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Missense and truncating variants in the X-chromosome-linked CLCN4 gene, resulting in reduced or complete loss-of-function (LOF) of the encoded chloride/proton exchanger ClC-4, were recently demonstrated to cause a neurocognitive phenotype in both males and females. Through international clinical matchmaking and interrogation of public variant databases we assembled a database of 90 rare CLCN4 missense variants in 90 families: 41 unique and 18 recurrent variants in 49 families. For 43 families, including 22 males and 33 females, we collated detailed clinical and segregation data. To confirm causality of variants and to obtain insight into disease mechanisms, we investigated the effect on electrophysiological properties of 59 of the variants in Xenopus oocytes using extended voltage and pH ranges. Detailed analyses revealed new pathophysiological mechanisms: 25% (15/59) of variants demonstrated LOF, characterized by a “shift” of the voltage-dependent activation to more positive voltages, and nine variants resulted in a toxic gain-of-function, associated with a disrupted gate allowing inward transport at negative voltages. Functional results were not always in line with in silico pathogenicity scores, highlighting the complexity of pathogenicity assessment for accurate genetic counselling. The complex neurocognitive and psychiatric manifestations of this condition, and hitherto under-recognized impacts on growth, gastrointestinal function, and motor control are discussed. Including published cases, we summarize features in 122 individuals from 67 families with CLCN4-related neurodevelopmental condition and suggest future research directions with the aim of improving the integrated care for individuals with this diagnosis.

Purpose: Neurodevelopmental disorders (NDDs) encompass a spectrum of genetically heterogeneous disorders with features that commonly include developmental delay, intellectual disability, and autism spectrum disorders. We sought to delineate the molecular and phenotypic spectrum of a novel neurodevelopmental disorder caused by variants in the GNAI1 gene. Methods: Through large cohort trio-based exome sequencing and international data-sharing, we identified 24 unrelated individuals with NDD phenotypes and a variant in GNAI1, which encodes the inhibitory Gαi1 subunit of heterotrimeric G-proteins. We collected detailed genotype and phenotype information for each affected individual. Results: We identified 16 unique variants in GNAI1 in 24 affected individuals; 23 occurred de novo and one was inherited from a mosaic parent. Most affected individuals have a severe neurodevelopmental disorder. Core features include global developmental delay, intellectual disability, hypotonia, and epilepsy. Conclusion: This collaboration establishes GNAI1 variants as a cause of NDDs. GNAI1-related NDD is most often characterized by severe to profound delays, hypotonia, epilepsy that ranges from self-limiting to intractable, behavior problems, and variable mild dysmorphic features.

KCNN2 encodes the small conductance calcium-activated potassium channel 2 (SK2). Rodent models with spontaneous Kcnn2 mutations show abnormal gait and locomotor activity, tremor and memory deficits, but human disorders related to KCNN2 variants are largely unknown. Using exome sequencing, we identified a de novo KCNN2 frameshift deletion in a patient with learning disabilities, cerebellar ataxia and white matter abnormalities on brain MRI. This discovery prompted us to collect data from nine additional patients with de novo KCNN2 variants (one nonsense, one splice site, six missense variants and one in-frame deletion) and one family with a missense variant inherited from the affected mother. We investigated the functional impact of six selected variants on SK2 channel function using the patch-clamp technique. All variants tested but one, which was reclassified to uncertain significance, led to a loss-of-function of SK2 channels. Patients with KCNN2 variants had motor and language developmental delay, intellectual disability often associated with early-onset movement disorders comprising cerebellar ataxia and/or extrapyramidal symptoms. Altogether, our findings provide evidence that heterozygous variants, likely causing a haploinsufficiency of the KCNN2 gene, lead to novel autosomal dominant neurodevelopmental movement disorders mirroring phenotypes previously described in rodents.

Item does not contain fulltext BACKGROUND: Cohesinopathies are rare neurodevelopmental disorders arising from a dysfunction in the cohesin pathway, which enables chromosome segregation and regulates gene transcription. So far, eight genes from this pathway have been reported in human disease. STAG1 belongs to the STAG subunit of the core cohesin complex, along with five other subunits. This work aimed to identify the phenotype ascribed to STAG1 mutations. METHODS: Among patients referred for intellectual disability (ID) in genetics departments worldwide, array-comparative genomic hybridisation (CGH), gene panel, whole-exome sequencing or whole-genome sequencing were performed following the local diagnostic standards. RESULTS: A mutation in STAG1 was identified in 17 individuals from 16 families, 9 males and 8 females aged 2-33 years. Four individuals harboured a small microdeletion encompassing STAG1; three individuals from two families had an intragenic STAG1 deletion. Six deletions were identified by array-CGH, one by whole-exome sequencing. Whole-exome sequencing found de novo heterozygous missense or frameshift STAG1 variants in eight patients, a panel of genes involved in ID identified a missense and a frameshift variant in two individuals. The 17 patients shared common facial features, with wide mouth and deep-set eyes. Four individuals had mild microcephaly, seven had epilepsy. CONCLUSIONS: We report an international series of 17 individuals from 16 families presenting with syndromic unspecific ID that could be attributed to a STAG1 deletion or point mutation. This first series reporting the phenotype ascribed to mutation in STAG1 highlights the importance of data sharing in the field of rare disorders.

