Performance of screening for aneuploidies by cell-free DNA analysis of maternal blood in twin pregnancies.

OBJECTIVES: To report clinical implementation of cell-free DNA (cfDNA) analysis of maternal blood in screening for trisomies 21, 18 and 13 in twin pregnancies and examine variables that could influence the failure rate of the test. METHODS: cfDNA testing was performed in 515 twin pregnancies at 10-28 weeks' gestation. The failure rate of the test to provide results was compared with that in 1847 singleton pregnancies, and logistic regression analysis was used to determine which factors among maternal and pregnancy characteristics were significant predictors of test failure. RESULTS: Failure rate of the cfDNA test at first sampling was 1.7% in singletons and 5.6% in twins. Of those with a test result, the median fetal fraction in twins was 8.7% (range, 4.1-30.0%), which was lower than that in singletons (11.7% (range, 4.0-38.9%)). Multivariable regression analysis demonstrated that twin pregnancy, higher maternal weight and conception by in-vitro fertilization provided significant independent prediction of test failure. Follow-up was available in 351 (68.2%) of the twin pregnancies and comprised 334 with euploid fetuses, 12 discordant for trisomy 21 and five discordant for trisomy 18. In all 323 euploid cases with a result, the risk score for each trisomy was < 1:10 000. In 11 of the 12 cases with trisomy 21 and in the five with trisomy 18, the cfDNA test gave a high-risk result, but in one case of trisomy 21, the score was < 1:10 000. CONCLUSION: In twin pregnancies screening by cfDNA testing is feasible, but the failure rate is higher and detection rate may be lower than in singletons.

The first 3,000 Non-Invasive Prenatal Tests (NIPT) with the Harmony test in Belgium and the Netherlands.

As the classical first trimester Down syndrome screening (FTS, combination test) has a false-negative rate of 20-25% and > 95% of the abnormal FTS results are false-positive, we evaluated the new Non-Invasive Prenatal Test (NIPT) in Belgium and the Netherlands. The study population consisted of 3000 consecutive pregnancies in Belgium and the Netherlands in which NIPT was performed using the Harmony test. In 57 (1.9%) of the 3000 pregnancies an abnormal NIPT result was found. This included 51 fetuses with trisomy 21, 4 fetuses with trisomy 18 and 2 fetuses with trisomy 13. In 47 of the 57 the NIPT result was confirmed by genetic testing of material obtained by amniocentesis or chorionic biopsy, and no false-positive results were recorded. The false-negative rate as determined on more than 2000 women that had delivered at the time of reporting was low, and so far only 2 false-negative results were reported (one trisomy 18 and one trisomy 21). The failure rate where no NIPT result could be obtained after repeated sampling was 0.90%. In this large clinical series, NIPT using the Harmony test proves to be a very reliable prenatal test to detect fetal trisomies 21, 18 and 13 in maternal blood in Belgium and the Netherlands.

Genetic factors in non-syndromic congenital heart malformations.

The genetic defect in most patients with non-syndromic congenital heart malformations (CHM) is unknown, although more than 40 different genes have already been implicated. Only a minority of CHM seems to be due to monogenetic mutations, and the majority occurs sporadically. The multifactorial inheritance hypothesis of common diseases suggesting that the cumulative effect of multiple genetic and environmental risk factors leads to disease, might also apply for CHM. We review here the monogenic disease genes with high-penetrance mutations, susceptibility genes with reduced-penetrance mutations, and somatic mutations implicated in non-syndromic CHM.

Sex-linked deafness.

Many human syndromes associated with hearing loss are caused by disease genes located on the X chromosome, but few X-linked loci for non-syndromic hearing loss have been reported. Surprisingly, a Y-linked locus has been identified, representing one of the only disease loci on the Y chromosome. This study reviews the different sex-linked genes and loci on the X- and Y chromosome leading to syndromic and especially non-syndromic hearing loss.

