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Purpose Short stature is a common condition of great concern to patients and their families. Mostly genetic in origin, the underlying cause often remains elusive due to clinical and genetic heterogeneity. Methods We systematically phenotyped 565 patients where common nongenetic causes of short stature were excluded, selected 200 representative patients for whole-exome sequencing, and analyzed the identified variants for pathogenicity and the affected genes regarding their functional relevance for growth. Results By standard targeted diagnostic and phenotype assessment, we identified a known disease cause in only 13.6% of the 565 patients. Whole-exome sequencing in 200 patients identified additional mutations in known short-stature genes in 16.5% of these patients who manifested only part of the symptomatology. In 15.5% of the 200 patients our findings were of significant clinical relevance. Heterozygous carriers of recessive skeletal dysplasia alleles represented 3.5% of the cases. Conclusion A combined approach of systematic phenotyping, targeted genetic testing, and whole-exome sequencing allows the identification of the underlying cause of short stature in at least 33% of cases, enabling physicians to improve diagnosis, treatment, and genetic counseling. Exome sequencing significantly increases the diagnostic yield and consequently care in patients with short stature.

Genetic integrity of induced pluripotent stem cells (iPSCs) is essential for their validity as disease models and for potential therapeutic use. We describe the comprehensive analysis in the ForIPS consortium: an iPSC collection from donors with neurological diseases and healthy controls. Characterization included pluripotency confirmation, fingerprinting, conventional and molecular karyotyping in all lines. In the majority, somatic copy number variants (CNVs) were identified. A subset with available matched donor DNA was selected for comparative exome sequencing. We identified single nucleotide variants (SNVs) at different allelic frequencies in each clone with high variability in mutational load. Low frequencies of variants in parental fibroblasts highlight the importance of germline samples. Somatic variant number was independent from reprogramming, cell type and passage. Comparison with disease genes and prediction scores suggest biological relevance for some variants. We show that high-throughput sequencing has value beyond SNV detection and the requirement to individually evaluate each clone.

Genetic integrity of induced pluripotent stem cells (iPSCs) is essential for their validity as disease models and for potential therapeutic use. We describe the comprehensive analysis in the ForIPS consortium: an iPSC collection from donors with neurological diseases and healthy controls. Characterization included pluripotency confirmation, fingerprinting, conventional and molecular karyotyping in all lines. In the majority, somatic copy number variants (CNVs) were identified. A subset with available matched donor DNA was selected for comparative exome sequencing. We identified single nucleotide variants (SNVs) at different allelic frequencies in each clone with high variability in mutational load. Low frequencies of variants in parental fibroblasts highlight the importance of germline samples. Somatic variant number was independent from reprogramming, cell type and passage. Comparison with disease genes and prediction scores suggest biological relevance for some variants. We show that high-throughput sequencing has value beyond SNV detection and the requirement to individually evaluate each clone.

Short stature is a common pediatric disorder affecting 3% of the population. However, the clinical variability and genetic heterogeneity prevents the identification of the underlying cause in about 80% of the patients. Recently, heterozygous mutations in the ACAN gene coding for the proteoglycan aggrecan, a main component of the cartilage matrix, were associated with idiopathic short stature. To ascertain the prevalence of ACAN mutations and broaden the phenotypic spectrum in patients with idiopathic short stature we performed sequence analyses in 428 families. We identified heterozygous nonsense mutations in four and potentially disease-causing missense variants in two families (1.4%). These patients presented with a mean of −3.2 SDS and some suggestive clinical characteristics. The results suggest heterozygous mutations in ACAN as a common cause of isolated as well as inherited idiopathic short stature.

Background Tetralogy of Fallot (ToF), the most frequent cyanotic congenital heart disease, is associated with a wide range of intra- and extracardiac phenotypes. In order to get further insight into genotype–phenotype correlation, a large cohort of 230 unselected patients with ToF was comprehensively investigated. Methods and results 230 patients with ToF were studied by karyotyping, comprehensive 22q11.2 deletion testing and sequencing of TBX1, NKX2.5 and JAG1 , as well as molecular karyotyping in selected patients. Pathogenic genetic aberrations were found in 42 patients (18%), with 22q11.2 deletion as the most common diagnosis (7.4%), followed by trisomy 21 (5.2%) and other chromosomal aberrations or submicroscopic copy number changes (3%). Mutations in JAG1 were detected in three patients with Alagille syndrome (1.3%), while NKX2.5 mutations were seen in two patients with non-syndromic ToF (0.9%). One patient showed a recurrent polyalanine stretch elongation within TBX1 which represents a true mutation resulting in loss of transcriptional activity due to cytoplasmatic protein aggregation. Conclusion This study shows that 22q11.2 deletion represents the most common known cause of ToF, and that the associated cardiac phenotype is distinct for obstruction of the proximal pulmonary artery, hypoplastic central pulmonary arteries and subclavian artery anomalies. Atrioventricular septal defect associated with ToF is very suggestive of trisomy 21 and almost excludes 22q11.2 deletion. We report a further patient with a recurrent polyalanine stretch elongation within TBX1 and for the first time link TBX1 cytoplasmatic protein aggregation to congenital heart defects.