Deletions in the middle portion of 11q are not as well described in the literature as terminal 11q deletions that result in Jacobsen syndrome. One confounding factor in the older literature is that the G-banding pattern of 11q13q21 is very similar to 11q21q23. The advent of fluorescence in situ hybridization and later microarray technologies have allowed for a better resolution of many of these deletions, but genotype-phenotype correlations are still difficult since these deletions are rare events. We present five individuals who presented with developmental delays with de novo 11q22.2q23.3 deletions. Deletions were observed by standard G-banded chromosome analysis with clarification of breakpoints and gene content by SNP microarray analysis. Of note, all individuals had identical distal breakpoints. All deletions include SDHD, which is implicated in hereditary paraganglioma/pheochromocytoma, for which the patients will need to be monitored in adulthood. In spite of the large deletions of 8.6 Mb (Patients 1 and 3), 13.98 Mb (Patient 2), and 12.6 Mb (Patients 4 and 5) all patients show somewhat mild intellectual disability and dysmorphism. © 2015 Wiley Periodicals, Inc.

Two revolutionary advances in molecular biology have enabled scanning of the entire human genome for genetic variation: 1. Array-comparative genomic hybridization-microarray analysis (aCGH, CMA, microarray analysis, referred to henceforth as aCGH) that identifies altered DNA dosage. 2. Whole genome or exome sequencing (WGS or WES) that identifies nucleotide sequence changes.

Discussion of DNA diagnosis using targeted DNA or whole exome sequencine (WES) is necessarily truncated because of two reasons.

Rapid incorporation of microarray analysis/aCGH for studies of children with developmental disabilities and its endorsement as a first-tier test for these children [1] has yielded a vast number of subtle chromosome changes that were unseen by routine karyotyping.

The following summaries include examples of human chromosome heteromorphisms that have been compiled by us, contributed by individual investigators specifically for this volume, or have been reprinted with permission from various published sources. Individual contributions have been identified by a numerical designation prefixed with the letter “c”, after the name of the contributor, i.e. Lauren Jenkins (c2).

Patients with KCNK9 imprinting syndrome demonstrate congenital hypotonia, variable cleft palate, normal MRIs and EEGs, delayed development, and feeding problems. Associated facial dysmorphic features include dolichocephaly with bitemporal narrowing, short philtrum, tented upper lip, palatal abnormalities, and small mandible. This disorder maps to chromosomal region 8q24, and it is caused by a specific missense mutation 770G>A in exon 2, replacing glycine at position 236 by arginine (G236R) in the maternal copy of KCNK9 within this locus. KCNK9 (also called TASK3) encodes a member of the two pore- domain potassium channel (K2P) subfamily. This gene is normally imprinted with paternal silencing, thus a mutation in the maternal copy of the gene will result in disease, whereas a mutation in the paternal copy will have no effect. Exome sequencing in four new patients with developmental delay and central hypotonia revealed de novo G236R mutations. Older members of a previously reported Arab-Israeli family have intellectual disability of variable severity, persistent feeding difficulties in infancy with dysphagia of liquids and dysphonia with a muffled voice in early adulthood, generalized hypotonia, weakness of proximal muscles, elongated face with narrow bitemporal diameter, and reduced facial movements. We describe the clinical features in four recently recognized younger patients and compare them with those found in members of the originally reported Arab-Israeli family and suggest this may be a treatable disorder. © 2016 Wiley Periodicals, Inc.

A dilemma of penetrance parallels the uncertainty principle as array analyses uncover a disturbing continuity of human molecular variation. Among mechanisms for altered penetrance is maternal genetic effect, hypothesized to explain mother-offspring discordance from 12/13 translocation [Wilson et al., 1991] and important to consider when confronting identical genetic changes in normal mothers and their abnormal children. A boy with developmental delays, autistic features, minimal dysmorphology, and maternally inherited 17p13.3 microduplication could exemplify maternal genetic effect, as could a boy with altered brain pattern and maternally inherited Sonic Hedgehog mutation reported by Schell-Apacik et al. [2009]. Maternal genetic effects, characterized in fly, fish, and mouse, are important to consider in human DNA analysis so that identical findings in mother and child are not routinely dismissed as benign variations. Suspect families can define relevant maternal-zygote transition (MZT) genes, provide assays for oocyte function and disruption, and shift anomaly emphasis from pattern recognition to pattern restoration. © 2012 Wiley Periodicals, Inc.