The load/capacity ratio affects the sit-to-stand movement strategy.

BACKGROUND: In this study the effect of a changed load/capacity ratio on sit-to-stand performance and on the underlying net joint moments was investigated. In subjects with muscle weakness the load/capacity ratio is increased due to reduced muscle capacity. In the current study this ratio was manipulated by changing the load. This approach allowed studying the isolated effect of an increased load/capacity ratio on sit-to-stand strategy. METHODS: Ten healthy women performed sit-to-stand movements under four load conditions. The load/capacity ratio was manipulated by adding 0%, 15%, 30% and 45% of the body mass to a weight vest. To determine changes in sit-to-stand strategy flexion of the trunk and temporal characteristics were assessed. Joint moments at ankle, knee and hip joints and activation patterns of major leg muscles were determined from the kinematics and kinetics. FINDINGS: Increasing the extra load from 30% to 45% changed the sit-to-stand performance. In the 45% condition maximal trunk flexion was increased and movement time significantly elongated. The strategy change was associated with a disproportionate increase of the net hip extension moment and a delayed peak of the net knee extension moment. INTERPRETATION: This study shows that experimentally observed changes in sit-to-stand strategy can be attributed to an increase in the load/capacity ratio. For treatment purposes this implies that increasing muscle strength, reducing body mass or a combination of these could be a suitable approach to improve sit-to-stand performance. The experimental model applied will be useful to study the isolated effect of the load/capacity ratio.

Mitochondrial deafness.

Non-syndromic deafness can be caused by mutations in both nuclear and mitochondrial genes. More than 50 nuclear genes have been shown to be involved in non-syndromic hearing loss, but mutations in mitochondrial DNA (mtDNA) might also cause hearing impairment. As mitochondria are responsible for oxidative phosphorylation, the primary energy-producing system in all eukaryotic cells, mitochondrial dysfunction has pleiotropic effects. Many mutations in mtDNA can lead to multisystem disorders, such as Kearns-Sayre syndrome, NARP, MELAS, or MERRF syndromes, the presentation of which may include hearing loss. A more specific association of mitochondrially inherited deafness and diabetes known as MIDD syndrome can be caused by a limited number of specific mitochondrial mutations. In addition, several rare mutations in the mitochondrial MTTS1 and MTRNR1 genes have been found to be responsible for non-syndromic hearing loss. The most frequent form of non-syndromic deafness is presbyacusis, affecting more than 50% of the elderly. This age-related hearing loss is a paradigm for multifactorial inheritance, involving a multitude of inherited and acquired mutations in the nuclear and mitochondrial genomes, each with a low penetrance, in complex interplay with environmental factors, such as ototoxic medication, that accumulate with age. This study reviews the different mitochondrial mutations, leading to syndromic and especially non-syndromic deafness.

A novel susceptibility locus for Hirschsprung's disease maps to 4q31.3-q32.3.

We report on a multigenerational family with isolated Hirschsprung's disease (HSCR). Five patients were affected by either short segment or long segment HSCR. The family consists of two main branches: one with four patients (three siblings and one maternal uncle) and one with one patient. Analysis of the RET gene, the major gene involved in HSCR susceptibility, revealed neither linkage nor mutations. A genome wide linkage analysis was performed, revealing suggestive linkage to a region on 4q31-q32 with a maximum parametric multipoint LOD score of 2.7. Furthermore, non-parametric linkage (NPL) analysis of the genome wide scan data revealed a NPL score of 2.54 (p = 0.003) for the same region on chromosome 4q (D4S413-D4S3351). The minimum linkage interval spans a region of 11.7 cM (12.2 Mb). No genes within this chromosomal interval have previously been implicated in HSCR. Considering the low penetrance of disease in this family, the 4q locus may be necessary but not sufficient to cause HSCR in the absence of modifying loci elsewhere in the genome. Our results suggest the existence of a new susceptibility locus for HSCR at 4q31.3-q32.3.

