Analysis of a non-lethal biallelic frameshift mutation in ZMPSTE24 reveals utilization of alternative translation initiation codons.

Restrictive dermopathy (RD) is a lethal condition caused by biallelic loss-of-function mutations in ZMPSTE24, whereas mutations preserving residual enzymatic activity of the ZMPSTE24 protein lead to the milder mandibuloacral dysplasia with type B lipodystrophy (MADB) phenotype. Remarkably, we identified a homozygous, presumably loss-of-function mutation in ZMPSTE24 [c.28_29insA, p.(Leu10Tyrfs*37)] in two consanguineous Pakistani families segregating MADB. To clarify how lethal consequences are prevented in affected individuals, functional analysis was performed. Expression experiments supported utilization of two alternative translation initiation sites, preventing complete loss of protein function consistent with the relatively mild phenotypic outcome in affected patients. One of these alternative start codons is newly formed at the insertion site. Our findings indicate that the creation of new potential start codons through N-terminal mutations in other disease-associated genes should generally be taken into consideration in the variant interpretation process.

An exceptional biallelic N-terminal frame shift mutation in ZMPSTE24 leads to non-lethal progeria due to possible utilization of a downstream alternative start codon.

Biallelic mutations in ZMPSTE24 are known to be associated with autosomal recessive mandibuloacral dysplasia with type B lipodystrophy (MADB) and lethal restrictive dermopathy (RD), respectively. Disease manifestation is depending on the remaining enzyme activity of the mutated ZMPSTE24 protein. To date, complete loss of function has exclusively been reported in RD cases. In this study, we identified a novel N-terminal homozygous frameshift mutation (c.28_29insA) in a consanguineous family segregating with MADB. An in-depth analysis of the mutated sequence revealed, that the one base pair insertion creates a novel downstream in-frame start codon, which supposedly serves as an alternative translation initiation site (TIS). This possible rescue mechanism would explain the relatively mild clinical outcome in the studied individuals. Our findings demonstrate the necessity for careful interpretation of N-terminal variants potentially effecting translation initiation.

A novel protein truncating mutation in L2HGDH causes L-2-hydroxyglutaric aciduria in a consanguineous Pakistani family.

BACKGROUND: L-2-hydroxyglutaric aciduria (L2HGA) is a rare neurometabolic disorder that occurs due to accumulation of L-2-hydroxyglutaric acid in the cerebrospinal fluid (CSF), plasma and urine. The clinical manifestation of L2HGA includes intellectual disability, cerebellar ataxia, epilepsy, speech problems and macrocephaly. METHODS: In the present study, we ascertained a multigenerational consanguineous Pakistani family with 5 affected individuals. Clinical studies were performed through biochemical tests and brain CT scan. Locus mapping was carried out through genome-wide SNP genotyping, whole exome sequencing and Sanger sequencing. For in silico studies protein structural modeling and docking was done using I-TASSER, Cluspro and AutoDock VINA tools. RESULTS: Affected individuals presented with cognitive impairment, gait disturbance, speech difficulties and psychomotor delay. Radiologic analysis of a male patient revealed leukoaraiosis with hypoattenuation of cerebral white matter, suggestive of hypomyelination. Homozygosity mapping in this family revealed a linkage region on chromosome 14 between markers rs2039791 and rs781354. Subsequent whole exome analysis identified a novel frameshift mutation NM_024884.3:c.180delG, p.(Ala62Profs*24) in the second exon of L2HGDH. Sanger sequencing confirmed segregation of this mutation with the disease phenotype. The identification of the most N-terminal loss of function mutation published thus far further expands the mutational spectrum of L2HGDH.

Cardio-pathogenic variants in unexplained intrauterine fetal death: a retrospective pilot study.

To describe the prevalence and spectrum of cardio-pathogenic variants in singleton fetuses after unexplained intrauterine fetal death (IUFD). DNA from post-mortem fibroblastic tissue samples of 16 fetuses after unexplained IUFD was retrieved at two tertiary university hospitals for clinical exome sequencing with subsequent filtering of 122 cardio-specific genes to elucidate underlying cardio-pathogenic variants. In total, we included 12 (75%) male and four (25%) female fetuses who were stillborn at a median gestational age of 34+6 (23+2-40+5) weeks. In two (12.5%) fetuses no cardio-pathogenic variants were found. In 14 (87.5%) fetuses, overall 33 variants were detected in 22 cardio-specific genes, involving 14 (63.63%) genes associated with cardiomyopathy, six (27.27%) arrhythmogenic susceptibility genes and two (9.09%) arrhythmia and cardiomyopathy associated genes. Among the 33 variants, five (15.2%) were classified as likely benign according to the American College of Medical Genetics and Genomics; 28 (84.8%) variants were considered as variants of uncertain significance. Compared to a cohort of explained IUFDs, the cases with and without fetal variants in cardiac genes differed not significantly regarding maternal age, previous history of stillbirth, time of stillbirth or fetal sex. Unexplained stillbirth may be caused by cardio-genetic pathologies, yet a high number of variants of uncertain significance merit a more detailed post-mortem examination including family segregation analysis.