Using whole-exome sequencing, we identified seven unrelated individuals with global developmental delay, hypotonia, dysmorphic facial features, and an increased frequency of short stature, ataxia, and autism with de novo heterozygous frameshift, nonsense, splice, and missense variants in the Early B-cell Transcription Factor Family Member 3 (EBF3) gene. EBF3 is a member of the collier/olfactory-1/early B-cell factor (COE) family of proteins, which are required for central nervous system (CNS) development. COE proteins are highly evolutionarily conserved and regulate neuronal specification, migration, axon guidance, and dendritogenesis during development and are essential for maintaining neuronal identity in adult neurons. Haploinsufficiency of EBF3 may affect brain development and function, resulting in developmental delay, intellectual disability, and behavioral differences observed in individuals with a deleterious variant in EBF3.

Autosomal dominant (AD) inheritance is determined by the presence of one abnormal gene on one of the autosome (chromosome 1–22).

Chromosome deletions involving 1p36 are the most common known terminal rearrangements occurring at a frequency of ∼1 in 5,000 live births. In contrast, duplications of the same region have been reported rarely. We describe a familial rearrangement der(1)del(1)(p36.33)dup(1)(p36.33p36.22) identified in a mother, daughter, and son. These individuals help define a syndrome with variable mental disability, attention deficit-hyperactivity disorder, and a distinctive facial appearance with wide palpebral fissures, broad nasal root, macrostomia, ear malformations, and prominent incisors. Based on our results we suggest that the complex rearrangement seen in our family could be the result of the breakage-fusion-bridge (BFB) cycles model of formation. © 2005 Wiley-Liss, Inc.

A 10-month old female is described with inv dup(8)(p12p23) who had macrocephaly with subtle changes in facial appearance and no structural birth defects. Her findings, together with those of 37 reported cases with inv dup (8), define a syndrome that emphasizes the importance of genes on the 8p region for brain development.

Roberts syndrome (RS) is a rare autosomal recessive disorder characterized by heterogeneous clinical features, the most notable being tetraphocomelia, cleft lip, and cleft palate. Cells derived from most RS patients exhibit abnormal cytogenetic and cellular phenotypes that include the premature separation of para- and pericentromeric heterochromatin visible on C-banded metaphase chromosomes, a phenomenon referred to as heterochromatic splaying. Previously, it was shown that these abnormal phenotypes can be complemented following somatic cell hybridization between RS cells and control cells. In the current study, a permanent cell line was established from a new RS patient with a more severe phenotype than represented by previously established cells in culture. With a newly developed assay designed to facilitate rapid evaluation of in vitro complementation, we assigned this new patient to the same genetic complementation group defined by other, less severely affected patients. The results demonstrate that a single complementation group defines RS patients with heterochromatic splaying regardless of clinical severity.

Coordinated development of heart and limbs is suggested by a review of human abortus, chromosomal, and teratogenic syndromes, and characterized by an analysis of Mendelian disorders that affect the limbs, heart, or both (672, 202, or 107, respectively). Mendelian syndromes with altered limb patterns often include cardiac anomalies, as shown by limb duplications (34%), deficiencies (30%), hypoplasias (23%), or dysplasias (9.3%). Syndromes with particular cardiac anomalies, illustrated by VSD (85%) or ASD (90%), frequently include limb defects. Positional correlations of anterior (preaxial/conotruncal), posterior (postaxial/atrial), or lateral (mirror hand/atrial isometry) heart/limb anomalies are consistent with the existence of a cardiomelic developmental field. Vertebrate comparisons suggest an early D-V limb-heart gradient, influenced by the neural crest, with distal limb segments (80% of syndromic defects) at its dorsal extreme. The proposed cardiomelic field relates the genetic heterogeneity of disorders such as Holt-Oram syndrome to a cascade of molecules, including the brachyury, sonic hedgehog, bone morphogenetic protein, retinoic acid receptor, and transforming growth factor-beta families.

Mammalian genes encoding a 35-kDa peroxisomal membrane protein (PMP35, peroxisome assembly factor-1) are compared using the polymerase chain reaction and DNA sequencing. DNA sequencing of the 915 bp of the PMP35 coding regions was in complete agreement with previously published rat data and showed 36 and 133 nucleotide substitutions, respectively, in mouse and man. The 12 and 35 respective amino acid changes encoded by these nucleotide substitutions are clustered and compatible with putative membrane-spanning regions. Rat/human and rat/mouse comparisons yield silent mutation rates of 0.33 and 0.21% per site per million years and replacement mutation rates of 0.082 and 0.076%; transitions account for 67% (human/mouse) and 83% (rat/mouse) of nucleotide replacements among PMP35 genes. PMP35 gene expression in mouse tissues as measured by reverse transcriptase-PCR was responsive to clofibrate and disproportionately high in neural tissue.