Non-syndromic, autosomal-recessive deafness.

Non-syndromic deafness is a paradigm of genetic heterogeneity with 85 loci and 39 nuclear disease genes reported so far. Autosomal-recessive genes are responsible for about 80% of the cases of hereditary non-syndromic deafness of pre-lingual onset with 23 different genes identified to date. In the present article, we review these 23 genes, their function, and their contribution to genetic deafness in different populations. The wide range of functions of these DFNB genes reflects the heterogeneity of the genes involved in hearing and hearing loss. Several of these genes are involved in both recessive and dominant deafness, or in both non-syndromic and syndromic deafness. Mutations in the GJB2 gene encoding connexin 26 are responsible for as much as 50% of pre-lingual, recessive deafness. By contrast, mutations in most of the other DFNB genes have so far been detected in only a small number of families, and their contribution to deafness on a population scale might therefore be limited. Identification of all genes involved in hereditary hearing loss will help in our understanding of the basic mechanisms underlying normal hearing, in early diagnosis and therapy.

The phenotypic spectrum in patients with arginine to cysteine mutations in the COL2A1 gene.

BACKGROUND: The majority of COL2A1 missense mutations are substitutions of obligatory glycine residues in the triple helical domain. Only a few non-glycine missense mutations have been reported and among these, the arginine to cysteine substitutions predominate. OBJECTIVE: To investigate in more detail the phenotype resulting from arginine to cysteine mutations in the COL2A1 gene. METHODS: The clinical and radiographic phenotype of all patients in whom an arginine to cysteine mutation in the COL2A1 gene was identified in our laboratory, was studied and correlated with the abnormal genotype. The COL2A1 genotyping involved DHPLC analysis with subsequent sequencing of the abnormal fragments. RESULTS: Six different mutations (R75C, R365C, R519C, R704C, R789C, R1076C) were found in 11 unrelated probands. Each mutation resulted in a rather constant and site-specific phenotype, but a perinatally lethal disorder was never observed. Spondyloarthropathy with normal stature and no ocular involvement were features of patients with the R75C, R519C, or R1076C mutation. Short third and/or fourth toes was a distinguishing feature of the R75C mutation and brachydactyly with enlarged finger joints a key feature of the R1076C substitution. Stickler dysplasia with brachydactyly was observed in patients with the R704C mutation. The R365C and R789C mutations resulted in classic Stickler dysplasia and spondyloepiphyseal dysplasia congenita (SEDC), respectively. CONCLUSIONS: Arginine to cysteine mutations are rather infrequent COL2A1 mutations which cause a spectrum of phenotypes including classic SEDC and Stickler dysplasia, but also some unusual entities that have not yet been recognised and described as type II collagenopathies.

Mutations in FLNB cause boomerang dysplasia.

Boomerang dysplasia (BD) is a perinatal lethal osteochondrodysplasia, characterised by absence or underossification of the limb bones and vertebrae. The BD phenotype is similar to a group of disorders including atelosteogenesis I, atelosteogenesis III, and dominantly inherited Larsen syndrome that we have recently shown to be associated with mutations in FLNB, the gene encoding the actin binding cytoskeletal protein, filamin B. We report the identification of mutations in FLNB in two unrelated individuals with boomerang dysplasia. The resultant substitutions, L171R and S235P, lie within the calponin homology 2 region of the actin binding domain of filamin B and occur at sites that are evolutionarily well conserved. These findings expand the phenotypic spectrum resulting from mutations in FLNB and underline the central role this protein plays during skeletogenesis in humans.

Homozygosity mapping of a gene for arterial tortuosity syndrome to chromosome 20q13.

