesity or overweight and/or a square-shaped stature without overt facial dysmorphic features. Combining our data with phenotypic and genotypic data of three patients from the literature we defined the minimal region of overlap which contained one gene, i.e., MYT1L. MYT1L is highly transcribed in the mouse embryonic brain where its expression is restricted to postmitotic differentiating neurons. In mouse-induced pluripotent stem cell (iPS) models, MYT1L is essential for inducing functional mature neurons. These resemble excitatory cortical neurons of the forebrain, suggesting a role for MYT1L in development of cognitive functions. Furthermore, MYT1L can directly convert human fibroblasts into functional neurons in conjunction with other transcription factors. MYT1L duplication was previously reported in schizophrenia, indicating that the gene is dosage-sensitive and that shared neurodevelopmental pathways may be affected in ID and schizophrenia. Finally, deletion of MYT1, another member of the Myelin Transcription Factor family involved in neurogenesis and highly similar to MYT1L, was recently described in ID as well. The identification of MYT1L as candidate gene for ID justifies further molecular studies aimed at detecting mutations and for mechanistic studies on its role in neuron development and on neuropathogenic effects of haploinsufficiency.

Formation of novel CENP-A domains on tandem repetitive DNA and across chromosome breakpoints on human chromosome 8q21 neocentromeres.

Endogenous human centromeres form on megabase-sized arrays of tandemly repeated alpha satellite DNA. Human neocentromeres form epigenetically at ectopic sites devoid of alpha satellite DNA and permit analysis of centromeric DNA and chromatin organization. In this study, we present molecular cytogenetic and CENP-A chromatin immunoprecipitation (ChIP) on CHIP analyses of two neocentromeres that have formed in chromosome band 8q21 each with a unique DNA and CENP-A chromatin configuration. The first neocentromere was found on a neodicentric chromosome 8 with an inactivated endogenous centromere, where the centromeric activity and CENP-A domain were repositioned to band 8q21 on a large tandemly repeated DNA. This is the first example of a neocentromere forming on repetitive DNA, as all other mapped neocentromeres have formed on single copy DNA. Quantitative fluorescent in situ hybridization (FISH) analysis showed a 60% reduction in the alpha satellite array size at the inactive centromere compared to the active centromere on the normal chromosome 8. This neodicentric chromosome may provide insight into centromere inactivation and the role of tandem DNA in centromere structure. The second neocentromere was found on a neocentric ring chromosome that contained the 8q21 tandemly repeated DNA, although the neocentromere was localized to a different genomic region. Interestingly, this neocentromere is composed of two distinct CENP-A domains in bands 8q21 and 8q24, which are brought into closer proximity on the ring chromosome. This neocentromere suggests that chromosomal rearrangement and DNA breakage may be involved in neocentromere formation. These novel examples provide insight into the formation and structure of human neocentromeres.

A translocation in acute lymphoblastic leukemia that cytogenetically mimics the recurrent MLL-AFF1 translocation and fuses SEPT11 to MLL.

A 55-year-old man sought care for aggressive acute lymphoblastic leukemia (ALL), which developed 8 years after he had received chemotherapeutic treatment for nephrotic syndrome. The sole cytogenetic abnormality observed in bone marrow-derived metaphases was a t(4;11)(q21;q23), which is a frequently occurring translocation in ALL. However, subsequent reverse transcriptase-polymerase chain reaction for the expected mixed lineage leukemia [trithorax homolog, Drosophila] (MLL)-AFF1 fusion transcript was negative. Further fluorescence in situ hybridization (FISH) analysis narrowed the 4q21 breakpoint down to a 250-kb region proximal of AFF1. This comprised four genes, of which septin11 (SEPT11) was further analyzed. Reverse transcriptase-polymerase chain reaction revealed expression of a chimeric MLL-SEPT11 transcript, thus identifying what is to our knowledge a hitherto undescribed translocation in ALL. Sequence analysis of cDNA showed in-frame fusion of MLL exon 11 to SEPT11 exon 2. This MLL-SEPT11 fusion is cytogenetically indistinguishable from the recurrent t(4;11)(q21;q23). Thus, it is crucial to characterize cytogenetic aberrations in leukemia by molecular methods, even in cases where a known recurrent translocation is presumed. This report expands the spectrum of ALL-related translocations and hypothesizes on the mechanism leading to the MLL-SEPT11 fusion. Five septins have been identified thus far as MLL fusion partners in leukemia. Their putative oncogenic role may be related to forced MLL dimerization by the septin coiled coil and GTP-binding domains, which could convert MLL to an oncogene.