Compelling evidence for genomic imprinting as a pathogenetic mechanism in humans mandates re-evaluation of every genetic or multifactorial disease for parent-of-origin effects. In an expanding list of malformation syndromes, cancers, growth abnormalities, and chromosomal disorders, phenotypes may be determined by source rather than content of transmitted DNA. A multidisciplinary conference held on April 13–14, 1992, reviewed the substantial impact of genomic imprinting in mouse development and discussed in role in human pregnancy, childhood cancers, chromosomal translocations, X-inactivations, and several disorders associated with mental retardation. Topics for future research include the mechanism, timing, reversibility, and homology of the imprinting process. © 1993 Wiley-Liss, Inc.

An 18-year-old female with mild mental disability (global IQ 69), febrile seizures with subsequent myoclonic/grand mal epilepsy, and subtle morphologic changes is described with del 5(q14.3q21.3) by karyotype and minimal DNA deletion of 21.08 Mb by array comparative genomic hybridization microarray analysis (arr chr5:83,592,798-104,671,993 X1) that encompasses at least 50 genes. Included in the deletion interval is the MEF2C gene that usually causes severe mental disability when haploinsufficient, illustrating the complexity of clinic-cytogenetic correlation even with defined segmental aneuploidy. Interaction of MEF2C with the deleted febrile seizure (FEB4) and juveline myoclonic epilepsy (EJM4) loci plus the G-protein receptor (GPR98/MASS1/Usher syndrome) gene may moderate the phenotype, perhaps through common regulation by calcium.

Genetic mechanisms for selective mutagenesis in female mammals might include alterations of genomic imprinting, maternally derived molecules, mitochondrial DNA or sex chromosome loci. None of these mechanisms provides an obvious explanation for the higher mutational rates observed for certain mutagens in mouse female pronuclei, but the association of DNA methylation with maternal genomic imprinting is an enticing avenue for research. Further characterization of the extent and homology of genomic imprinting among mammals is required before its relevance to mutagenesis can be determined. The existence of maternal effect mutations in mammals merits evaluation but is not yet proven. The relevance of mitochondrial DNA to female-specific mutagenesis will be greatest in multi-generational studies.

A survey of Mendelian Inheritance in Man emphasizes the large Mendelian contribution to human dysmorphogenesis and contrasts single gene conditions with chromosomal disorders. There were 1761 conditions that involved altered morphogenesis (49% of disease entries), including 1040 multiple defect syndromes and 721 inherited single birth defects. Premature death (36-57%), mental retardation (20-59%), and growth retardation (37-59%) are more frequent in autosomal recessive or X-linked syndromes, while predisposition to tumorigenesis was more common in dominant (16%) than recessive (3.4%) syndromes. Comparison of the Mendelian conditions with 100 chromosomal disorders showed a strikingly similar spectrum of malformation, with skeletal, craniofacial, eye, epidermal, and neuromuscular systems being most frequently affected. Chromosomal syndromes average 10.6 systems affected per disorder, in contrast to 3.55 for Mendelian syndromes, and pleiotropy does correlate weakly with aneuploid segment length. Genomic understanding of these relationships is still primitive, with 74 of 1609 (4.6%) autosomal conditions and 43 of 152 (29%) X-linked conditions mapped to specific chromosomal regions. The societal toll of human dysmorphogenesis and the evident progress with X-linked disorders provide a powerful rationale for the Human Genome Project.

Peroxisomal membrane proteins (PMPs) from the Swiss-Webster mouse are analyzed and compared to those of rats and humans using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. A purification procedure for fresh mouse, rat, or human biopsy liver which enriches peroxisomal/mitochondrial marker enzyme ratios over 100-fold is characterized. When analyzed by SDS-PAGE, membranes of purified liver peroxisomes are shown to contain the same complement of 145-, 70-, 55-, 36-, and 22-kDa PMPs in rats, mice, and humans. A rabbit polyclonal antibody raised against mouse peroxisomal membranes demonstrates immunoreactivity to 145- and 70-kDa proteins in fresh liver homogenates from all three species and in control or Zellweger syndrome fibroblasts from humans. Human autopsy or placental tissues which were refrigerated before analysis exhibited 105-, 55-, and 36-kDa peptides which may be derived from the 145- and 70-kDa peptides. Such conversions, if related to degradation, may explain difficulties in purifying peroxisomes from human autopsy specimens. Variable amounts of the 55-kDa peptide also occurred in mouse adrenal and lung, and the conversion of higher to lower molecular weight PMPs could not be demonstrated by in vitro incubation of mouse liver. Further definition of the structure and variability of mammalian PMPs should be helpful in understanding polyenzymopathies such as Zellweger syndrome.