Identification of a novel protein truncating mutation p.Asp98* in XPC associated with xeroderma pigmentosum in a consanguineous Pakistani family.

BACKGROUND: Xeroderma pigmentosum (XP) is a rare genetic disorder, which is characterized by hyper-sensitivity to solar ultraviolet (UV) radiation. Clinical consequences of sun exposure are skin lesions and an increased risk of developing skin cancer. Genetic studies have identified eight genes associated with xeroderma pigmentosum. The proteins encoded by these genes are mainly involved in DNA repair mechanisms. METHODS: Molecular genetic characterization of patients with xeroderma pigmentosum involved positional cloning methods such as homozygosity mapping and subsequent candidate gene analysis. Mutation screening was performed through Sanger DNA sequencing. RESULTS AND DISCUSSION: In this case study, we report a novel protein truncating mutation in XPC associated with autosomal recessive xeroderma pigmentosum in a consanguineous Pakistani family. Genetic mapping revealed a novel single base insertion of a thymine nucleotide NM_004628.4: c.291dupT (c.291_292insT) in the second exon of XPC. The identified mutation leads to a premature stop codon (TGA) at amino acid position 98 (p.Asp98*) and thus presumably results in a truncated protein. The Xeroderma pigmentosum, complementation group C (XPC) is located on 3p25.1 and encodes a protein involved in nucleotide excision repair. The identified mutation presumably truncates all functional domains of the XPC protein, which likely results in the loss of protein function. CONCLUSION: The study expands the knowledge of the mutational spectrum of XPC and is valuable for genetic counseling of affected individuals and their families.

Genetic study of Khyber-Pukhtunkhwa resident Pakistani families presenting primary microcephaly with intellectual disability.

OBJECTIVE: To investigate the genetic factor responsible for causing microcephaly and determine allelic heterogeneity of Abnormal spindle microtubule gene. METHODS: The genetic study was conducted at the Kohat University of Science and Technology, Kohat, and Gomal University, D.I.Khan, Pakistan, during 2017-18, and comprised 5 consanguineous families from South Waziristan, Kurram Agency, Karak, Bannu and Dera Ismail Khan regions of the country's Khyber Pakhtukhwa province. Blood samples from all available and cooperative family members (including normal and affected) were obtained, and molecular analysis was carried out through whole genome single nucleotide polymorphisms genotyping, exome sequencing and Sanger sequencing. RESULTS: Of the 15 patients, 9(60%) were males and 6(40%) were females. Genetic mapping revealed linkage to the MCPH5 locus which harbours the microcephaly-associated abnormal spindle-like microcephaly gene. Mutation analysis of the gene identified missense mutation c.3978G>A (p.Trp1326*) in families A, B and C, a deletion mutation c.7782_7783delGA (p.(Lys2595Serfs*6)) in family D, and a splice site defect c.2936+5G>A in family E. CONCLUSIONS: There was suggestion of strong founder effect of mutation c.3978G>A (p.Trp1326*).

Prenatal Detection of Chromosome Aneuploidy by Quantitative Fluorescence PCR.

Autosomal chromosome aneuploid pregnancies that survive to term, namely, trisomies 13, 18, and 21, account for 89% of chromosome abnormalities with a severe phenotype identified in prenatal samples. They are traditionally detected by full karyotype analysis of cultured cells. The average reporting time for a prenatal karyotype analysis is approximately 14 days, and in recent years, there has been increasing demand for more rapid prenatal results with respect to the common chromosome aneuploidies, to relieve maternal anxiety and facilitate options in pregnancy. The rapid tests that have been developed negate the requirement for cultured cells, instead directly testing cells from the amniotic fluid or chorionic villus sample, with the aim of generating results within 48 h of sample receipt. Interphase fluorescence in situ hybridization is the method of choice in some genetic laboratories, usually because the expertise and equipment are readily available. However, a quantitative fluorescence (QF)-PCR-based approach is now widely used and reported as a clinical diagnostic service in many studies. It may be used as a stand-alone test or as an adjunct test to full karyotype or array CGH analysis, which scan for other chromosome abnormalities not detected by the QF-PCR assay.