The authors observed a patient with a cryptic subtelomeric de novo balanced translocation 46,XY.ish t(11;20)(p15.4;q13.2) presenting with severe mental retardation, muscular hypotonia, seizures, bilateral sensorineural hearing loss, submucous cleft palate, persistent ductus Botalli, unilateral cystic kidney dysplasia and frequent infections.Fluorescence in situ hybridisation mapping and sequencing of the translocation breakpoints showed that no known genes are disrupted at 20q13.2 and that ST5 (suppression of tumorigenicity 5; MIM 140750) is disrupted on 11p15.4. By quantitative PCR from different human tissues, the authors found ST5 to be relatively evenly expressed in fetal tissues. ST5 expression was more pronounced in adult brain, kidney and muscle than in the corresponding fetal tissues, whereas expression in other tissues was generally lower than in the fetal tissue. Using RNA in situ hybridisation in mouse, the authors found that St5 is expressed in the frontal cortex during embryonic development. In adult mouse brain, expression of St5 was especially high in the hippocampal area and cerebellum.Hence, the authors suppose that ST5 plays an important role in central nervous system development probably due to disturbance of DENN-domain-mediated vesicle formation and neurotransmitter trafficking. Thus, these findings implicate ST5 in the aetiology of mental retardation, seizures and multiple congenital anomalies.

We report on a healthy man with high normal intelligence, minor dysmorphic features and infertility due to hypogonadism and azoospermia. Cytogenetic analysis showed a 6.7 Mb duplication in chromosome band 11q24.2q25, which could be confirmed with FISH and molecular karyotyping using an Affymetrix GeneChip Human Mapping 250 K Nsp SNP array.

In seven cases additional minute chromosomes studied by FISH were identified as no. 3, 11, 15, 18, 21 and X. Findings were unexpected except for partial trisomy 21 in an adolescent with minor features of Down's syndrome. Moreover, an i(18p) in a mentally retarded dysmorphic child and an idic(15) in a child with Fallot tetralogy was confirmed. In a child with r(21), a supernumerary marker was shown to be derived from no. 21, while in the mother an additional marker idic(22) was noted.

Two new cases of del(2)(q37.1) and one case of partial trp(2)(q37) are studied by FISH with cosmid probes from the COL6A3 and PAX3 genes mapped in 2q37.3 and 2q36, respectively. While the PAX3 gene dosage appeared unaffected, the COL6A3 gene was found to be deleted and triplicated, respectively. This finding could explain features of connective tissue disorders such as joint laxity and hypotonia or joint stiffness and epiphyseal dysplasia, particularly documented by congenital dislocation of the radial head. Karyotype-phenotype correlations with reference to published cases are discussed.

Males with deletions of or within Xp22.3-pter display variable contiguous gene syndromes including manifestations of Léri-Weill syndrome, chondrodysplasia punctata, mental retardation, ichthyosis, Kallmann syndrome, and ocular albinism. Herein, we report on a male infant with a large, cytogenetically visible, terminal Xp deletion defined by extensive FISH and STS marker analysis to encompass 9.6 Mb, and findings of all of the disorders mentioned above. His deletion approximates the largest Xp terminal deletion ever reported in a male individual. Since the extent of terminal Xp deletions viable in males is limited by the position of male lethal genes in Xp22.2 at about 10-11 Mb from the telomere, this patient falls into the category of the most severe male terminal Xp deletion phenotype.

We describe the clinical course, as well as cytogenetic and molecular findings, of a 3-year-old obese boy with psychomotor retardation who exhibited two rare conditions: succinic semialdehyde dehydrogenase deficiency (SSADH deficiency, MIM 271980), a disorder of gamma-aminobutyric acid metabolism with a heterogeneous clinical spectrum, and partial Wilms' tumor, aniridia, genital abnormalities, and mental retardation (WAGR) syndrome, an association between Wilms' tumor, aniridia, genitourinary malformations, and mental retardation due to mutations involving the short arm of chromosome 11, particularly deletions at the chromosomal region 11p13 (MIM 194072). Diagnosis of SSADH deficiency in our patient was established by demonstration of absent enzyme activity in isolated leucocytes, and was associated with a novel missense mutation (c.587G>A; p.Gly196Asp) in the SSADH coding sequence. We further confirmed an incomplete WAGR syndrome in this boy [karyotype 46, XY, del (11) (p13p14.2)] with a normal WT1 (Wilms' tumor) gene and an absence of pathology in the genitourinary tract, but with obesity (WAGR syndrome with obesity, WAGRO syndrome). The patient also exhibited distinctive cerebral anomalies such as increased signals of the globi pallidi, internal hydrocephalus and cerebellar vermian atrophy. However, treatment options for this patient are limited, including supportive treatment, physiotherapy, special educational training, and vigabatrin. In summary, we report the first patient with the exceptional rare findings of both SSADH deficiency and partial WAGR/WAGRO syndrome.