Studies in "lower" organisms have identified a set of homologous sequences expressed in oocytes and early embryos that is critical for pattern formation. Mutations in such genes may exhibit maternal effect--they cause abnormalities in the fetus only when present in the mother. We report on a mother and child with identical, apparently balanced translocations having the breakpoints 12q13.1 and 13p13. The fetus had multiple anomalies including bilateral trilobar lungs, complex heart defect, malrotation of the gut, and asplenia, while the mother was entirely normal. Several hypotheses are advanced to explain this variable expression including transection of a gene with maternal effect--lateral asymmetry in the fetus is influenced by the maternal genotype. This explanation would account for the higher transmission of congenital heart disease to offspring by affected females noted in several studies. The human counterparts of 2 loci (int-1 and HOX 3) involved in Drosophila early pattern formation are located near the translocation breakpoint 12q13.1. If one of these genes is responsible for situs abnormality, then university of positional code (but not of embryologic mechanism) is suggested for higher metazoans.

Alternative explanations for aneuploid phenotypes are evaluated according to clinical and experimental predictions. The additive hypothesis views the diverse manifestations of aneuploidy as the simple sum of individual gene dosage effects. The interactive hypothesis views aneuploid characteristics as network properties of genes within and outside the aneuploid segment. The importance and historical context of this debate are emphasized, and the 2 hypotheses are contrasted in terms of their experimental predictions and explanatory power. In particular, the concepts of colinearity and continuity derived from single gene action are explicitly examined in human aneuploidy with the conclusion they are likely to be false assumptions.

A newborn female presented with costovertebral dysplasia (CVD), subtle facial anomalies, and neonatal respiratory distress. Her karyotype demonstrated a small supernumerary NOR-positive marker that was subsequently identified as del(22)(q11.2). This extra structurally abnormal chromosome was found by DNA microsatellite marker analyses to be derived from a paternal chromosome 22. The child has had severe growth and developmental delay along with pulmonary insufficiency and hypoxia but is presently stable at age 20 months. Findings in our patient correlate with similar observations in children with small markers derived from D/G and D/D translocations reported before banding technology was available. These reports and recent mapping results suggest that a pericentric gene family, distributed on one or more acrocentric chromosomes, may have played a role in the development of the human axial skeleton. Data from additional studies will be needed to confirm or refute this hypothesis.

A balanced Robertsonian translocation 45,XY,t(15q15q) was detected in a patient with mental retardation, microcephaly, and hypertonia. Deletion of the 15q11q13 region was unlikely based on fluorescence in situ hybridization studies that revealed hybridization of appropriate DNA probes to both arms of the Robertsonian chromosome. Inheritance of alleles from 13 highly polymorphic DNA markers on chromosome 15 showed paternal uniparental isodisomy. The clinical, cytogenetic, and molecular results are consistent with a diagnosis of Angelman syndrome.

Two families and 3 patients with dup(10p)/del(10q) syndrome segregating from a maternal pericentric inversion are described, including a stillborn female with Potter sequence and multicystic renal dysplasia. Comparison of 32 dup(10p) patients to 11 del(10)(q25) patients emphasized dolichocephaly, wide sutures, frontal bossing, micrognathia, and renal defects as distinguishing characteristics of the dup(10p) syndrome. The 3 new and 6 previously reported dup(10p)/del(10q) patients had several manifestations in common with the dup(10p) and del(10q) syndromes, but were more typical of dup(10p) syndromes, with respect to all 5 distinguishing characters. © 1993 Wiley-Liss, Inc.

A heteromorphism of the short arm of 16 (16p+) was discovered in 2 unrelated infants. By G banding, the euchromatic variant appears as a light and a medium dark band just distal to the centromere. This results in an increase of the short arm by about 1/3. The same variant was present in the normal father and the normal paternal grandmother in one family and mildly retarded mother in the 2nd family. The anomalies of the 2 infants are not similar and are apparently unrelated to the 16p + variant. Though the discovery of such euchromatic variants is highly significant for clinical diagnosis, their genetic significance and mode of origin remain to be elucidated.

