Femoral facial syndrome associated with a de novo complex chromosome 2q37 rearrangement.

The femoral facial syndrome (FFS) is a rare congenital anomaly syndrome characterized by bilateral femoral hypoplasia and facial dysmorphism. The etiology of FFS is currently unknown but maternal/gestational diabetes has been proposed as a strong risk factor for syndromic femoral hypoplasia. In affected children born to non-diabetic mothers, a genetic contribution to FFS is suspected; however, no chromosomal anomalies or gene mutations have been identified so far. Here, we report on a girl with FFS and a de novo complex chromosome rearrangement of terminal chromosome 2q37.2. Radiographs of the pelvis and lower limbs showed bilateral shortening and bowing of the femur and radiographs of hands and feet revealed a brachydactyly type E (BDE). Using high resolution array-CGH, qPCR, and FISH, we detected a ~1.9 Mb duplication in the chromosomal region 2q37.2 and a ~5.4 Mb deletion on chromosome 2q37.3 that were absent in the parents. The duplication contains six genes and the deletion encompasses 68 genes; the latter has previously been shown to cause BDE (through haploinsufficiency for HDAC4) but not femoral hypoplasia. Therefore, we propose that the duplication 2q37.2 could be causative for the femur phenotype. To the best of our knowledge, our report is the first to propose a genetic cause in a case of FFS.

STIL mutation causes autosomal recessive microcephalic lobar holoprosencephaly.

Holoprosencephaly is a clinically and genetically heterogeneous midline brain malformation associated with neurologic manifestations including developmental delay, intellectual disability and seizures. Although mutations in the sonic hedgehog gene SHH and more than 10 other genes are known to cause holoprosencephaly, many patients remain without a molecular diagnosis. Here we show that a homozygous truncating mutation of STIL not only causes severe autosomal recessive microcephaly, but also lobar holoprosencephaly in an extended consanguineous Pakistani family. STIL mutations have previously been linked to centrosomal defects in primary microcephaly at the MCPH7 locus. Our results thus expand the clinical phenotypes associated with biallellic STIL mutations to include holoprosencephaly.

Autism-associated SHANK3 mutations impair maturation of neuromuscular junctions and striated muscles.

Heterozygous mutations of the gene encoding the postsynaptic protein SHANK3 are associated with syndromic forms of autism spectrum disorders (ASDs). One of the earliest clinical symptoms in SHANK3-associated ASD is neonatal skeletal muscle hypotonia. This symptom can be critical for the early diagnosis of affected children; however, the mechanism mediating hypotonia in ASD is not completely understood. Here, we used a combination of patient-derived human induced pluripotent stem cells (hiPSCs), Shank3Δ11(-/-) mice, and Phelan-McDermid syndrome (PMDS) muscle biopsies from patients of different ages to analyze the role of SHANK3 on motor unit development. Our results suggest that the hypotonia in SHANK3 deficiency might be caused by dysfunctions in all elements of the voluntary motor system: motoneurons, neuromuscular junctions (NMJs), and striated muscles. We found that SHANK3 localizes in Z-discs in the skeletal muscle sarcomere and co-immunoprecipitates with α-ACTININ. SHANK3 deficiency lead to shortened Z-discs and severe impairment of acetylcholine receptor clustering in hiPSC-derived myotubes and in muscle from Shank3Δ11(-/-) mice and patients with PMDS, indicating a crucial role for SHANK3 in the maturation of NMJs and striated muscle. Functional motor defects in Shank3Δ11(-/-) mice could be rescued with the troponin activator Tirasemtiv that sensitizes muscle fibers to calcium. Our observations give insight into the function of SHANK3 besides the central nervous system and imply potential treatment strategies for SHANK3-associated ASD.

Retinoic acid worsens ATG10-dependent autophagy impairment in TBK1-mutant hiPSC-derived motoneurons through SQSTM1/p62 accumulation.