The underlying cause of mental retardation remains unknown in up to 80% of patients. As chromosomal aberrations are the most common known cause of mental retardation, several new methods based on FISH, PCR, and array techniques have been developed over recent years to increase detection rate of subtle aneusomies initially of the gene rich subtelomeric regions, but nowadays also genome wide. As the reported detection rates vary widely between different reports and in order to compare the diagnostic yield of various investigations, we analyzed the diagnostic yield of conventional karyotyping, subtelomeric screening, molecular karyotyping, X-inactivation studies, and dysmorphological evaluation with targeted laboratory testing in unselected patients referred for developmental delay or mental retardation to our cytogenetic laboratory (n = 600) and to our genetic clinic (n = 570). In the cytogenetic group, 15% of patients showed a disease-related aberration, while various targeted analyses after dysmorphological investigation led to a diagnosis in about 20% in the genetic clinic group. When adding the patients with a cytogenetic aberration to the patient group seen in genetic clinic, an etiological diagnosis was established in about 40% of the combined study group. A conventional cytogenetic diagnosis was present in 16% of combined patients and a microdeletion syndrome was diagnosed in 5.3%, while subtelomeric screening revealed only 1.3% of causes. Molecular karyotyping with a 10 K SNP array in addition revealed 5% of underlying causes, but 29% of all diagnoses would have been detectable by molecular karyotyping. In those patients without a clear diagnosis, 5.6% of mothers of affected boys showed significant (>95%) skewing of X-inactivation suggesting X-linked mental retardation. The most common diagnoses with a frequency of more than 0.5% were Down syndrome (9.2%), common microdeletion 22q11.2 (2.4%), Williams-Beuren syndrome (1.3%), Fragile-X syndrome (1.2%), Cohen syndrome (0.7%), and monosomy 1p36.3 (0.6%). From our data, we suggest the following diagnostic procedure in patients with unexplained developmental delay or mental retardation: (1) Clinical/dysmorphological investigation with respective targeted analyses; (2) In the remaining patients without an etiological diagnosis, we suggest conventional karyotyping, X-inactivation screening in mothers of boys, and molecular karyotyping, if available. If molecular karyotyping is not available, subtelomeric screening should be performed.

OTC deficiency, the most common urea cycle defect, is transmitted as a partially dominant X-linked trait. The most severe form of the disease, however, is usually restricted to males. We report on monozygotic female twins with severe neonatal-onset OTC deficiency and a de novo balanced reciprocal translocation t(X;5)(p21.1;q11). Disruption of the OTC gene on the derivative X-chromosome was confirmed by FISH analysis. Consistent inactivation of the normal X could be demonstrated by RGB staining. Manifestation of X-linked recessive disorders in females due to a balanced reciprocal X-autosome translocation has previously been described in Duchenne muscular dystrophy and several other disorders but not in OTC deficiency. This report emphasizes the importance of chromosome analysis in any female manifesting severe OTC deficiency.

We observed a novel 3.5 Mb 5q subtelomeric deletion in a 3-year-old girl with developmental delay, hypotonia and multiple minor anomalies. Comparison of her phenotype with the few published patients with terminal 5q35 deletions revealed several overlapping features, but also showed remarkable differences such as shortness of stature versus macrosomia. After the report of 5q35.3 microdeletions in Sotos syndrome we integrated the published BACs into the public draft sequence and exactly mapped the deletion size in our patient by FISH analysis with 15 BAC probes. We demonstrated that the deletion in our patient is immediately adjacent to the reported Sotos syndrome deletion site. Subtracting the symptoms of Sotos syndrome from the published patients with larger 5q35.3 deletions allowed us to delineate a distinct phenotype of prenatal lymphedema with increased nuchal translucency, pronounced muscular hypotonia and delay of reaching motor milestones, but speech development within normal limits, wide fontanels, failure to thrive with postnatal short stature, and multiple minor anomalies such as mildly bell-shaped chest, minor congenital heart disease, and a distinct facial gestalt, associated with the novel 3.5 Mb cryptic deletion. We further showed in our patient that the deletion of the LCT(4) synthase gene results in a reduction of cysteinyl leukotriene synthesis to about 65% compared to normal values. The prenatal nuchal lymphedema associated with this deletion syndrome my be related to the deletion of the FLT4 gene causing autosomal dominant primary lymphedema and contributes to the differential diagnosis of increased fetal nuchal translucency.

Monosomy 1p36 is a recently delineated contiguous gene syndrome, which is now considered to be one of the most common subtelomeric microdeletion syndromes. We report four unrelated patients with subtle deletions within 1p36 confirmed by high resolution karyotyping and FISH. All exhibited severe psychomotor retardation. Microcephaly, seizures, and visual impairment occurred in three subjects. Results of a first routine karyotyping were unrevealing in three probands. The diagnosis was primarily suggested on the basis of a distinct pattern of facial anomalies in all except the first case. This report illustrates that monosomy 1p36 may be recognized clinically, at least in some patients, whereas the diagnosis is easily missed on routine karyotype.