BACKGROUND: Arterial tortuosity syndrome (ATS) is an uncommon connective tissue disorder of unknown aetiology. The most prominent feature is tortuosity of the large arteries, but lengthening, stenosis, and aneurysm formation are also frequent. METHODS: We performed a genomewide screen by homozygosity mapping of three consanguineous multiplex families, two from Morocco, and one from Italy, which included 11 ATS patients. The two families from Morocco may possibly have a common ancestor. RESULTS: We mapped the ATS gene to chromosome 20q13. Recombinations within an extended haplotype of 11 microsatellite markers localised the ATS gene between markers D20S836 and D20S109, an interval of 9.5 cM. CONCLUSIONS: Cloning and completing functional and structural analysis of the ATS gene may provide new insights into the molecular mechanisms of elastogenesis.

Spatial and temporal heterogeneity of superficial muscle strain during in situ fixed-end contractions.

Numerical models of contracting muscle offer a powerful tool to study local mechanical load. For validation of these models, the spatial and temporal distribution of strain was quantified in fixed-end contracting rat tibialis anterior muscle in situ at optimal muscle length (L(o)) and at 120 degrees plantar flexion as well as at 125 and 33Hz stimulation frequency. We studied the hypothesis that after termination of stimulation in situ muscle segments near the motor endplates elongate while segments away from the endplates shorten. We show that both spatial and temporal inhomogeneities in muscle deformation occurred during contraction. Muscle plateau shortening strain equalled 4.1%. Maximal plateau shortening of a muscle segment was much larger (9.6%) and occurred distally (at 0.26 of the scaled length of the muscle). Manipulating torque levels by decreasing the stimulation frequency at the same muscle length induced a decrease in torque ( approximately 20%) with a smaller effect on the level and no effect on the pattern of muscle deformation. During relaxation, distal segments actively shortened at the expense of proximal muscle segments, which elongated. The segments undergoing lengthening were nearer to motor endplates than segments undergoing shortening. In conclusion, the present study provides experimental data on magnitude of contraction-induced deformation needed for validation of numerical models. Local muscle deformation is heterogeneous both temporally and spatially and may be related to proximity to the motor endplates.

Genetic epidemiology of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a late onset, rapidly progressive and ultimately fatal neurological disorder, caused by the loss of motor neurons in the brain and spinal cord. Familial aggregation of ALS, with an age-dependent but high penetrance, is a major risk factor for ALS. Familial ALS (FALS) is clinically and genetically heterogeneous. Three genes and linkage to four additional gene loci have been identified so far and may either predominantly lead to ALS (ALSI-ALS6) or cause multisystem neurodegeneration with ALS as an occasional symptom (tauopathies, ALS-dementia complex). This review presents a tentative classification of the "major" ALS genes and ALS "susceptibility" genes, that may act as susceptibility factors for neurodegeneration in interaction with other genetic or environmental risk factors. Considering that mutations in ALS genes explain approximately 10% of familial as well as sporadic ALS, and most remaining cases of the discase are thought to result form the interaction of several genes and environmental factors, ALS is a paradigm for multifactorial discases.

Poor outcome in Down syndrome fetuses with cardiac anomalies or growth retardation.

The outcome of Down syndrome fetuses presenting with sonographic abnormalities in the second or third trimester is unclear. Therefore, we studied 55 pregnancies referred because of sonographically suspected fetal structural anomalies or growth retardation due to trisomy 21. A detailed ultrasound scan was performed in all cases to delineate the structural anomalies. Congenital heart malformations (CHMs) were diagnosed pre- and postnatally in 29 out of 55 Down fetuses (53%), with complete or incomplete atrioventricular septal defects (AVSDs) and ventricular septal defects (VSDs) being the most frequent anomalies. The most frequent noncardiac findings were a short femur (45%) and a small-for-gestational age (SGA) fetus (27%). Termination of pregnancy was carried out in 25 out of 55 pregnancies (45%). Of the 30 continued pregnancies, 10 ended with intrauterine death. The remaining 20 pregnancies resulted in the delivery of a live-born infant whose prognosis was poor, with a 1-year survival of only 60%. Combining intrauterine death and death in the first year indicated that the overall survival rate was only 40%. Fatal outcome was noted in 68% (13/19) in the presence of CHM, in 83% (10/12) in SGA fetuses, in 86% (6/7) in combined CHM and SGA, but only in 17% (1/6) in the absence of CHM and SGA. This study indictes that second- and third-trimester in utero diagnosis of Down syndrome has a poor outcome when associated with CHM and/or SGA. This is important in the genetic counseling of the parents.