FG syndrome is an X-linked recessive condition in which mental retardation is associated with congenital hypotonia, macrocephaly, characteristic face, and constipation. This syndrome was mapped by Zhu et al. [Cytogenet Cell Genet 1991;58:2091A] to Xq21.31-q22 by linkage analysis with a max lod score of 1.2 for the DXYS1X, DXS178, DXS101, and DXS94 loci and crossovers at DXS16 (Xp22.31) and DXS287 (Xq22.3). However, this mapping was only provisional and needed to be refined. In this paper, we report the results of a new linkage analysis performed on 10 families including that studied by Zhu et al. [1991]. Two-point analysis demonstrated linkage with DXS441 (Zmax = 3.39 at θ = 0.12) at Xq13. In addition, separate analysis of the lod scores obtained for the Xq13 markers suggested linkage exclusion for three families. Genetic heterogeneity was confirmed by analysis of the linkage results with the HOMOG program (max logL = 4.07, θ = 0, α = 0.65). Localization of one FG gene between DXS135 and DXS1066 was suggested by analysis of crossovers found in those three families which were assumed to be linked to Xq13 with a probability of 0.95 or more. This region could be reduced to the DXS135-DXS72 interval after combining our data with those from deletions previously described in males in the Xq13-q21 region. Am. J. Med. Genet. 73:87–90, 1997. © 1997 Wiley-Liss, Inc.

Alterations of human laterality range from situs reversal or consensus isometry to isolated anomalies of the cardiac, respiratory, gastrointestinal, skeletal, and central nervous systems. A mechanism for human situs determination has been derived from the general model of Brown and Wolpert (1990) , with steps involving A-P axis, D-V axis, midline, global situs, and local situs specification. Comparison with Drosophila segmentation is supported by maternal transmission of certain human situs defects and mutation of appropriate Drosophila gene homologues in the human Waardenburg and Greig syndromes. Anteroposterior gradients in expression of vertebrate homeotic genes may relate to a proposed hierarchy of regional laterality decisions. Early alterations in A-P or D-V axis polarity would produce situs reversal in 100 of individuals, as observed in pure situs inversus, homozygous inv mice or manipulated Xenopus embryos. Later alterations would permit random right-left decisions and account for heterotaxy in 50 of affected individuals, as observed in poly asplenia or homozygous iv mice. Randomisation of brain asymmetry could explain why situs but not brain laterality may be reversed in humans, with forebrain situs reversal or isometry leading to brain anomalies. Homologues of Drosophila genes regulating axis polarity, heart and gut development are attractive candidates in human laterality disorders, but none is uniquely localised to the 6q14-q21 or9q32-q34, 7q22, 10q21-22, 11q13 or 11q25, 12q13, 13qter, or Xq24-q27.1 chromosome regions highlighted by heterotaxic patients or mutant mice.

Gene segments encoding the 70 and 22-kDa peroxisomal membrane proteins (PMP) have been characterized in mice and compared with other peroxisomal proteins in terms of evolution and expression. The mouse PMP22 gene sequence predicts A 16 G and I 136 V substitutions that agree with those defined by cyanogen bromide cleavage analysis, providing additional evidence that this gene encodes the major 22-kDa membrane protein visualized by SDS-polyacrylamide electrophoresis. Mammalian PMP22 genes exhibit higher evolutionary rates (0.17% amino acid substitution per million years) than PMP35 (0.14%), PMP70 (0.07%), or catalase genes (0.13%). PMP70 gene regions are conserved throughout vertebrate phylas based on Southern analysis, while PMP22 sequences were only detected in rodents. Amino acid substitutions are clustered in both PMP22 and PMP70 genes, and their pattern supports membrane topologies derived from hydropathy profiles. Northern blot analysis identifies single mRNAs of 4.6 kb (PMP70), 1.4 kb (PMP22), and 2.3 kb (catalase) in several rodent tissues. Quantitative or competitive RT-PCR assays detected two- to three-fold greater numbers of catalase mRNA molecules relative to PMP mRNA molecules in brain, liver, and kidney; PMP22 and PMP35 mRNAs were two-fold more abundant than PMP70 mRNA in these tissues. Steady-state levels of PMP22 mRNA were highest in rodent liver kidney, spinal cord, and duodenum with low levels in colon, adrenal, thymus, and spleen. We conclude that PMP genes exhibit independent evolutionary rates and tissue regulation suggesting that they have unique roles in peroxisome biogenesis aod tissue differentiation.

We describe clinical and chromosomal findings in two patients with del(4q). Patient 1, with interstitial deletion (4)(q21.1q25), had craniofacial and skeletal anomalies and died at 8 months of hydrocephalus. Patient 2, with interstitial deletion (4)(q25q27), had craniofacial and skeletal anomalies with congenital hypotonia and developmental delay. These patients shared certain manifestations with other del(4q) patients but did not have Rieger anomaly. Clinical variability among patients with interstitial deletions of 4q may be related to variable expression, variable deletion, or imprinting of genes within the 4q region.