Deletions within HSA band 4p16.3 cause Wolf-Hirschhorn syndrome (WHS), which comprises mental retardation and developmental defects. A WHS critical region (WHSCR) of approximately 165 kb has been defined on the basis of 2 atypical interstitial deletions; however, genotype–phenotype correlation remains controversial, due to the large size of deletion usually involving several megabases. We report on the first known patient with a small de novo interstitial deletion restricted to the WHSCR who presented with a partial WHS phenotype consisting only of low body weight for height, speech delay, and minor facial anomalies; shortness of stature, microcephaly, seizures and mental retardation were absent. The deletion was initially demonstrated by FISH analysis, and breakpoints were narrowed with a “mini-FISH” technique using 3–5 kb amplicons. A breakpoint-spanning PCR assay defined the distal breakpoint as disrupting the WHSC1 gene within intron 5, exactly after an AluJb repeat. The proximal breakpoint was not found to be associated with a repeated sequence or a known gene. The deletion encompasses 191.5 kb and includes WHSC2, but not LETM1. Thus, manifestations attributable to this deletion are reduced weight for height, minor facial anomalies, ADHD and some learning and fine motor deficiencies, while seizures may be associated with deletions of LETM1. © 2001 Wiley-Liss, Inc.

Both double aneuploidy and trisomy 10 are rare chromosome findings. All five published cases of trisomy 10 in liveborns were found to be mosaic with an euploid cell line. In a liveborn female twin, double aneuploidy mosaicism 47,XX, + 10/45,X was detected prenatally by amniocentesis performed because of severe intrauterine growth retardation and malformations. Chromosome analysis from neonatal lymphocyte cultures revealed exclusively the 45,X cell line. Double aneuploidy mosaicism trisomy 10/monosomy X was confirmed from skin fibroblasts. The child died at the age of 7 weeks. This is the first reported case of double aneuploidy involving trisomy 10, and the first case of trisomy 10 without a normal cell line in a liveborn. Prenatal diagnosis of trisomy 10 in a liveborn has not been published so far. The case illustrates that in specific cases amniotic fluid cells may reflect the karyotype of the fetus better than blood.

We report the prenatal findings of mos 46,XY/46,XY,i(20q) after amniocentesis. The propositus presented with two epidermal scalp scars, retrobulbar orbital cysts, and dyssegmentation of the thoracic spine. The abnormal cell line was discovered in cells cultured from the proximal umbilical cord and—by fluorescence in situ hybridization (FISH)—in interphase nuclei from buccal epithelium and urinary sediment but not from the placenta, lymphocytes, or skin fibroblasts. © 1997 John Wiley & Sons, Ltd.

Fragestellung: Die Diagnose des Williams-Beuren-Syndroms (WBS) wird durch die klinische Variabilitat erschwert. Daher sollen der diagnostische Wert sowohl des von Preus aufgestellten klinischen Scores als auch der Elastingenhemizygotie gepruft und miteinander verglichen werden. Methodik: Bei 13 Kindern mit Verdacht auf WBS wurden der Preus-Score erhoben und eine Fluoreszenz-in situ-Hybridisierungs-Analyse des Elastingens sowie eine Chromosomenanalyse durchgefuhrt. Ergebnisse: Neun der 13 Patienten zeigten eine Deletion eines Elastingens bei normalem Karyotyp. Zwei Patienten wiesen bei normaler Elastingendosis eine Chromosomenaberration auf. Der Preus-Score lag bei allen Patienten mit Elastindeletion >6 und war damit hoch positiv. Bei den ubrigen Kindern wurde ein Preus-Score von 0,18; 0,37, 1,00 und 6,4 ermittelt. Schlusfolgerung: Bei allen Patienten mit WBS lagen in dieser Studie eine Elastingendeletion und ein hoch positiver Preus-Score vor. Ein positiver Preus-Score wurde jedoch auch bei Patienten mit Chromosomenaberrationen ermittelt. Unter Berucksichtigung dieser Ergebnisse und der Daten aus der Literatur sollte daher die Diagnose Wiliams-Beuren-Syndrom nur unter Vorbehalt gestellt werden, wenn keine Elastingendeletion nachweisbar ist. Da aber bisher unbekannte Mutationen bei den wenigen Patienten ohne Elastingendeletion denkbar sind, kann ein WBS auf molekularer Ebene nicht mit letzter Sicherheit ausgeschlossen werden. Bei diesen Patienten konnte der Preus-Score zur klinischen Diagnose beitragen. Setzt man fur die Diagnose eines WBS im Preus-Score einen Endsummenwert von >1 voraus, so wird eine Sensitivitat von 100% bei einer Spezifitat von 92% erreicht.

9 Mb deletion including chromosome band 3q24 associated with unsuspicious facial gestalt, persistent ductus omphaloentericus, mild mental retardation and tic.

Two unpublished cases with partial tandem duplication of 12p and one previously published case were studied by fluorescence in situ hybridization using 11 cosmid DNA probes from 12p. We propose that the smallest duplications of 12(p13.2pter) and 12(p13.1p13.33) produce the "trisomy 12p syndrome" which is characterized by heavy birth weight, macrocephaly, muscular hypotonia, short neck, flat face, high forehead, prominent cheeks, large philtrum, short nose with anteverted nostrils, and broad everted lower lip. From a review of the published cases we conclude that gross malformations are lacking in "pure" trisomy 12p, and mental retardation is severe in complete and moderate in partial trisomy 12p. Polydactyly and accessory nipples were found only with almost complete trisomy 12p. Abnormalities of hair growth may be related to a gene at 12p. The sub-band 12p11.21 may be critical for acrocallosal syndrome. Macrocephaly may be due to a metabolic disorder.

