Herdit M. Schüler

Is maternal duplication of 11p15 associated with Silver- Russell syndrome?

Background: Silver-Russell syndrome (SRS) is a heterogeneous malformation syndrome characterised by intrauterine and postnatal growth retardation (IUGR, PGR) and dysmorphisms. The basic causes are unknown, however in approximately 10% of patients a maternal uniparental disomy (UPD) of chromosome 7 or chromosomal aberrations can be detected. Four growth retarded children, two with SRS-like features, associated with maternal duplications of 11p15 have been described. Considering the involvement of this genomic region in Beckwith-Wiedemann overgrowth syndrome (BWS), we postulated that some cases of SRS—with an opposite phenotype to BWS—might also be caused by genomic disturbances in 11p15. Methods: A total of 46 SRS patients were screened for genomic rearrangements in 11p15 by STR typing and FISH analysis. Results: Two SRS patients with duplications of maternal 11p material in our study population (n = 46) were detected. In patient SR46, the duplicated region covered at least 9 Mb; FISH analysis revealed a translocation of 11p15 onto 10q. In patient SR90, additional 11p15 material (approximately 5 Mb) was translocated to the short arm of chromosome 15. Conclusions: We suggest that diagnostic testing for duplication in 11p15 should be offered to patients with severe IUGR and PGR with clinical signs reminiscent of SRS. SRS is a genetically heterogeneous condition and patients with a maternal duplication of 11p15.5 may form an important subgroup.

Genetic counseling in Robertsonian translocations der(13;14): Frequencies of reproductive outcomes and infertility in 101 pedigrees

Robertsonian translocations 13/14 are the most common chromosome rearrangements in humans. However, most studies aimed at determining risk figures are more than 20 years old. Their results are often contradictory regarding important topics in genetic counseling such as infertility and unfavorable pregnancy outcomes. Here, we present a study on a sample of 101 previously unreported pedigrees of der(13;14)(q10;q10). In order to minimize problems of partial ascertainment, we included families with a wide range of reasons of ascertainment such as birth of a child with congenital anomalies, prenatal diagnosis due to maternal age, fertility problems and recurrent pregnancy loss. No evidence of increased infertility rates of female and male carriers was found. The detected miscarriage frequency of female carriers was higher than previously reported (27.6 ± 4.0% of all spontaneous pregnancies). This may be explained by an over‐correction of earlier studies, which excluded all unkaryotyped miscarriages. In three out of 42 amniocenteses, translocation trisomies 13 were diagnosed (7.1 ± 4.0% of all amniocenteses). The frequency of stillbirths was 3.3 ± 1.6% for female carriers and 1.4 ± 1.4% for male carriers. A low risk for the live birth of translocation trisomy 13 children was confirmed since no live born children with trisomy 13 or Pätau syndrome were detected in the ascertainment‐corrected sample.

To clone or not to clone? Induced pluripotent stem cells can be generated in bulk culture

Induced pluripotent stem cells (iPSCs) are usually clonally derived. The selection of fully reprogrammed cells generally involves picking of individual colonies with morphology similar to embryonic stem cells (ESCs). Given that fully reprogrammed cells are highly proliferative and escape from cellular senescence, it is conceivable that they outgrow non-pluripotent and partially reprogrammed cells during culture expansion without the need of clonal selection. In this study, we have reprogrammed human dermal fibroblasts (HDFs) with episomal plasmid vectors. Colony frequency was higher and size was larger when using murine embryonic fibroblasts (MEFs) as stromal support instead of HDFs or human mesenchymal stromal cells (MSCs). We have then compared iPSCs which were either clonally derived by manual selection of a single colony, or derived from bulk-cultures of all initial colonies. After few passages their morphology, expression of pluripotency markers, and gene expression profiles did not reveal any significant differences. Furthermore, clonally-derived and bulk-cultured iPSCs revealed similar in vitro differentiation potential towards the three germ layers. Therefore, manual selection of individual colonies does not appear to be necessary for the generation of iPSCs – this is of relevance for standardization and automation of cell culture procedures.