Prenatal diagnosis and confirmation of the acrofacial dysostosis syndrome type Rodriguez.

The group of acrofacial dysostosis (AFD) syndromes is very heterogeneous and contains many different entities. In 1990, Rodriguez et al. [1990: Am J Med Genet 35:484-489] described a new type of AFD characterized by severe mandibular hypoplasia, phocomelia and oligodactyly of the upper limbs, absence of fibulae, microtia, cleft palate, internal organ anomalies including arrhinencephaly and abnormal lung lobulation, and early lethality. We describe another case of AFD type Rodriguez, identified by prenatal ultrasonography at 25 weeks of gestation.

Identification of a 52 kb deletion downstream of the SOST gene in patients with van Buchem disease.

Van Buchem disease is an autosomal recessive skeletal dysplasia characterised by generalised bone overgrowth, predominantly in the skull and mandible. Clinical complications including facial nerve palsy, optic atrophy, and impaired hearing occur in most patients. These features are very similar to those of sclerosteosis and the two conditions are only differentiated by the hand malformations and the tall stature appearing in sclerosteosis. Using an extended Dutch inbred van Buchem family and two inbred sclerosteosis families, we mapped both disease genes to the same region on chromosome 17q12-q21, supporting the hypothesis that van Buchem disease and sclerosteosis are caused by mutations in the same gene. In a previous study, we positionally cloned a novel gene, called SOST, from the linkage interval and identified three different, homozygous mutations in the SOST gene in sclerosteosis patients leading to loss of function of the underlying protein. The present study focuses on the identification of a 52 kb deletion in all patients from the van Buchem family. The deletion, which results from a homologous recombination between Alu sequences, starts approximately 35 kb downstream of the SOST gene. Since no evidence was found for the presence of a gene within the deleted region, we hypothesise that the presence of the deletion leads to a down regulation of the transcription of the SOST gene by a cis regulatory action or a position effect.

Hereditary multiple exostoses: from genetics to clinical syndrome and complications.

OBJECTIVE: To give an overview of genetic, clinical and radiological aspects in two families over four generations with known hereditary multiple exostoses (HME). METHODS AND MATERIAL: After linkage analysis in both families to localize the defective gene, mutation analysis was performed in these genes to identify the underlying mutation. In the 31 affected individuals, location, number and morphology and evolution of exostosis, evolution of remodeling defects at the metaphysis, and the extent of possible complications were evaluated on clinical and imaging (plain radiography, computed tomography (CT), and magnetic resonance imaging (MRI)) data over a lifetime period. RESULTS AND CONCLUSIONS: Both families demonstrate the gene defect in the same EXT-2 gene locus on chromosome 11p. Exostoses are preferentially located in the lower extremity (hip, knee and lower leg), humerus, and forearm. Any other bone may be involved, except for the calvaria of the skull and the mandible. Exostoses are rather sessile than pedunculated. Exostosis is rarely present at birth but develops gradually and may persist to grow slowly after closure of the growth plates. Preferential expression of the remodeling defect was seen in the hip, distal femur (trumpet-shaped metaphysis) and forearm (shortening of the ulna with secondary bowing of the radius and development of a pseudo-Madelung deformity). These radiological manifestations start at the age of 4-5 years and become more obvious as the enchondral bone formation progresses with age. Reported complications in these families consist of local entrapment phenomenons (vessel, tendon, nerve), frictional bursitis, and sarcomatous transformation. MRI was able to suggest these complications and is the imaging technique of choice in the evaluation of symptomatic exostoses.