The cosegregation of a reciprocal translocation t(17;19) (q11.2;13.2) with neurofibromatosis type 1 in three generations suggested that the breakpoint on chromosome 17 involved the NF1 gene. In order to map the breakpoint, we analysed DNAs of patients using parts of the NF1 gene as probes. Southern analysis revealed that the chromosome 17 breakpoint lies within intron 23 of the NF1 gene. One of the patients of the family developed a non-Hodgkin lymphoma. An additional translocation t(14;20) (q32;13.1) in his B lymphocytes points to a gene on chromosome 20 that is juxtaposed to the IGH locus on 14q32, and that may be of relevance for the development of this tumor type.

A cell line, designated SEM, was established from the peripheral blood of a 5-year-old girl in relapse with acute lymphoblastic leukaemia (ALL). Both the lymphoblasts of the patient and the cells of the cell line SEM showed the t(4;11) chromosomal rearrangement. The analysis of the immunophenotype of the SEM cell line revealed the B-cell differentiation antigens CD19, CD22 and CDw75 in the absence of CD20, CD24 and immunoglobulin expression. Besides B-lineage antigens, SEM cells were positive for the myeloid antigens CD13, CD15, CD33 and CDw65. Immunogenotypic analysis of SEM cells showed a monoclonal rearrangement of immunoglobulin heavy-chain (IgH). T-cell receptor (TCR) gamma and delta genes. Addition of interleukin (IL)-7 promoted the growth of the patient's lymphoblasts in culture and enhanced the proliferation of SEM cells. The SEM cells also express messenger RNA (mRNA) for the IL-7 receptor (IL-7R), but no evidence for autocrine production of IL-7 by the cell line was found. Addition of IL-4, tumour necrosis factor (TNF)-alpha, interferon (IFN)-alpha, or IFN-gamma resulted in a profound inhibition of SEM growth. Thus, these cytokines may have important growth regulatory activities for biphenotypic leukaemic ALL cells.

Epigenetic regulation of gene expression, through covalent modification of histones, is a key process controlling growth and development. Accordingly, the transcription factors regulating these processes are important targets of genetic diseases. However, surprisingly little is known about the relationship between aberrant epigenetic states, the cellular process affected, and their phenotypic consequences. By chromosomal breakpoint mapping in a patient with a Noonan syndrome-like phenotype that encompassed short stature, blepharoptosis, and attention deficit hyperactivity disorder, we identified haploinsufficiency of the histone acetyltransferase gene MYST histone acetyltransferase (monocytic leukemia) 4 (MYST4), as the underlying cause of the phenotype. Using acetylation, whole genome expression, and ChIP studies in cells from the patient, cell lines in which MYST4 expression was knocked down using siRNA, and the Myst4 querkopf mouse, we found that H3 acetylation is important for neural, craniofacial, and skeletal morphogenesis, mainly through its ability to specifically regulating the MAPK signaling pathway. This finding further elucidates the complex role of histone modifications in mammalian development and adds what we believe to be a new mechanism to the pathogenic phenotypes resulting from misregulation of the RAS signaling pathway. usc Refereed/Peer-reviewed

We report on a female patient carrying a de novo 15 Mb duplication of 6q24.1–q25.3 detected by conventional karyotyping and fine-mapped by molecular karyotyping with a 250 K SNP array. Pure interstitial duplications of 6q are rarely reported in the literature and none of them exactly mapped by array technique so far. Our patient shows typical aspects of the “duplication 6q” syndrome such as hypertelorism, downslanting palpebral fissures, carp shaped mouth and joint contractures, but milder mental retardation and no growth retardation.

We delineate a pure "distal 14q duplication" phenotype, characterized by primordial short stature, mild developmental delay, and distinct facial dysmorphism with high forehead, mild hypertelorism, broad nasal bridge, dysplastic ear helices, short philtrum, thin and "cupid bow" upper lip, broad mouth, and micrognathia.

The microphthalmia with linear skin defects (MLS or MIDAS) syndrome is a rare X-linked dominant inherited disorder with male lethality, associated with segmental aneuploidy of the Xp22.2 region in most of the cases. However, we recently described heterozygous sequence alterations in a single gene, HCCS, in females with MLS. Beside the classical MLS phenotype, occasional features such as sclerocornea, agenesis of the corpus callosum, and congenital heart defects can occur. Although the majority of cases are sporadic, mother-to-daughter transmission has been observed and a high intra- and interfamilial phenotypic variability exists. We describe an asymptomatic mother and her daughter presenting with the typical features of MLS syndrome. By cytogenetic analysis both females were found to have a terminal Xp deletion with the breakpoint in Xp22.2, mapping near to or within the MSL3L1 gene which is located centromeric to HCCS. FISH analysis revealed that the mother is a mosaic with 45,X(11)/46,X,del(X)(p22.2)(89), while in all cells of the MLS-affected daughter a hybridization pattern consistent with a 46,X,del(X)(p22.2) karyotype was detected. By haplotype analysis we identified the paternal X chromosome of the mother to carry the terminal Xp deletion. X-inactivation studies showed a completely skewed pattern in mother and daughter with the deleted X chromosome to be preferentially inactivated in their peripheral blood cells. We suggest that both chromosomal mosaicism as well as functional X chromosome mosaicism could contribute to the lack of any typical MLS feature in individuals with a heterozygous MLS-associated mutation. The 45,X cell population, that most likely is also present in other tissues of the mother, might have protected her from developing MLS. Nonetheless, a non-random X-inactivation pattern in favor of activity of the wild-type X chromosome in the early blastocyte could also account for the apparent lack of any disease sign in this female.