Is the molecular clock ticking differently in bipolar disorder? Methylation analysis of the clock gene ARNTL.

OBJECTIVES: The clock gene ARNTL is associated with the transcription activation of monoamine oxidase A according to previous literature. Thus, we hypothesised that methylation of ARNTL may differ between bipolar disorder (BD) and controls. METHODS: The methylation status of one CpG island covering the first exon of ARNTL (PS2) and one site in the 5' region of ARNTL (cg05733463) were analysed in patients with BD (n = 151) versus controls (n = 66). Methylation analysis was performed by bisulphite-conversion of DNA from fasting blood with the EpiTect Bisulfite Kit, PCR and pyrosequencing. Analysis of covariances considering the covariates age, body mass index, sex, smoking, lithium and anticonvulsant intake were performed to test methylation differences between BD and controls. RESULTS: Methylation at cg05733463 of ARNTL was significantly higher in BD than in controls (F(1,209) = 44.500, P < .001). In contrast, methylation was significantly lower in BD at PS2_POS1 compared to controls (F(1,128) = 5.787, P = .018) and by trend at PS2_POS2 (F(1,128) = 3.033, P = .084) and POS7 (F(1,34) = 3.425, P = .073). CONCLUSIONS: Methylation of ARNTL differed significantly between BD and controls. Thus, our study suggests that altered epigenetic regulation of ARNTL might provide a mechanistic basis for better understanding circadian rhythms and mood swings in BD.

Time-lapse imaging provides further evidence that planar arrangement of blastomeres is highly abnormal.

PURPOSE: Recently, guidelines on the annotation of dynamic human embryo monitoring recommended screening for the presence of planar blastomere arrangement at the 4-cell stage. This observational study was set up in order to analyze whether developmental kinetics of planar human embryos are different from tetrahedral ones. METHODS: Therefore, embryos of 115 consecutive ICSI patients (showing 32 planar and 554 tetrahedral embryos) were cultured in a new time-lapse system (Miri TL) and their embryos were annotated for morphokinetic development and screened for irregular cleavages and morphological dysmorphisms. RESULTS: Significantly less planar embryos reached blastocyst stage and showed worse quality as compared to regular tetrahedral embryos. The rate of bi- and/or multinucleation was also significantly higher in the affected group. Irregular cleavages, particularly embryo rolling, were more often seen in planar embryos. Morphokinetics between planar and tetrahedral were distinguishable up to 4-cell stage (t2-t4), thereafter the observed delay in planar embryos (t8) was more likely the result of a higher rate of arrested embryos in the planar group. CONCLUSIONS: Planar embryos are associated with both a significant increase in irregular cleavage as well as a delay in preimplantation development. This indicates that planar embryos are rather abnormal and should only be considered for transfer if no other embryos are available.

Oocyte competence in in vitro fertilization and intracytoplasmic sperm injection patients suffering from endometriosis and its possible association with subsequent treatment outcome: a matched case-control study.

INTRODUCTION: Endometriosis affects up to 15% of women of reproductive age. There is an obvious lack of studies dealing with morphological parameters of oocyte morphology in endometriosis patients in assisted reproduction. One aim of the study is to describe oocyte morphology in patients undergoing intracytoplasmic sperm injection suffering from endometriosis. In addition, the impact of endometriosis on in vitro fertilization results is analyzed. Both in vitro fertilization and intracytoplasmic sperm injection patients are then matched with an endometriosis-free control group for highlighting the possible association of endometriosis with pregnancy outcome. MATERIAL AND METHODS: Oocyte morphology of endometriosis patients was assessed in two groups. Both study group and control group consisted of 129 in vitro fertilization/intracytoplasmic sperm injection cycles each. Patients were matched according to anti-Müllerian hormone, female age, previous treatment cycles, and method of fertilization. Endometriosis was graded according to the revised American Society for Reproductive Medicine guidelines of 1997. RESULTS: Patients with endometriosis had a significantly lower rate of mature oocytes (p < 0.03) and morphologically normal oocytes (p < 0.001). In particular, brownish oocytes (p < 0.009; stage I-IV) and the presence of refractile bodies (p < 0.001; stage IV) were found to be increased. Endometriosis stage IV was associated with significantly worse-quality oocytes than stages I-III (p < 0.01). Fertilization was significantly reduced in conventional in vitro fertilization but not in intracytoplasmic sperm injection (p < 0.03). This was due to lower fertilization rates in stage III-IV endometriosis compared with stage I-II (p < 0.04). No difference was observed with respect to rates of implantation, clinical pregnancy, miscarriage, live birth, and malformation. CONCLUSIONS: Endometriosis patients, in particular those with severe endometriosis, present lower-quality oocytes. Once fertilized, no impairment of further preimplantation embryo development and pregnancy outcome right up to healthy live birth rate has to be expected.