Mutations in the TBK1 (TANK binding kinase 1) gene are causally linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). TBK1 phosphorylates the cargo receptors OPTN and SQSTM1 regulating a critical step in macroautophagy/autophagy. Disruption of the autophagic flux leads to accumulation of cytosolic protein aggregates, which are a hallmark of ALS. hiPSC-derived TBK1-mutant motoneurons (MNs) showed reduced TBK1 levels and accumulation of cytosolic SQSTM1-positive aggresomes. By screening a library of nuclear-receptor-agonists for modifiers of the SQSTM1 aggregates, we identified 4-hydroxy(phenyl)retinamide (4HPR) as a potent modifier exerting detrimental effects on mutant-TBK1 motoneurons fitness exacerbating the autophagy overload. We have shown by TEM that TBK1-mutant motoneurons accumulate immature phagophores due a failure in the elongation phase, and 4HPR further worsens the burden of dysfunctional phagophores. 4HPR-increased toxicity was associated with the upregulation of SQSTM1 in a context of strongly reduced ATG10, while rescue of ATG10 levels abolished 4HPR toxicity. Finally, we showed that 4HPR leads to a downregulation of ATG10 and to an accumulation of SQSTM1+ aggresomes also in hiPSC-derived C9orf72-mutant motoneurons. Our data show that cultured human motoneurons harboring mutations in TBK1 gene display typical ALS features, like decreased viability and accumulation of cytosolic SQSTM1-positive aggresomes. The retinoid 4HPR appears a strong negative modifier of the fitness of TBK1 and C9orf72-mutant MNs, through a pathway converging on the mismatch of initiated autophagy and ATG10 levels. Thus, autophagy induction appears not to be a therapeutic strategy for ALS unless the specific underlying pathway alterations are properly addressed. Abbreviations: 4HPR: 4-hydroxy(phenyl)retinamide; AKT: AKT1 serine/threonine kinase 1; ALS: amyotrophic lateral sclerosis; ATG: autophagy related; AVs: autophagic vesicle; C9orf72: chromosome 9 open reading frame 72; CASP3: caspase 3; CHAT: choline O-acetyltransferase; CYCS: cytochrome c, somatic; DIV: day in vitro; FTD: frontotemporal dementia; FUS: FUS RNA binding protein; GFP: green fluorescent protein; hiPSCs: human induced pluripotent stem cells; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MNs: motoneurons; mRFP: monomeric red fluorescent protein; MTOR: mechanistic target of rapamycin kinase; NFE2L2/NRF2: nuclear factor, erythroid 2 like 2; RARA: retinoic acid receptor alpha; SLC18A3/VACHT: solute carrier family 18 (vesicular acetylcholine transporter), member 3; SQSTM1/p62: sequestosome 1; TBK1: TANK binding kinase 1; TEM: transmission electron microscopy.

NEK1 loss-of-function mutation induces DNA damage accumulation in ALS patient-derived motoneurons.

Mutations in genes coding for proteins involved in DNA damage response (DDR) and repair, such as C9orf72 and FUS (Fused in Sarcoma), are associated with neurodegenerative diseases and lead to amyotrophic lateral sclerosis (ALS). Heterozygous loss-of-function mutations in NEK1 (NIMA-related kinase 1) have also been recently found to cause ALS. NEK1 codes for a multifunctional protein, crucially involved in mitotic checkpoint control and DDR. To resolve pathological alterations associated with NEK1 mutation, we compared hiPSC-derived motoneurons carrying a NEK1 mutation with mutant C9orf72 and wild type neurons at basal level and after DNA damage induction. Motoneurons carrying a C9orf72 mutation exhibited cell specific signs of increased DNA damage. This phenotype was even more severe in NEK1c.2434A>T neurons that showed significantly increased DNA damage at basal level and impaired DDR after induction of DNA damage in an maturation-dependent manner. Our results provide first mechanistic insight in pathophysiological alterations induced by NEK1 mutations and point to a converging pathomechanism of different gene mutations causative for ALS. Therefore, our study contributes to the development of novel therapeutic strategies to reduce DNA damage accumulation in neurodegenerative diseases and ALS.

Mosaic genome-wide maternal isodiploidy: an extreme form of imprinting disorder presenting as prenatal diagnostic challenge.