Sixteen newly established cell lines with small supernumerary marker chromosomes (sSMC) derived from chromosomes 1, 2, 4, 6, 7, 8, 14, 15, 16, 18, 19, 21, and 22 are reported. Two sSMC are neocentric and derived from 15q24.1-qter and 2q35-q36, respectively. Two further cases each present with two sSMC of different chromosomal origin. sSMC were characterized by multicolor fluorescence in situ hybridization for their chromosomal origin and genetic content. Moreover, uniparental disomy of the sister chromosomes of the sSMC was excluded in all nine cases studied for that reason. The 16 cases provide information to establish a refined genotype-phenotype correlation of sSMC and are available for future studies.

Background: Chromosomal imbalances are a major cause of developmental defects as well as cancer and often constitute the key in identification of novel disease related genes. Classical cytogenetic methods are limited in resolution and dependent on highly skilled labour, while methods with higher resolution, based on molecular cytogenetics approaches such as matrix CGH, are not widely available. Methods: We have developed and evaluated a method we term "molecular karyotyping", using readily available and easy to handle oligonucleotide arrays originally designed for parallel genomewide analysis of over 10 000 SNPs. We show that we can easily and reliably detect unbalanced chromosomal aberrations of various sizes from as little as 250 ng of DNA on a single microarray, based on fluorescence intensity information from clusters of SNPs. Results: We determined the resolution of this method through analysis of 20 trios with 21 previously confirmed subtle aberrations sizing between 0.2 and 13 Mb. Duplications and deletions of at least 5 Mb in size were reliably detectable, but detection of smaller aberrations was dependent on the number of SNPs they contained, thus seven of 10 different deletions analysed, with sizes ranging from 0.2 to 3.7 Mb, were not detectable due to insufficient SNP densitiy in the respective region. Conclusions: Deduction of reliable cut off levels for array peaks in our series of well characterised patients allows the use of the GeneChip Mapping 10K SNP array for performing rapid molecular karyotyping from small amounts of DNA for the detection of even subtle deletions and duplications with high sensitivity and specificity.

We observed a novel 3.5 Mb 5q subtelomeric deletion in a 3-year-old girl with developmental delay, hypotonia and multiple minor anomalies. Comparison of her phenotype with the few published patients with terminal 5q35 deletions revealed several overlapping features, but also showed remarkable differences such as shortness of stature versus macrosomia. After the report of 5q35.3 microdeletions in Sotos syndrome we integrated the published BACs into the public draft sequence and exactly mapped the deletion size in our patient by FISH analysis with 15 BAC probes. We demonstrated that the deletion in our patient is immediately adjacent to the reported Sotos syndrome deletion site. Subtracting the symptoms of Sotos syndrome from the published patients with larger 5q35.3 deletions allowed us to delineate a distinct phenotype of prenatal lymphedema with increased nuchal translucency, pronounced muscular hypotonia and delay of reaching motor milestones, but speech development within normal limits, wide fontanels, failure to thrive with postnatal short stature, and multiple minor anomalies such as mildly bell-shaped chest, minor congenital heart disease, and a distinct facial gestalt, associated with the novel 3.5 Mb cryptic deletion. We further showed in our patient that the deletion of the LCT(4) synthase gene results in a reduction of cysteinyl leukotriene synthesis to about 65% compared to normal values. The prenatal nuchal lymphedema associated with this deletion syndrome my be related to the deletion of the FLT4 gene causing autosomal dominant primary lymphedema and contributes to the differential diagnosis of increased fetal nuchal translucency.

Glutaric aciduria type III is a rare metabolic abnormality leading to persistent isolated glutaric acid excretion. We report the clinical and biochemical phenotypes of three affected children. The first patient is a boy with dysmorphic features and a chromosomal deletion (monosomy 6q26-qter) in whom a persistent glutaric aciduria (500 mmol/mol creatinine, normal