Modelling IRF8 Deficient Human Hematopoiesis and Dendritic Cell Development with Engineered iPS Cells

Human induced pluripotent stem (iPS) cells can differentiate into cells of all three germ layers, including hematopoietic stem cells and their progeny. Interferon regulatory factor 8 (IRF8) is a transcription factor, which acts in hematopoiesis as lineage determining factor for myeloid cells, including dendritic cells (DC). Autosomal recessive or dominant IRF8 mutations occurring in patients cause severe monocytic and DC immunodeficiency. To study IRF8 in human hematopoiesis we generated human IRF8−/− iPS cells and IRF8−/− embryonic stem (ES) cells using RNA guided CRISPR/Cas9n genome editing. Upon induction of hematopoietic differentiation, we demonstrate that IRF8 is dispensable for iPS cell and ES cell differentiation into hemogenic endothelium and for endothelial‐to‐hematopoietic transition, and thus development of hematopoietic progenitors. We differentiated iPS cell and ES cell derived progenitors into CD141+ cross‐presenting cDC1 and CD1c+ classical cDC2 and CD303+ plasmacytoid DC (pDC). We found that IRF8 deficiency compromised cDC1 and pDC development, while cDC2 development was largely unaffected. Additionally, in an unrestricted differentiation regimen, IRF8−/− iPS cells and ES cells exhibited a clear bias toward granulocytes at the expense of monocytes. IRF8−/− DC showed reduced MHC class II expression and were impaired in cytokine responses, migration, and antigen presentation. Taken together, we engineered a human IRF8 knockout model that allows studying molecular mechanisms of human immunodeficiencies in vitro, including the pathophysiology of IRF8 deficient DC. Stem Cells 2017;35:898–908

A new case of proximal monosomy 1p36, extending the phenotype.

The monosomy 1p36 deletion syndrome is due to the loss of the most distal 10.5 Mb of chromosome region 1p due to pure terminal deletions, interstitial deletions, derivative chromosomes, and complex rearrangements. Rearrangements of 1p36 resulting in monosomy have been estimated to occur in 1 in 5,000 live births [Shaffer and Lupski, 2000; Heilstedt et al., 2003a]. Therefore, this segmental aneuploidy is among the most common microdeletions in humans. The syndrome of ‘‘pure’’ terminal deletion 1p36 comprises mental and growth retardation, hypotonia, seizures, hearing impairment, and vision problems. Craniofacial characteristics include a tower skull with a large and late closing anterior fontanelle, deep-set eyes with straight eyebrows, midface hypoplasia, long philtrum, low-set ears with thick helices, pointed chin, and a prominent forehead [Battaglia, 2005; Gajecka et al., 2007; Battaglia et al., 2008]. Organ malformations are infrequent as are specific structural defects of the brain. Recently, more proximally located 1p36 deletions of varying size were reported in five patients who manifested different clinical characteristics such as growth deficiency, microcephaly, limb anomalies, cardiovascular anomalies, developmental delay, seizures, frontal and parietal bossing, hypertelorism, prominent nose, abnormally shaped and malrotated ears, and arched eyebrows [Kang et al., 2007]. Here we report on a girl with microcephaly and a midline brain malformation and an interstitial deletion of 1p36 on conventional chromosome analysis. Further characterization was carried out by FISH and arrayCGH, which documented a deletion ranging form band 1p36.13 to 1p36.23, comprising a loss of 8.7Mb. The girl was the second child of healthy parents, mother of German, father of Polish origin. The elder sister was healthy. Pregnancy and delivery were uneventful; the girl presented with microcephaly and low birth weight (weight 2,800 g, 3rd centile; length 50 cm, 25th centile; head circumference 32 cm,<3rd centile). Follow-up at 2, 5, and 8 months, respectively, showed microcephaly, low body weight, a broad nasal bridge, hypertelorism, long eyelashes, infraorbital bulging, fleshy ears and a large mouth (Fig. 1). At 2 months, the anterior fontanelle was closed. An MRI at 5 months demonstrated agenesis of the anterior commissure and of the rostral corpus callosum and partial agenesis of the septum pellucidum (Fig. 2). There were no other malformations or organ dysfunctions, such as malformations of the visual system, interhemispheric cysts or signs for pituitary dysfunction. At age 6 months, she developed social smiling. She was able to turn from back to side at 4–5 months but she was not able to lift her head and to put weight on her arms before 7 months. At 8 months she showed a clear developmental delay corresponding to a developmental age of 4–5 months. Conventional chromosome analysis documented an interstitial deletion of the short arm of chromosome arm 1p (Fig. 3a). Subsequent fluorescence in situ hybridization (FISH) with the wcp1 probe (Qbiogene, Inc., Carlsbad, CA) excluded a reciprocal