MASA syndrome is a recessive X-linked disorder characterized by mental retardation, adducted thumbs, shuffling gait, aphasia and, in some cases, hydrocephalus. Since it has been shown that X-linked hydrocephalus can be caused by mutations in L1CAM, a neuronal cell adhesion molecule, we performed an L1CAM mutation analysis in eight unrelated patients with MASA syndrome. Three different L1CAM mutations were identified: a deletion removing part of the open reading frame and two point mutations resulting in amino acid substitutions. L1CAM, therefore, harbours mutations leading to either MASA syndrome or HSAS, and might be frequently implicated in X-linked mental retardation with or without hydrocephalus.

An 11-year-old female with early feeding problems, mild motor delays, normal speech, subtle facial changes, social difficulties, anxiety and a diagnosis of Asperger disorder was found to have deletion of 10q26.3 by subtelomere fluorescent in situ hybridization (stF) analysis. Our patient and others with 10q26 aneuploidy add this region to 11 other autism susceptibility loci qualified by converging genome linkage/association, high resolution chromo - some, and mutation studies in our review. We summarize these loci and the current spectrum of terminal 10q deletion cases.

Progress relevant to human peroxisomal disorders over the past 3 years includes improved biochemical delineation of disease phenotypes and new insights into peroxisomal structure and biogenesis. Immunoblotting studies using antibodies to peroxisomal β-oxidation enzymes have defined mutations affecting each step of the pathway, some with clinical phenotypes as severe as disorders with global peroxisome deficiency. The latter disorders, typified by Zellweger syndrome, often lack matrix proteins but retain major membrane species of 150, 70, 35, and 22 kDa in empty peroxisomal “ghost” structures. The hypothesis that peroxisomal deficiency disorders result from altered targeting or import of peroxisomal matrix proteins has been strengthened by the demonstration of a carboxy terminal peroxisome-targeting signal which is distinct from amino terminal signals directing proteins to mitochondria. A mutation which mistargets alanine/glyoxylate aminotransferase from peroxisomes to mitochondria in primary hyperoxaluria provides a graphic example of these signals. The structural significance of membrane function is supported by the primacy of membrane assembly in normal ontogeny or regenerating liver. The coordinate control, targeting, and striking inducibility of peroxisomal proteins suggests a potential vehicle for gene and enzyme therapy.

To study the effects of peroxisome proliferators on fetal biogenesis, 400 mg/kg oral clofibrate was administered to pregnant mice beginning at d 6 of gestation. Maternal/fetal tissues from three separate experiments were harvested between gestational d 13 and 19 and maternal/fetal tissues were assayed for peroxisomal matrix, membrane-associated, and integral membrane proteins. By comparison to control pregnancies that received vehicle only, clofibrate produced a 4− to 5-fold increase in the levels of peroxisomal membrane protein 70, a 1.5− to 2-fold increase of dihydroxyacetone phosphate acyltransferase (DHAP-AT) sp act, and a 1.2− to 1.8-fold increase of catalase sp act in fetal liver of 19 d gestation. Fetal liver endoplasmic reticulum and peroxisomes were also amplified as visualized by electron microscopy. Clofibrate exhibited maximal effects on maternal tissues by 6 d of oral treatment with increases in peroxisomal membrane protein 70 occurring most clearly in maternal liver; DHAP-AT activity was increased in maternal liver, kidney, and brain but not in lung. Slight increases in DHAP-AT activities were evident in fetal brain, lung, and placenta as compared with the greater increases in liver and kidney. There was a general increase in peroxisomal proteins between 13 and 19 gestational d in all fetal tissues except placenta, and the effect of clofibrate was evident by gestational d 13 in lung and placenta. Minimal changes in the activities of acid phosphatase (lysosomal enzyme) or glycerol-3-phosphate acyltransferase (microsomal enzyme) were observed in maternal or fetal tissues although the latter enzyme was increased 10–30% by clofibrate in maternal brain, fetal lung, and placenta. The degree of amplification peroxisomal membrane portion 70 > DHAP-AT > catalase observed in several tissues after clofibrate treatment is consistent with a similar order in the sequence of peroxisomal assembly. The ability to manipulate fetal peroxisomes by maternal pharmacology provides a potentially useful system for studying the regulation of peroxisomal biogenesis.

An 11-year-old patient with incomplete testicular feminization and a 47,XXY karyotype is described. The patient had female external genitalia, clitoromegaly, and some features of Klinefelter's syndrome, including speech delay and delayed intellectual development. DNA analysis using X chromosomal DNA sequences suggests that the supernumerary X chromosome in the patient resulted from maternal nondisjunction during meiosis II. The M II error thereby provides the basis for homozygosity of a mutation in the androgen receptor locus.