BACKGROUND: Uniparental disomy of certain chromosomes are associated with a group of well-known genetic syndromes referred to as imprinting disorders. However, the extreme form of uniparental disomy affecting the whole genome is usually not compatible with life, with the exception of very rare cases of patients with mosaic genome-wide uniparental disomy reported in the literature. RESULTS: We here report on a fetus with intrauterine growth retardation and malformations observed on prenatal ultrasound leading to invasive prenatal testing. By cytogenetic (conventional karyotyping), molecular cytogenetic (QF-PCR, FISH, array), and methylation (MS-MLPA) analyses of amniotic fluid, we detected mosaicism for one cell line with genome-wide maternal uniparental disomy and a second diploid cell line of biparental inheritance with trisomy X due to paternal isodisomy X. As expected for this constellation, we observed DNA methylation changes at all imprinted loci investigated. CONCLUSIONS: This report adds new information on phenotypic outcome of mosaic genome-wide maternal uniparental disomy leading to an extreme form of multilocus imprinting disturbance. Moreover, the findings highlight the technical challenges of detecting these rare chromosome disorders prenatally.

FUS Mislocalization and Vulnerability to DNA Damage in ALS Patients Derived hiPSCs and Aging Motoneurons.

Mutations within the FUS gene (Fused in Sarcoma) are known to cause Amyotrophic Lateral Sclerosis (ALS), a neurodegenerative disease affecting upper and lower motoneurons. The FUS gene codes for a multifunctional RNA/DNA-binding protein that is primarily localized in the nucleus and is involved in cellular processes such as splicing, translation, mRNA transport and DNA damage response. In this study, we analyzed pathophysiological alterations associated with ALS related FUS mutations (mFUS) in human induced pluripotent stem cells (hiPSCs) and hiPSC derived motoneurons. To that end, we compared cells carrying a mild or severe mFUS in physiological- and/or stress conditions as well as after induced DNA damage. Following hyperosmolar stress or irradiation, mFUS hiPS cells recruited significantly more cytoplasmatic FUS into stress granules accompanied by impaired DNA-damage repair. In motoneurons wild-type FUS was localized in the nucleus but also deposited as small punctae within neurites. In motoneurons expressing mFUS the protein was additionally detected in the cytoplasm and a significantly increased number of large, densely packed FUS positive stress granules were seen along neurites. The amount of FUS mislocalization correlated positively with both the onset of the human disease (the earlier the onset the higher the FUS mislocalization) and the maturation status of the motoneurons. Moreover, even in non-stressed post-mitotic mFUS motoneurons clear signs of DNA-damage could be detected. In summary, we found that the susceptibility to cell stress was higher in mFUS hiPSCs and hiPSC derived motoneurons than in controls and the degree of FUS mislocalization correlated well with the clinical severity of the underlying ALS related mFUS. The accumulation of DNA damage and the cellular response to DNA damage stressors was more pronounced in post-mitotic mFUS motoneurons than in dividing hiPSCs suggesting that mFUS motoneurons accumulate foci of DNA damage, which in turn might be directly linked to neurodegeneration.

Uniparental Disomy in Somatic Mosaicism 45,X/46,XY/46,XX Associated with Ambiguous Genitalia.

Disorders of sex development (DSD) affect the development of chromosomal, gonadal and/or anatomical sex. We analyzed a patient with ambiguous genitalia aiming to correlate the genetic findings with the phenotype. Blood and tissue samples from a male patient with penoscrotal hypospadias were analyzed by immunohistochemistry, karyotyping and FISH. DNA was sequenced for the AR, SRY and DHH genes, and further 26 loci in different sex chromosomes were analyzed by MLPA. The gonosomal origin was evaluated by simple tandem repeat (STR) analysis and SNP array. Histopathology revealed a streak gonad, a fallopian tube and a rudimentary uterus, positive for placental alkaline phosphatase, cytokeratin-7 and c-kit, and negative for estrogen, androgen and progesterone receptors, alpha-inhibin, alpha-1-fetoprotein, ß-hCG, and oct-4. Karyotyping showed a 45,X/46,XY mosaicism, yet FISH showed both 46,XX/46,XY mosaicism (gonad and urethral plate), 46,XX (uterus and tube) and 46,XY karyotypes (rudimentary testicular tissue). DNA sequencing revealed intact sequences in SOX9, WNT4, NR0B1, NR5A1, CYP21A2, SRY, AR, and DHH. STR analysis showed only one maternal allele for all X chromosome markers (uniparental isodisomy, UPD), with a weaker SRY signal and a 4:1 ratio in the X:Y signal. Our findings suggest that the observed complex DSD phenotype is the result of somatic gonosomal mosaicism and UPD despite a normal blood karyotype. The presence of UPD warrants adequate genetic counseling for the family and frequent, lifelong, preventive follow-up controls in the patient.