Characterization of the injection funnel during intracytoplasmic sperm injection reflects cytoplasmic maturity of the oocyte.

OBJECTIVE: To quantify cytoplasmic maturity on the basis of intracytoplasmic sperm injection (ICSI) injection funnel manifestation and to evaluate influence factors of the latter. DESIGN: Prospective study. SETTING: Private fertility center. PATIENT(S): A total of 31 patients with good ovarian response. INTERVENTION(S): Mature and immature oocytes were injected intracytoplasmatically. Formation and persistence of an injection funnel was documented and measured. MAIN OUTCOME MEASURE(S): ICSI funnel size, persistence of injection funnel, rates of degeneration and fertilization, embryo quality. RESULT(S): Funnel volume in germinal vesicle stage oocytes (prophase I [PI]) was significantly smaller than that of metaphase I (MI) and MII oocytes. Immature eggs (PI, MI) almost never showed a persistent funnel 2-4 minutes after ICSI, whereas in MII eggs the funnel was still observable in 35% (117/334) of the cases. Uni- and multivariate analysis revealed that pipette type and stimulation protocol significantly influenced appearance of injection funnel. Funnel volume in oocytes that fertilized regularly was significantly higher compared with three-polar body and degenerated oocytes. CONCLUSION(S): Oocyte maturation within the follicle is closely associated with a remarkable change in cytoplasm viscosity from an aqueous to a more viscous subtype. Precise evaluation of the injection funnel may help to explain deviations from expected ICSI outcome and could also assist in optimizing controlled ovarian hyperstimulation.

Quantitative and qualitative trophectoderm grading allows for prediction of live birth and gender.

PURPOSE: Prolonged in vitro culture is thought to affect pre- and postnatal development of the embryo. This prospective study was set up to determine whether quality/size of inner cell mass (ICM) (from which the fetus ultimately develops) and trophectoderm (TE) (from which the placenta ultimately develops) is reflected in birth and placental weight, healthy live-birth rate, and gender after fresh and frozen single blastocyst transfer. METHODS: In 225 patients, qualitative scoring of blastocysts was done according to the criteria expansion, ICM, and TE appearance. In parallel, all three parameters were quantified semi-automatically. RESULTS: TE quality and cell number were the only parameters that predicted treatment outcome. In detail, pregnancies that continued on to a live birth could be distinguished from those pregnancies that aborted on the basis of TE grade and cell number. Male blastocysts had a 2.53 higher chance of showing TE of quality A compared to female ones. There was no correlation between the appearance of both cell lineages and birth or placental weight, respectively. CONCLUSIONS: The presented correlation of TE with outcome indicates that TE scoring could replace ICM scoring in terms of priority. This would automatically require a rethinking process in terms of blastocyst selection and cryopreservation strategy.

Yellow laser acupuncture - A new option for prevention and early intervention of lifestyle-related diseases: A randomized, placebo-controlled trial in volunteers.

BACKGROUND AND AIMS: The yellow laser constitutes a totally new option in the field of laser acupuncture, in addition to the already existing red, near infrared, green and violet lasers. Especially for so called lifestyle-related diseases, this could open up new methods of integrative therapy. The goal of the present study was to investigate among other parameters blood pressure (BP), heart rate (HR), heart rate variability (HRV), and temperature effects before, during, and after stimulation of different acupoints with yellow laser. SUBJECTS AND METHODS: We recruited 26 healthy volunteers (13 female, 13 male; mean age ± SD 24.1 ± 3.3 years) at the Medical University of Graz. The acupoints Baihui, Neiguan, Taichong and a placebo point were stimulated with a 589 nm (50 mW, 500 µm; 5 min) yellow laser. Blood pressure was measured noninvasively at the wrist; for the registration of the electrocardiogram a medilog AR12 HRV system was used. Effects on temperature were measured with a Flir i7 infrared camera. RESULTS: There were significant decreases after yellow laser acupuncture in the systolic BP, diastolic BP also decreased (n.s.). HRV in both (men and women) increased. The temperature during the yellow laser stimulation decreased significantly in all measured points. After the stimulation it increased again significantly. Based on a questionnaire volunteers reported a significantly decreased level of stress after yellow laser stimulation. CONCLUSION: Significant positive effects on BP and well-being were found after yellow laser stimulation. The results are very promising and can be very important especially for the treatment of lifestyle related diseases.