Mosaic tetrasomy 14pter-q13 due to a supernumerary isodicentric derivate of proximal chromosome 14q

Tetrasomy of proximal 14q is an extremely rare condition and has never been reported to be associated with survival. We here report on the first case of mosaic tetrasomy of 14pter‐q13 due to a de‐novo supernumerary pseudoisodicentric chromosome in a 2‐year‐old boy with multiple dysmorphisms and malformations. The marker was detectable in nearly 25% of lymphocytes as well as in cells from buccal mucosa. Detailed fluorescence in situ hybridization (FISH) analyses allowed the characterization of the marker to entirely consist of proximal 14q material and to be symmetric. The pattern of clinical features in our patient only slightly correspond to that of patients with trisomy of proximal 14q, but further cases are needed to define whether tetrasomy of proximal 14q is a separate entity.

Rare proximal interstitial deletion of chromosome 4q, del(4)(q13.2q21.22): New case and comparison with the literature

Interstitial deletions in the long arm of chromosome 4 have been reported in more than 70 patients with variable clinical features [for review: Strehle et al., 2001]. Themajority of cases are terminal distal 4q deletions with loss of the bands (4)(q31q33) resulting in a characteristic, well-described phenotype including mild facial and digital dysmorphisms, cardiac, renal, gastrointestinal and skeletal anomalies, developmental delay, learning difficulties, and failure to thrive. In contrast, deletions within the proximal long arm of chromosome 4 have rarely been reported (Table I). Due to the variable extent of the deletions in proximal chromosome 4q, a distinct phenotype remains difficult to define. Even in patients sharing similar breakpoints the clinical pictures vary significantly. Here, we report on a girl carrying a deletion spanning from 4q13.2 to 4q21.22. The girl showed several signs observed in recently published cases with similar but not identical cytogenetic aberrations. The girl was born at term to a 25-year-oldG1P1. The parents were nonconsanguinous and of German descent. Family history was unremarkable. Birth weight (3,050 g), length (49 cm), and OFC (37 cm) were within normal limits. Post partum, growth delay was noted and feeding problems persisted throughout the neonatal period. Hypotonia, however, was never present. Visual and audiologic functions were shown to be normal. At age 18 months, a cranial MRT was performed that showed slight callosal hypoplasia andmultiple periventricular lesions. While epileptic seizures have never been observed, anticonvulsive treatment with valproate had been started due to EEG irregularities. At age 31⁄2 years medication was stopped as no seizure occurred and EEG was normal. Moderate growth retardation (length 93 cm, 3rd centile; weight 13 kg, 10th centile) and relative macrocephaly (OFC 50 cm, 50th centile) were noted. Psychomotormilestones were significantly delayed. Unaided sitting was possible at 11 months, unstable walking at 31⁄2 years. At age 5 years, the patient was able to climb stairs without help and speak about ten single words. Dysmorphic features in the child included a prominent forehead, a left pre-auricular tag, low-set ears, a thin upper lip, a high arched palate, short fingers and toes, low-set thumbs, pes planus on both feet, and a nevus on her back (Fig. 1). Piebaldism, other pigmentary changes or skin abnormalities were not observed. Notably, at routine examination glycosuria and hyperglycemia were detected. Diabetes mellitus was excluded as the underlying reason. Intermittently, episodes of lactic acidosis and hypoand hyperglycemia were noted. However, in spite of thorough examinations, a definite diagnosis could not bemade. Thus, the blood glucose level has been monitored every few months since then without showing any irregularities. By G-banding on stimulated peripheral lymphocytes a deletion in the proximal long arm of chromosome 4 could be identified (karyotype: 46,XX,del(4)(q13.2q21.22) (Fig. 2). Application of fluorescence in-situ hybridization using a whole chromosome 4 painting probe excluded a more complex rearrangement. Maternal chromosomes were normal: a paternal blood sample was not available for analysis. The paternal origin and the extent of the deletion was determined by typing short tandem repeats as listed in Table II (online version). Genomic DNA of the patient and her mother was extracted from peripheral lymphocytes, primer sequences and PCR conditions were obtained from the Genome Data base. Visualization of the amplification products was performed by silver-staining after denaturing sequencing gel electrophoresis. It is a fundamental concept that patients with overlapping chromosomal deletions share certain clinical findings. Thus autosomal deletions—in particular those that are not combined with a partial trisomy of an autosomal region—may be useful for human gene mapping. We report on a new rare case of a deletion in the proximal short arm of chromosome 4. While 13 of the cases reported so far share a commoncytogenetic breakpoint in 4q12, ourpatient is to the best of our knowledge the fourth patient with the proximal breakpoint in 4q13 [McDemort et al., 1980; Mascari et al., 1989; Nowaczyk et al., 1997]. The distal deletion breakpoint in the presented case is in 4q21, the deletion is therefore the smallest described so far. By additional molecular analysis we aimed at precisely defining the deleted region: Owing to absence of paternal DNA we could not confirm the de-novo origin of the deletion. However, short tandem repeat typing did not show lack of anymaternal alleles in the region of interest; it can thereforebe postulated that the deletion affected the paternal chromosome. Based on homo-/hemizygosity of the loci in the region of interest, we assume that the deletion is flanked by the STRs D4S398andD4S1517. This region covers approximately 12Mb in maximum. Comparison of the clinical signs in our patient with those published by other groups shows that our patient has the key *Correspondence to: Katja Eggermann, Ph.D., Institute of Human Genetics, University Hospital, RWTH Aachen, Pauwelsstr. 30, D-52074 Aachen. E-mail: keggermann@ukaachen.de