An excess of chromosome 1 breakpoints in male infertility.

In a search for potential infertility loci, which might be revealed by clustering of chromosomal breakpoints, we compiled 464 infertile males with a balanced rearrangement from Mendelian Cytogenetics Network database (MCNdb) and compared their karyotypes with those of a Danish nation-wide cohort. We excluded Robertsonian translocations, rearrangements involving sex chromosomes and common variants. We identified 10 autosomal bands, five of which were on chromosome 1, with a large excess of breakpoints in the infertility group. Some of these could potentially harbour a male-specific infertility locus. However, a general excess of breakpoints almost everywhere on chromosome 1 was observed among the infertile males: 26.5 versus 14.5% in the cohort. This excess was observed both for translocation and inversion carriers, especially pericentric inversions, both for published and unpublished cases, and was significantly associated with azoospermia. The largest number of breakpoints was reported in 1q21; FISH mapping of four of these breakpoints revealed that they did not involve the same region at the molecular level. We suggest that chromosome 1 harbours a critical domain whose integrity is essential for male fertility.

Mutations in SPRTN cause early onset hepatocellular carcinoma, genomic instability and progeroid features.

Age-related degenerative and malignant diseases represent major challenges for health care systems. Elucidation of the molecular mechanisms underlying carcinogenesis and age-associated pathologies is thus of growing biomedical relevance. We identified biallelic germline mutations in SPRTN (also called C1orf124 or DVC1) in three patients from two unrelated families. All three patients are affected by a new segmental progeroid syndrome characterized by genomic instability and susceptibility toward early onset hepatocellular carcinoma. SPRTN was recently proposed to have a function in translesional DNA synthesis and the prevention of mutagenesis. Our in vivo and in vitro characterization of identified mutations has uncovered an essential role for SPRTN in the prevention of DNA replication stress during general DNA replication and in replication-related G2/M-checkpoint regulation. In addition to demonstrating the pathogenicity of identified SPRTN mutations, our findings provide a molecular explanation of how SPRTN dysfunction causes accelerated aging and susceptibility toward carcinoma.

Rat embryonic fibroblasts improve reprogramming of human keratinocytes into induced pluripotent stem cells.

Patient-specific human induced pluripotent stem (hiPS) cells not only provide a promising tool for cellular disease models in general, but also open up the opportunity to establish cell-type-specific systems for personalized medicine. One of the crucial prerequisites for these strategies, however, is a fast and efficient reprogramming strategy from easy accessible somatic cell populations. Keratinocytes from plucked human hair had been introduced as a superior cell source for reprogramming purposes compared with the widely used skin fibroblasts. The starting cell population is, however, limited and thereby further optimization in terms of time, efficiency, and quality is inevitable. Here we show that rat embryonic fibroblasts (REFs) should replace mouse embryonic fibroblasts as feeder cells in the reprogramming process. REFs enable a significantly more efficient reprogramming procedure as shown by colony number and total amount of SSEA4-positive cells. We successfully produced keratinocyte-derived hiPS (k-hiPS) cells from various donors. The arising k-hiPS cells display the hallmarks of pluripotency such as expression of stem cell markers and differentiation into all 3 germ layers. The increased reprogramming efficiency using REFs as a feeder layer occurred independent of the proliferation rate in the parental keratinocytes and acts, at least in part, in a non-cell autonomous way by secreting factors known to facilitate pluripotency such as Tgfb1, Inhba and Grem1. Hence, we provide an easy to use and highly efficient reprogramming system that could be very useful for a broad application to generate human iPS cells.