Contains fulltext : 185102.pdf (Publisher’s version ) (Closed access) Nonsyndromic X-linked mental retardation (MRX) is defined by an X-linked inheritance pattern of low IQ, problems with adaptive behavior, and the absence of additional specific clinical features. The 13 MRX genes identified to date account for less than one-fifth of all MRX, suggesting that numerous gene defects cause the disorder in other families. In a female patient with severe nonsyndromic mental retardation and a de novo balanced translocation t(X;7)(p11.3;q11.21), we have cloned the DNA fragment that contains the X-chromosomal and the autosomal breakpoint. In silico sequence analysis provided no indication of a causative role for the chromosome 7 breakpoint in mental retardation (MR), whereas, on the X chromosome, a zinc-finger gene, ZNF41, was found to be disrupted. Expression studies indicated that ZNF41 transcripts are absent in the patient cell line, suggesting that the mental disorder in this patient results from loss of functional ZNF41. Moreover, screening of a panel of patients with MRX led to the identification of two other ZNF41 mutations that were not found in healthy control individuals. A proline-to-leucine amino acid exchange is present in affected members of one family with MRX. A second family carries an intronic splice-site mutation that results in loss of specific ZNF41 splice variants. Wild-type ZNF41 contains a highly conserved transcriptional repressor domain that is linked to mechanisms of chromatin remodeling, a process that is defective in various other forms of MR. Our results suggest that ZNF41 is critical for cognitive development; further studies aim to elucidate the specific mechanisms by which ZNF41 alterations lead to MR.

Hematopoietic disorders can be used as a suitable tool of additional information on the actual resolving power of comparative genomic hybridization (CGH). Therefore, CGH examination was performed of DNA extracted from 23 acute and 15 chronic myeloproliferative disorders which had just been analyzed using classical cytogenetic techniques. In nearly all cases CGH analysis was repeated with reversely labeled probes. A Zeiss axioplan microscope was equipped with the ISIS 3 system for photometric evaluation of the CGH data. A main group was selected of 34 cases showing karyotypic mosaics when routinely diagnosed by classical cytogenetics. The grade of mosaicism was basically determined from the classical cytogenetic analysis and was additionally defined examining target anomalies by I-FISH analysis in 28 of the cases. The second group included 23 cases with deletions, and in 1 case another informative genomic imbalance could be analyzed. Every target anomaly irrespective of its type could be detected in all cases with an affected cell population equalling or exceeding about 25%, but in none was it below 23%. This value was the lowest and was found in a case, with CGH-detected 20q deletion. The smallest deletions of two bands on 20q which could visually be detected by CGH were estimated in the range of 5-7 Mb. CGH was also suitable to detect imbalances which were not clearly detected by routine cytogenetics. Reverse labelling, performed in nearly all cases, confirmed the result of the original CGH analysis. These data not only document the readiness and reliability of CGH studies on human leukemia, but also further contribute to a clearer definition of the limits of the resolving power of this technique.

We report on a male infant with ambiguous genitalia (scrotal hypospadias, sinus urogenitalis) trisomic for 8q23-ter and monosomic for 9p23-ter, who shared craniofacial and other abnormalities with either phenotype. Gonadal histology was nearly normal for age. Normal endocrinological findings and exclusion of mutations in SRY, androgen receptor and alpha-reductase genes point to supplementary gene(s) located in 9p2305-ter, haplo-insufficiency (by deletion) of which is expected to cause defective male morphogenesis.This observation lends further support to the hypothesis that genetic factors are located at 9p23-ter which are involved in normal sex determination.

Interruption of the aortic arch (IAA) is a severe malformation of the heart with known association to DiGeorge syndrome (DGS) and 22q11.2 hemizygosity. The aim of this study was to establish incidence and significance of 22q11.2 hemizygosity in an unbiased sample of patients with IAA. All 15 children with IAA who were referred to our hospital in a 3-year period were tested by chromosome and fluorescence in situ hybridization (FISH) analysis with the probes D22S75, Tuplel, and cHKAD26 and by a set of 10 simple tandem repeat polymorphic (STRP) markers. In nine of 11 children with IAA type B, 22q11.2 hemizygosity was demonstrated by FISH and STRP analysis, but in none of the four children with type A. In all but one child, deletion size was approximately 3 Mb. The girl with the smaller deletion of approximately 1.5 Mb differed because of an Ullrich-Turner syndrome-like phenotype and severe T-cell defect. Additionally, in one patient with phenotypic signs of DGS, a small deletion distal to the known DGS region containing the marker D22S308 was suspected by STRP analysis. One deletion was shown to be inherited from a healthy father and one IAA type A recurred in a sib. T-cell anomalies were evident in eight of the nine children with classical deletion, five of whom suffered also from hypoparathyroidism. With respect to cause and clinical course, IAA type A and B were shown to represent different entities. This study showed that variable symptoms of 22q11.2 hemizygosity may cluster.

Deletions within chromosome band 22q11.2 are associated with a variety of conditions, although a simple genotype-phenotype correlation has not been established so far. Environmental factors, chance events, or a second hit theory were supported by two observations of monozygotic twins with 22q11.2 deletions and discordant phenotypes [Goodship et al., J Med Genet 1995;32:746-748; Fryer, J Med Genet 1996;33:173]. We present monozygotic twins concordant for 22q11.2 deletion and Cayler syndrome, favoring the view that there exists a predominant genetic determination of the del 22q11.2 phenotype. As these twins are diamniotic and dichorionic, they may offer a more reliable insight in genetic phenotype determination than the other published, probably monochorionic, twins who may have a discordant malformation by twinning itself.