Viability of cumulus cells is associated with basal AMH levels in assisted reproduction.

OBJECTIVE(S): An interesting non-invasive approach to select embryos for transfer is analyzing the health state of somatic granulosa cells surrounding the oocyte addressing their mutual dependence. This prospective study was set up to analyse whether the DNA integrity of cumulus cells correlates with preimplantation development and basal AMH levels. STUDY DESIGN: Therefore, 56 patients who gave written consent were enrolled. Sequential denudation of the cumulus-oocyte-complexes was performed in order to separate corona radiata from outer cumulus cells. DNA integrity of both cell types was analysed using a modified chromatin dispersion test. RESULTS: The percentage of viable corona radiata cells per patient showed a linear correlation to blastulation (P<0.05). These innermost cells showed significantly lower rates of strand breaks (P<0.01) as compared to outer cumulus cells. Age-corrected AMH was significantly associated with the DNA integrity of outer cumulus cells (P<0.05). CONCLUSION(S): For the first time it could be shown that in fact clinical embryologists deal with two different entities of cumulus cells, inner and outer ones. It seems that any protective mechanism of the female gamete follows an outward gradient, so that negative effects, e.g. apoptosis, may impair outer cumulus cells first. Age-corrected AMH reflects quality of these outer cumulus cells. KEYWORDS: AMH; Corona radiata cells; DNA fragmentation; Outer cumulus cells; SCD test.

Identification of risk genes for autism spectrum disorder through copy number variation analysis in Austrian families.

Autism or autism spectrum disorder (ASD) is a range of neurodevelopmental disorders starting in early childhood and is characterized by impairments in communication and reciprocal social interaction and presence of restricted and repetitive patterns of behavior. The contribution of genetic factors to autism is clear in twin and family studies. It is apparent that, overall, ASD is a complex non-Mendelian disorder. Recent studies suggest that copy number variations (CNVs) play a significant role in the etiology of ASD. For the current work, we recruited 245 family members from 73 ASD families from Styria, Austria. The DNA from probands was genotyped with Affymetrix single nucleotide polymorphism (SNP) 6.0 microarrays to screen for CNVs in their genomes. Analysis of the microarray data was performed using three different algorithms, and a list of stringent calls was compared to existing CNV data from over 2,357 controls of European ancestry. For stringent calls not present in controls, quantitative real-time PCR (qRT-PCR) was used to validate the CNVs in the probands and in their family members. Twenty-two CNVs were validated from this set (five of which are apparently de novo), many of which appear likely to disrupt genes that may be considered as good candidates for neuropsychiatric disorders, including DLG2, S100B, ARX, DIP2A, HPCAL1, and GPHN. Several others disrupt genes that have previously been implicated in autism, such as BDNF, AUTS2, DPP6, and C18orf22, and our data add to the growing evidence of their involvement in ASD.

Three different profiles: early socio-communicative capacities in typical Rett syndrome, the preserved speech variant and normal development.

BACKGROUND AND AIMS: This is the first study aiming to compare pre-diagnostic socio-communicative development of a female with typical Rett syndrome (RTT), a female with the preserved speech variant of RTT (PSV) and a control toddler. METHODS: We analysed 1275 min of family videos at the participants' age between 9 and 24 months and used the Inventory of Potential Communicative Acts (IPCA) to delineate their repertoires of communicative forms and functions. RESULTS: The results revealed different profiles for the three different conditions. The repertoire of communicative gestures and (pre)linguistic vocalizations was most comprehensive in the control toddler, followed by the female with PSV and the female with RTT. CONCLUSION: These findings contribute to the growing knowledge about early developmental abnormalities in RTT. In order to define distinctive profiles for typical and atypical RTT and evaluate their specificity, a larger body of evidence is needed.

Rare exonic deletions implicate the synaptic organizer Gephyrin (GPHN) in risk for autism, schizophrenia and seizures.