Characterization of a t(5;8)(q31;q21) translocation in a patient with mental retardation and congenital heart disease: implications for involvement of RUNX1T1 in human brain and heart development.

The chromosome break points of the t(8;21)(q21.3;q22.12) translocation associated with acute myeloid leukemia disrupt the RUNX1 gene (also known as AML1) and the RUNX1T1 gene (also known as CBFA2T3, MTG8 and ETO) and generate a RUNX1-RUNX1T1 fusion protein. Molecular characterization of the translocation break points in a t(5;8)(q32;q21.3) patient with mild-to-moderate mental retardation and congenital heart disease revealed that one of the break points was within the RUNX1T1 gene. Analysis of RUNX1T1 expression in human embryonic and fetal tissues suggests a role of RUNX1T1 in brain and heart development and support the notion that disruption of the RUNX1T1 gene is associated with the patient's phenotype.

Molecular cytogenetic characterization of two independent karyotypic anomalies in a patient with severe mental retardation and juvenile idiopathic arthritis.

We report on a patient with severe mental retardation, dysmorphic features as well as juvenile idiopathic arthritis. G-banding indicated two independent karyotypic anomalies in this patient: an interstitial deletion del(X)(p21p22.3) and a rearrangement involving chromosomes 1 and 7, which represents a direct insertion, ins(7;1)(q36;p13.2p31.2). Non-random inactivation of the paternally derived del(X) chromosome was observed in blood lymphocytes and fibroblasts. High resolution analysis of the rearrangement involving chromosomes 1 and 7 subsequently revealed the additional submicroscopic deletion of at least 5 Mb at the 1p13.2 breakpoint. The deletion occurred on the paternal chromosome and encompasses the PTPN22 gene, already known to be associated with juvenile idiopathic arthritis. Our findings underline the importance of closely investigating the breakpoint regions of apparently balanced rearrangements in patients with abnormal phenotypes since complex chromosomal rearrangements (CCRs) may turn out to be unbalanced.

Characterization of a 16 Mb interstitial chromosome 7q21 deletion by tiling path array CGH.

We report on a 42-year-old female patient with an interstitial 16 Mb deletion in 7q21.1-21.3 and a balanced reciprocal translocation between chromosomes 6 and 7 [karyotype 46,XX,t(6;7)(q23.3;q32.3)del(7)(q21.1q21.3)de novo]. We characterized the size and position of the deletion by tiling path array comparative genomic hybridization (CGH), and we mapped the translocation breakpoints on chromosomes 6 and 7 by FISH. The clinical features of this patient-severe mental retardation, short stature, microcephaly and deafness-are in accordance with previously reported patients with 7q21 deletions. Chromosome band 7q21.3 harbors a locus for split hand/split foot malformation (SHFM1), and part of this locus, including the SHFM1 candidate genes SHFM1, DLX5, and DLX6, is deleted. The absence of limb abnormalities in this patient suggests either a location of the SHFM1 causing factor distal to this deletion, or reduced penetrance of haploinsufficiency of a SHFM1 factor within the deleted interval.

Molecular cytogenetic analysis of a familial interstitial deletion Xp22.2-22.3 with a highly variable phenotype in female carriers.

We describe a familial interstitial deletion of 7.7-Mb involving Xp22.2-22.3. The deletion was transmitted from an asymptomatic mother to her two children with severe developmental delay, no speech development and autistic behavior. Assessment of the deletion boundaries by FISH and PCR analyses indicated that the deletions encompasses 27 genes. Several of these genes are associated with known disorders, like KAL1 (Kallmann syndrome), steroid sulfatase (STS) (X-linked ichtyosis), and arylsulfatase E (ARSE) (chondrodysplasia punctata). The deletion also includes all four VCX genes (VCX-A, VCX-B1, VCX-B, and VCX-C) and the neuroligin 4 (NLGN4) gene. VCX-A deficiency has been shown previously to be associated with mental retardation and NLGN4 mutations lead to mental retardation in conjunction with autism. Functional deficiency of both MRX genes, VCX-A and NLGN4, are most likely associated with the impaired cognitive development of the patients described here. The phenotype associated with the Xp deletion was highly variable in female carriers and might be attributed to unfavorable X inactivation. However, all the 27 genes included in the deleted interval escape X inactivation and are expressed at variable levels from the normal X chromosome. Thus, the overall X inactivation pattern and inter-individual expression variability of the genes in distal Xp might be determinants of the phenotype associated with the deletion.