Maternal uniparental isodisomy is responsible for serious molybdenum cofactor deficiency.

Molybdenum cofactor (MoCo) deficiency is a rare autosomal recessive inherited metabolic disorder resulting in the combined deficiency of aldehyde oxidase, xanthine dehydrogenase, and sulfite oxidase. We report a male infant with MoCo deficiency whose clinical findings consisted of microcephaly, intractable seizures soon after birth, feeding difficulties, and developmental delay. Sequencing of MOCS1, MOCS2, and GEPH genes, and single nucleotide polymorphism genotyping array analysis showed, to our knowledge, unusual inheritance of MoCo deficiency/maternal uniparental isodisomy for the first time in the literature. At 10 months of age, he now has microcephaly and developmental delay, and his seizures are controlled with phenobarbital, clonozepam, and vigabatrin therapy.

The unfolding clinical spectrum of holoprosencephaly due to mutations in SHH, ZIC2, SIX3 and TGIF genes.

Holoprosencephaly is a severe malformation of the brain characterized by abnormal formation and separation of the developing central nervous system. The prevalence is 1:250 during early embryogenesis, the live-born prevalence is 1:16 000. The etiology of HPE is extremely heterogeneous and can be teratogenic or genetic. We screened four known HPE genes in a Dutch cohort of 86 non-syndromic HPE index cases, including 53 family members. We detected 21 mutations (24.4%), 3 in SHH, 9 in ZIC2 and 9 in SIX3. Eight mutations involved amino-acid substitutions, 7 ins/del mutations, 1 frame-shift, 3 identical poly-alanine tract expansions and 2 gene deletions. Pathogenicity of mutations was presumed based on de novo character, predicted non-functionality of mutated proteins, segregation of mutations with affected family-members or combinations of these features. Two mutations were reported previously. SNP array confirmed detected deletions; one spanning the ZIC2/ZIC5 genes (approx. 100 kb) the other a 1.45 Mb deletion including SIX2/SIX3 genes. The mutation percentage (24%) is comparable with previous reports, but we detected significantly less mutations in SHH: 3.5 vs 10.7% (P=0.043) and significantly more in SIX3: 10.5 vs 4.3% (P=0.018). For TGIF1 and ZIC2 mutation the rate was in conformity with earlier reports. About half of the mutations were de novo, one was a germ line mosaic. The familial mutations displayed extensive heterogeneity in clinical manifestation. Of seven familial index patients only two parental carriers showed minor HPE signs, five were completely asymptomatic. Therefore, each novel mutation should be considered as a risk factor for clinically manifest HPE, with the caveat of reduced clinical penetrance.

Different distribution of the genetic subtypes of the Prader-Willi syndrome in the elderly.

The Prader-Willi syndrome (PWS) is a genetic disorder caused by the absent expression of the paternal copy of maternally imprinted genes in chromosome region 15q11-13. The frequencies of different subtypes in PWS are usually given in literature as 70% deletion, 25-30% maternal uniparental disomy (mUPD) and 3-5% others (imprinting centre (IC) defects and translocations). Little is known about factors that influence the frequency of genetic subtypes in PWS. The study sample comprised 102 adults with clinically and genetically confirmed PWS, contacted through the Dutch Prader-Willi Parent Association and through physicians specialized in treating persons with intellectual disabilities. Genetic testing showed 55 persons (54%) with a paternal deletion, 44 persons (43%) with an mUPD and 3 persons (3%) with a defect of the IC. The observed distribution in our study differed from that in literature (70% deletion, 30% mUPD), which was statistically significant (z-score: P<0.05). This was mainly caused by a higher proportion of mUPD in the advanced age groups. Differences in maternal age and BMI of persons with PWS could not explain the differences in distribution across the age groups. Our study population had a much broader age range, compared with other studies, because of a predominance of elderly people (40+ years) with PWS. In other studies, these elderly persons might have been undiagnosed and/or underreported because of a lack of genetic diagnosis. The results underline both the need for correct genetic diagnosis in all persons with PWS and adjustment of the guidelines for preventive management in adulthood.