A 10.7 Mb interstitial deletion of 13q21 without phenotypic effect defines a further non-pathogenic euchromatic variant.

Chromosome 13 deletions are associated with widely varying phenotypes but the clinical picture nearly almost includes mental and growth retardation, craniofacial dysmorphisms, and/or malformations. Several attempts have been made to link monosomy 13q intervals with specific clinical features, but a genotype–phenotype correlation could not be delineated. We report on a woman with a normal phenotype and intelligence referred for chromosomal analysis because of recurrent abortions followed by reproductive loss. Conventional karyotyping revealed an interstitial deletion of chromosome 13q21. By SNP array analysis and FISH the deletion was shown to comprise nearly 10.7 Mb of euchromatic material. This region harbors several genes but an association with recurrent miscarriages has not yet been reported. This is the second report of a 13q21 deletion without psychomotoric retardation, dysmorphisms and malformations. Both cases indicate that this 13q21 deletion can be added to the growing list of euchromatic imbalances without obvious phenotypic abnormalities.

A phenotype map for 14q32.3 terminal deletions.

Detailed molecular‐cytogenetic studies combined with thorough clinical characterization are needed to establish genotype–phenotype correlations for specific chromosome deletion syndromes. Although many patients with subtelomeric deletions have been reported, the phenotype maps for many of the corresponding syndromes, including the terminal deletion 14q syndrome, are only slowly emerging. Here, we report on five patients with terminal partial monosomy of 14q32.3 and characteristic features of terminal deletion 14q syndrome. Four of the patients carry de novo terminal deletions of 14q, three of which have not yet been reported. One patient carries an unbalanced translocation der(14)t(9;14)(q34.3;q32.3). Minimum deletion sizes as determined by molecular karyotyping and FISH are 5.82, 5.56, 4.17, 3.54, and 3.29 Mb, respectively. Based on our findings and a comprehensive review of the literature, we refine the phenotype map for typical clinical findings of the terminal deletion 14q syndrome (i.e., intellectual disability/developmental delay, muscular hypotonia, postnatal growth retardation, microcephaly, congenital heart defects, genitourinary malformations, ocular coloboma, and several dysmorphic signs). Combining this phenotype map with benign copy‐number variation data available from the Database of Genomic Variants, we propose a small region critical for certain features of the terminal deletion 14q syndrome which contains only seven RefSeq genes.

Pure distal trisomy 2q: a rare chromosomal abnormality with recognizable phenotype.

We present clinical and molecular cytogenetic results of two unrelated patients with isolated distal trisomy of 2q33‐qter and 2q35‐q37.3 and a remarkable similar facial appearance. Common craniofacial features included a high hairline, broad nasal bridge, prominent nasal tip, thin upper lip vermillion, and large ears. Contrary to patients with duplications proximal to 2q33, the children with pure trisomy distal to 2q35 have normal or increased body measurements and show no major malformations. Moderate psychomotor delay was a constant finding.