We report a child with a duplication-deficiency subsequent to t(15;20)(q25.2;p12.2), transmitted in at least 5 generations, who showed features of 15q-syndrome. We speculate that brachydactyly - most likely because of brachymesophalangism - is a feature of the phenotype of this chromosomal aberration and points to candidate gene(s) in this region. A similar brachydactyly was, however, reported with dup(20pl-pter).

Interphase-FISH (fluorescence in situ hybridization) studies have been devoted to the determination of clonality of aberrant karyotypes in human leukemia. Various levels of its extent have been examined, including the meaning of a single aberrant karyotype as representing a microclone, the use of FISH to confirm clonality in bi- or multiclonal leukemia, the estimation of the residual (aberrant) clone after contrasexual bone marrow transplantation, and the redetectability in interphase of the abl/bcr rearrangement. The quantitative findings of all these lines of interphase FISH analyses were based on the comparison with data from a large-scale "control" study on normal cells using the same DNA probes which have been chosen for the determination of clonality, i.e. centromeric DNA probes for chromosomes #1, #3, from #6 to #12, from #15 to #18, #20, X and Y, and a specific probe for the abl/bcr rearrangement. In addition, the validity of interphase-FISH analysis on classical bone marrow smears was examined. As a common outcome it was concluded that interphase-FISH technique is a valuable tool for defining clonality of karyotypic changes and, as a consequence, yields additional prognostic information in many human leukemias. It is recommended to perform interphase FISH in routine cytogenetics of leukemia, whenever reasonable.

The sizes of leukemic cell clones with hypersomies of the chromosome #8 or monosomy #7, which primarily were detected by classical karyotyping, were estimated by fluorescence in situ hybridization (FISH) with centromeric DNA probes in interphase cell populations of the bone marrow of 31 leukemia patients. Particularly, the data obtained by both routes of analysis were compared quantitatively. As most prominent result, all aberrations found by classical karyotyping were redetected by interphase cytogenetics, but additional aberrant clones could be observed among the interphase cell populations. The frequencies of the cell clones with hypersomies #8, in general, were higher in metaphase than' in interphase, and, vice versa, cells monosomic for #7, in the majority of cases, were found to be more frequent in interphase than in metaphase. These data support the idea that metaphase data per se may not sufficiently reflect the actual portions of aneuploid cell clones in the whole leukemic cell population. This may be of practical importance in diagnostic and prognostic respects but also for the choice of specific therapies.

We report on a retarded infant with minor dysmorphic features in whom deletion 16 and duplication 19q were discovered. The karyotype is 46,XX,del(16) (q13.08-21.05),dup(19)(q13.13-13.2). The origin and significance of the aberrant chromosomes are unknown.

Chromosomal analysis in a child with ambiguous sex showed mosaicism of at least two cell lines with one or more marker chromosomes or none at all. They were shown to be derived from the Y chromosome by fluorescent in situ hybridisation (FISH) using different DNA probes that cover parts of the long and the short arm.

Three children are reported with typical cat eye syndrome (CES) and three more children with partial CES because of absence of coloboma, in which the supernumerary marker chromosome was studied by FISH. Using a genomic library, and also a centromeric and particularly a cosmid probe of 22q11, partial tetrasomy was shown in all cases.

Several subclones were identified in unstimulated peripheral blood cells from a patient with chronic myeloproliferative disease, which was classified as myelofibrosis by morphologic terms. These subclones were characterized by an unusual number of different karyotype anomalies. Some of the more complex chromosomal rearrangements could be clearly defined by fluorescence in situ hybridization. Chromosome arms involved in clonal aberrations were 1q, 3p, 6p, 7q, 11q, 13q, 15q, 17q, 18p, and 20q. Reconstruction of karyotype evolution was attempted by karyotypic analysis of 100 metaphase spreads each in two separate investigations.

The gene associated with adenomatous polyposis coli (APC) has been mapped to the long arm of chromosome 5. To saturate the APC region with DNA markers, two independent microdissection libraries with an emphasis on 5q21.2–21.3 and 5q22 have been constructed from GTG-banded human metaphase chromosomes. PCR-amplified insert DNA of the primary amplificate used as a probe in chromosomal in situ suppression (CISS) hybridization of human metaphase spreads revealed region-specific signals at the chromosomal site that was excised for cloning. One hundred forty-two inserts, derived from both libraries, have been characterized in more detail. Deletion mapping analysis was performed with 17 single-copy clones on a hamster-human hybrid cell panel. Seven of these clones were located within two interstitial deletions of 6–8 Mb from APC-affected individuals around chromosome bands 5q21–22. The identification of new microclones mapping into these deletions and their use in isolating YAC clones should contribute to the construction of a contiguous physical map of the APC region.

The chromosome region 5q22 harbouring the putative gene associated with adenomatous polyposis coli (APC) was microdissected and microcloned from GTG-banded human metaphase chromosomes. In order to determine the precise regional localization of the microdissected material, we used polymerase chain reaction amplified microclones as a bulk-probe in nonradioactive chromosomal in situ suppression hybridization of human metaphase spreads. Specific in situ hybridization signals were obtained on the long arm of chromosome 5 in accordance with the chromosomal region excised for the cloning procedure. The application of this detection system should provide a rapid and powerful tool for analyzing patients with translocations or microdeletions of a given chromosome region.