Identical cryptic partial monosomy 20pter and trisomy 20qter in three adult siblings due to a large maternal pericentric inversion: detection by MLPA and breakpoint mapping by SNP array analysis.

Genotypic and phenotypic data are presented on three adult siblings with mild to moderate mental retardation and mild dysmorphic features. All three siblings showed a chromosome 20 gain at the q-telomere and loss at the p-telomere in routine subtelomeric MLPA screening. Analysis of GTG-banded chromosomes did not detect any abnormalities, but subtelomeric fluorescent in situ hybridization (FISH) confirmed cryptic partial monosomy of chromosome region 20p13 --> 20pter and cryptic partial trisomy of chromosome region 20q13.33 --> 20qter. Furthermore, FISH analysis in the mother showed a cryptic inv(20)(p13q13.33). This explained the cytogenetic mechanism underlying the chromosomal imbalance in the three children, that is, the meiotic formation of a recombinant chromosome 20 due to crossing-over in the inverted segment. All three children thus carried a rec(20)dup(20q)inv(20)(p13q13.33)mat chromosome. SNP array analysis enabled rapid and detailed imbalance sizing and showed a 1.06 Mb loss in 20p13 and a 2.51 Mb gain in 20q13.33, comprising 21 and 78 genes, respectively. The maternal inversion is the largest described thus far for chromosome 20, comprising 94.4% of its length. Such large inversions result in a particularly high risk for live-born unbalanced offspring because the partial monosomy and trisomy segments are small. Moreover, the inversion size is directly related to the percentage of unbalanced gametes due to high crossing-over change within the inverted segment. The fact that all three children carry an identical chromosomal rearrangement has consequences for genetic counseling for carriers of large pericentric inversions, as the recurrence risk is very high.

Array analysis and karyotyping: workflow consequences based on a retrospective study of 36,325 patients with idiopathic developmental delay in the Netherlands.

Anomalies of chromosome number and structure are considered to be the most frequent cause of unexplained, non-syndromic developmental delay and mental retardation (DD/MR). High-resolution, genome-wide, array-based segmental aneusomy profiling has emerged as a highly sensitive technique for detecting pathogenic genomic imbalances. A review of 29 array-based studies of DD/MR patients showed that a yield of at least approximately 19% pathogenic aberrations is attainable in unselected, consecutive DD/MR referrals if array platforms with 30-70 kb median probe spacing are used as an initial genetic testing method. This corresponds to roughly twice the rate of classical cytogenetics. This raises the question whether chromosome banding studies, combined with targeted approaches, such as fluorescence in situ hybridisation for the detection of microdeletions, still hold substantial relevance for the clinical investigation of these patients. To address this question, we reviewed the outcome of cytogenetic studies in all 36,325 DD/MR referrals in the Netherlands during the period 1996-2005, a period before the advent of array-based genome investigation. We estimate that in a minimum of 0.78% of all referrals a balanced chromosomal rearrangement would have remained undetected by array-based investigation. These include familial rearrangements (0.48% of all referrals), de novo reciprocal translocations and inversions (0.23% of all referrals), de novo Robertsonian translocations (0.04% of all referrals), and 69,XXX triploidy (0.03% of all referrals). We conclude that karyotyping, following an initial array-based investigation, would give only a limited increase in the number of pathogenic abnormalities, i.e. 0.23% of all referrals with a de novo, apparently balanced, reciprocal translocation or inversion (assuming that all of these are pathogenic), and 0.03% of all referrals with 69,XXX triploidy. We propose that, because of its high diagnostic yield, high-resolution array-based genome investigation should be the first investigation performed in cases of DD/MR, detecting >99% of all pathogenic abnormalities. Performing both array investigation and karyotyping may not be a feasible option when laboratories are faced with a need to limit the number of genetic tests available for each patient. However, laboratories that supplant karyotyping by array-based investigation should be aware that, as shown here, a chromosomal abnormality, with possible pathogenic consequences for the patient or the family, will escape detection in about 0.78% of all DD/MR referrals.