The GPHN gene codes for gephyrin, a key scaffolding protein in the neuronal postsynaptic membrane, responsible for the clustering and localization of glycine and GABA receptors at inhibitory synapses. Gephyrin has well-established functional links with several synaptic proteins that have been implicated in genetic risk for neurodevelopmental disorders such as autism spectrum disorder (ASD), schizophrenia and epilepsy including the neuroligins (NLGN2, NLGN4), the neurexins (NRXN1, NRXN2, NRXN3) and collybistin (ARHGEF9). Moreover, temporal lobe epilepsy has been linked to abnormally spliced GPHN mRNA lacking exons encoding the G-domain of the gephyrin protein, potentially arising due to cellular stress associated with epileptogenesis such as temperature and alkalosis. Here, we present clinical and genomic characterization of six unrelated subjects, with a range of neurodevelopmental diagnoses including ASD, schizophrenia or seizures, who possess rare de novo or inherited hemizygous microdeletions overlapping exons of GPHN at chromosome 14q23.3. The region of common overlap across the deletions encompasses exons 3-5, corresponding to the G-domain of the gephyrin protein. These findings, together with previous reports of homozygous GPHN mutations in connection with autosomal recessive molybdenum cofactor deficiency, will aid in clinical genetic interpretation of the GPHN mutation spectrum. Our data also add to the accumulating evidence implicating neuronal synaptic gene products as key molecular factors underlying the etiologies of a diverse range of neurodevelopmental conditions.

MODY type 2 in Greig cephalopolysyndactyly syndrome (GCPS) as part of a contiguous gene deletion syndrome.

Maturity-onset diabetes of the young type 2 (MODY2) is a form of monogenic diabetes, characterized by mild fasting hyperglycemia. MODY2 is caused by heterozygous mutations in the GCK gene that encodes the glucokinase enzyme. We describe the clinical features and the underlying genetic defect of MODY2 in a patient with atypical Greig cephalopolysyndactyly syndrome (GCPS). The patient presented with the limb formation and the craniofacial developmental abnormalities typical to GCPS, in addition to mental retardation and epilepsy (assigned as atypical syndrome). Fasting hyperglycemia in the diabetic range, impaired glucose tolerance, and lack of diabetes autoantibodies were compatible with MODY2. In order to delineate the genetic aberrations relevant both to MODY2 and Greig syndrome in this patient, we performed cytogenetic analysis, real-time PCR of the GCK gene, and comparative genomic hybridization (CGH) array. Cytogenetic study has shown a microscopic detectable deletion in the 7p13-15 chromosomal region. Real-time PCR demonstrated a deletion of the GCK gene in the patient but not her parents, and CGH array revealed a deleted region of approximately 12 Mb in the 7p13-15 region. This deleted region included GLI3 and GCK genes (where heterozygous mutations cause GCPS and MODY2, respectively), and many other contiguous genes. Our patient manifests a unique form of MODY2, where GCK gene deletion is part of a large deleted segment in the 7p13-15 chromosomal region.

Prenatal detection of chromosome aneuploidy by quantitative-fluorescence PCR.

QF-PCR refers to the amplification of chromosome-specific polymorphic microsatellite markers using fluorescence-labelled primers, followed by semi-quantitative analysis of the products on a genetic analyser to determine copy number and/or imbalances of specific chromosomal material. This approach is now widely used for rapid prenatal diagnosis of the common trisomies. In addition, it can successfully detect maternal cell contamination and mosaicism in prenatal material.

Automatic retrieval of single microchimeric cells and verification of identity by on-chip multiplex PCR.

The analysis of rare cells is not an easy task. This is especially true when cells representing a fetal microchimerism are to be utilized for the purpose of non-invasive prenatal diagnosis because it is both imperative and difficult to avoid contaminating the minority of fetal cells with maternal ones. Under these conditions, even highly specific biochemical markers are not perfectly reliable. We have developed a method to verify the genomic identity of rare cells that combines automatic screening for enriched target cells (based on immunofluorescence labelling) with isolation of single candidate microchimeric cells (by laser microdissection and subsequent laser catapulting) and low-volume on-chip multiplex PCR for DNA fingerprint analysis. The power of the method was tested using samples containing mixed cells of related and non-related individuals. Single-cell DNA fingerprinting was successful in 74% of the cells analysed (55/74), with a PCR efficiency of 59.2% (860/1452) for heterozygous loci. The identification of cells by means of DNA profiling was achieved in 100% (12/12) of non-related cells in artificial mixtures and in 86% (37/43) of cells sharing a haploid set of chromosomes and was performed on cells enriched from blood and cells isolated from tissue. We suggest DNA profiling as a standard for the identification of microchimerism on a single-cell basis.