The Zellweger cerebro-hepato-renal syndrome is a genetic disease characterized by the absence of peroxisomes and deficiency of glycerol-ether lipids in several tissues. We measured the activity of dihydroxyacetone phosphate (DHAP) acyltransferase, a peroxisomal enzyme with a major role in ether lipid synthesis, in fibroblasts and leukocytes from patients with Zellweger syndrome. Control skin and amniotic-fluid fibroblasts had normal activity of DHAP acyltransferase (0.28 to 0.3 nmol per minute per milligram of protein), whereas fibroblasts from three patients with Zellweger syndrome had deficient activity (0.013 +/- 0.006 nmol per minute per milligram of protein). The activity of the enzyme in leukocytes and levels of plasmalogens (the major class of cellular glycerol-ether lipids) in erythrocytes were also deficient in a patient, but normal levels of leukocyte enzyme and erythrocyte plasmalogens were found in her parents. Other enzymes of the acyl DHAP pathway exhibited alterations in fibroblasts from patients with Zellweger syndrome, and the activity of the glycerophosphate acyltransferase was also reduced. These results support prior studies emphasizing the role of peroxisomes and the acyl DHAP pathway in cellular ether lipid synthesis, establish Zellweger syndrome cells as valuable for elucidating peroxisomal functions, and provide prenatal and postnatal diagnostic assays as well as potential therapeutic strategies for Zellweger syndrome.

Diabetes induced by alloxan at day 6 of gestation in Wistar rats produced decreased fetal growth, delayed skeletal ossification, decreased fetal kidney beta-glucuronidase, and an increased frequency of fetal birth defects which correlated with the degree of diabetic control. Offspring of severely diabetic mothers (mean blood glucose greater than 501 mg/dl) sacrificed at 20 days had a mean weight of 2.12 +/- 0.16 g, a mean of 1.8 +/- 0.46 caudal ossification centers, and a 28% incidence of birth defects as compared to 3.70 +/- 0.22 g, 5.9 +/- 0.42 caudal centers, and 1.1% defects for controls. Offspring of severely diabetic mothers sacrificed at 21 days had mean numbers of caudal and sternal ossification centers which did not significantly differ from controls, indicating that decreased ossification observed at 20 days of gestation is a delayed developmental sequence which is mostly corrected by 21 days. Offspring of moderately diabetic (mean blood glucose 300-500 mg/dl) and insulin-treated dams (mean blood glucose 152-168 mg/dl) had intermediate degrees of growth or ossification delay and birth defect frequency at both the 20- and 21-day sacrifices. Maternal diabetes also retards the developmental increase in fetal kidney beta-glucuronidase such than 20-day offspring of severely diabetic mothers had a mean specific activity of 1.1 nmol/min/mg compared to 3.0 nmol/min/mg for controls. The results support prior studies in rodents suggesting a progression of early growth delay, altered developmental sequences, and birth defects in diabetic pregnancy. This progression is suggested as a common teratogenic mechanism which has implications for evaluating analogous pregnancies in man.

Eco-RI-A fragments of the human ribosomal RNA gene family from two types of tissue and three individuals were cloned in λ vectors and compared by restriction enzyme digestion and electron microscopy. The EcoRI fragment A contains (i) 0.2 kb of the 3' end of the 18S rDNA, (ii) 2.5 kb of internal transcribed spacer and the 5.8S rDNA, and (iii) 4.6 kb of the 28S rDNA gene. All of the six cloned rDNA fragments isolated are identical by these analyses. Moreover, all contain a HincII site that is absent in about 50% of the rDNA identified by genomic blotting. Polymorphism in the nontranscribed spacer rDNA was studied on genomic blots of BamHI-digested DNA, using the 3' end of the 28S rDNA as a probe. The boundaries between the 18S rDNA, internal transcribed spacer, 28S rDNA, and external nontranscribed spacer were determined by R-loop analysis, further defining the organization of the ribosomal RNA precursor.

We report on a 3-month-old girl with unusual facial appearance, short neck with low posterior hairline, wide chest, valvular pulmonic stenosis, abnormal fingernails, and diploid-tetraploid mosaicism (46,XX/92,XXXX in 7.2% of peripheral leucocytes and in 29% of skin fibroblasts). Comparison with 11 previously reported cases with mosaic or complete tetraploidy does not establish an easily recognizable syndrome. However, a malformation pattern is apparent when tetraploidy patients are compared with 14 cases of triploid mosaicism and 44 previously reported cases of nonmosaic triploidy. A history of sex hormone exposure was present in 5 of 11 pregnancies resulting in tetraploidy; this exposure may correlate with the occurrence of tetraploidy in polycystic ovary syndrome and in tumors of the female reproductive tract. The mechanism of dysmorphogenesis involved in polyploidy is considered, including hypotheses of altered nuclear/cytoplasmic ratio, of trophoblastic alteration, of delayed cell division, or of altered autosome/active X chromosome ratio.