Prenatal identification of a marker chromosome 16 by chromosome microdissection and reverse FISH.

Prenatal cytogenetic analysis of cultured amniocytes was performed after an increased foetal nuchal translucency thickness was detected by ultrasound in week 17 of a pregnancy. Analysis of GTG-banded chromosomes showed a small marker chromosome in six of the 12 colonies analysed. The supernumerary abnormal chromosome appeared to be positive with DA/DAPI staining and C-banding. The parents' karyotypes were normal. Using microFISH and FISH with band-specific probes, we found the marker appeared to be derived from chromosome region (16)(p13.1-->q12.2). Accurate identification of the marker chromosome was important for prenatal counselling: the marker chromosome contained euchromatic sequences, the foetus was carrying mosaic trisomy 16, and based on the literature the prognosis for the foetus was unfavourable and the pregnancy was terminated.

Toward positional cloning of the curly tail gene.

BACKGROUND: The curly tail (ct) mutant mouse is one of the best-studied mouse models of spina bifida. The ct mutation has been localized to distal chromosome 4 in two independent studies and was recently postulated to be in the Grhl-3 gene. METHODS: A recombinant BALB/c-ct strain was generated and used to precisely map the ct gene. RESULTS: We report the absence of gross chromosomal abnormalities and the precise mapping of the ct gene to a 3-Mb region at 135 Mb (66 cM) from the centromere, closely linked to the polymorphic microsatellite marker D4Mit148. Candidate genes, Idb3, Wnt4, Cdc42, and perlecan, all localized in the critical region, were studied by sequence and expression analyses. Our data indicate that these genes in all probability do not account for the ct phenotype. In addition, our expression data do not provide strong evidence that Grhl-3 is indeed the ct gene. CONCLUSIONS: The ct gene has not yet been identified. A total of 29 candidate genes remain present in the critical region. Refined mapping studies need to be performed to further narrow the region and additional candidate genes need to be examined. Supplementary material for this article can be found on the Birth Defects Research (Part A) website (http://www.mrw.interscience.wiley.com/suppmat/1542-0752/suppmat/2005/73/tables_S3-S6.doc).

Kabuki syndrome is not caused by an 8p duplication: a cytogenetic study in 20 patients.

Kabuki syndrome is characterized by a typical facial gestalt in combination with hypotonia and joint laxity, developmental delay, persistent fetal fingertip pads, and structural abnormalities mainly of the palate and the heart. Cytogenetic conditions may present with features of the syndrome. Recently, Milunsky and Huang [2003], reported an 8p duplication at chromosome 8p22-8p23.1 in 6 patients with Kabuki syndrome. We studied 20 individuals with Kabuki syndrome and were not able to confirm this finding. Kabuki syndrome remains a clinical diagnosis in the majority of cases.

Familial cryptic translocation with deletion 4q33-->4qter and duplication 7q34-->7qter in brothers with mental retardation, macrocephaly and iris coloboma.

The association of moderate mental retardation, behavioural problems, macrocephaly, dysmorphic features with iris coloboma, and supernumerary nipples was observed in two brothers with a terminal deletion 4q33-->4qter and a terminal duplication 7q34-->7qter. The aberration was detected by subtelomere FISH screening and (probably) resulted from a cryptic familial translocation (4;7)(q33;q34).