Disruptions of the novel KIAA1202 gene are associated with X-linked mental retardation.

The extensive heterogeneity underlying the genetic component of mental retardation (MR) is the main cause for our limited understanding of the aetiology of this highly prevalent condition. Hence we set out to identify genes involved in MR. We investigated the breakpoints of two balanced X;autosome translocations in two unrelated female patients with mild/moderate MR and found that the Xp11.2 breakpoints disrupt the novel human KIAA1202 (hKIAA1202) gene in both cases. We also identified a missense exchange in this gene, segregating with the Stocco dos Santos XLMR syndrome in a large four-generation pedigree but absent in >1,000 control X-chromosomes. Among other phenotypic characteristics, the affected males in this family present with severe MR, delayed or no speech, seizures and hyperactivity. Molecular studies of hKIAA1202 determined its genomic organisation, its expression throughout the brain and the regulation of expression of its mouse homologue during development. Transient expression of the wild-type KIAA1202 protein in HeLa cells showed partial colocalisation with the F-actin based cytoskeleton. On the basis of its domain structure, we argue that hKIAA1202 is a new member of the APX/Shroom protein family. Members of this family contain a PDZ and two ASD domains of unknown function and have been shown to localise at the cytoskeleton, and play a role in neurulation, cellular architecture, actin remodelling and ion channel function. Our results suggest that hKIAA1202 may be important in cognitive function and/or development.

Interstitial deletion del(10)(q25.2q25.3 approximately 26.11)--case report and review of the literature.

OBJECTIVES: To present the clinical, cytogenetic, and molecular cytogenetic findings of prenatally diagnosed interstitial deletion 10q25.2-q26.1. The majority of distal 10q deletions are pure terminal deletions with breakpoints in 10q25 and 10q26. Only four patients have been described so far with interstitial deletions involving bands 10q25.2-q26.1. METHODS: Postmortem physical examination and autopsy of the foetus after medically terminated pregnancy. GTG-banding, reverse painting, and FISH analysis with BAC clones on amniocyte metaphases were performed to determine the extent of the deletion. RESULTS: At 20 weeks the eutrophic female foetus showed pronounced microretrogeny and hypertelorism, clubfeet as well as minor internal anomalies like pancreas anulare, atypically lobed liver, and missing choleocystis. Cardiac anomalies were not observed and the genitalia were of a normal female. The deletion encompasses 6-Mb and is associated with hemizygosity for 30 genes, including the genes for beta-tectorin, the beta-1 adrenergic receptor, and the alpha-2A adrenergic receptor. CONCLUSION: An interstitial deletion del(10)(q25.2q25.3 approximately 26.11) was confirmed by FISH with mapped BAC clones. Clinical and molecular cytogenetic analyses of further interstitial 10q deletions are necessary to assess whether the phenotypic manifestations differ between deletions that are interstitial compared to those that include also the terminal region of chromosome 10.

Molecular cytogenetic analysis of a constitutional de novo interstitial deletion of chromosome 12p in a boy with developmental delay and congenital anomalies.

We describe the case of a 6-month-old boy with psychomotor retardation, craniofacial dysmorphism, cleft lip and palate, as well as hearing and visual impairment. Analysis of G-banded chromosomes of the propositus showed a de novo interstitial deletion of the short arm of chromosome 12, del(12)(p12.1p12.3). Molecular cytogenetic analysis with bacterial artificial chromosomes (BAC) clones was used to refine the extent of the deletion. The deleted segment encompasses about 12.5 Mb between markers D12S1832 and G62375. The phenotypic consequences of the deletion are discussed and compared with other cases of interstitial deletions of proximal chromosome 12p.