Dysmorphic syndrome of hereditary neuralgic amyotrophy associated with a SEPT9 gene mutation--a family study.

We report a family in which two siblings presented with an apparent dysmorphic syndrome, including hypotelorism, blepharophimosis, slight ptosis, epicanthal folds, microstomia and dysmorphic ears. One sibling had a cleft palate. Initially, blepharophimosis, ptosis, and epicanthus inversus syndrome (BPES) was suspected; however, mutation of the FOXL2 gene was not detected. Moreover, the patients' father and paternal grandmother had experienced recurrent episodes of unilateral brachial neuritis and were diagnosed to have hereditary neuralgic amyotrophy (HNA). HNA is a rare, inherited form of brachial neuritis whose phenotypic spectrum may include hypotelorism, cleft palate and other minor dysmorphisms. HNA maps to chromosome 17q25 and is associated with mutations in the SEPT9 gene. After confirming a heterozygous SEPT9 mutation (R88W) in the father and his mother, it became apparent that the dysmorphic features in the children were part of HNA and that previous complaints of the daughter, erroneously diagnosed as pronatio dolorosa and then epiphysiolysis of the capitellum humeri, were in fact a first neuralgic pain attack. Both children were shown to have inherited the paternal SEPT9 mutation. Wider recognition of HNA as a syndromic disorder may facilitate its diagnosis in affected young persons who may not yet have manifested episodes of brachial neuritis.

Ultrastructural changes in the cilia of experimental mice.

Mice lacking dynein arms in the cilia were examined; the strain was obtained by inactivation of dynein heavy chain gene in chromosome 7. The cilia of these mice were examined by electron microscopy and compared to the cilia of random-bred mice. No statistically significant differences or typical disorders in the outer or inner dynein arms were detected. The number of inner dynein arms was lower, in some cilia secondary changes presenting as swelling of the outer part of the ciliary membrane or formation of complex cilia were seen.

Novel mutations in the Atlastin gene (SPG3A) in families with autosomal dominant hereditary spastic paraplegia and evidence for late onset forms of HSP linked to the SPG3A locus.

Hereditary spastic paraplegias (HSP) comprise a genetically and clinically heterogeneous group of neurodegenerative disorders characterised by progressive spasticity and hyperreflexia of the lower limbs. Autosomal dominant hereditary spastic paraplegia linked to the SPG3A locus on chromosome 14q11-21 accounts for approximately 10% of autosomal dominant hereditary spastic paraplegia (ADHSP). It is caused by mutations in the SPG3A gene encoding the protein atlastin. To date, only five disease-causing mutations in the SPG3A gene have been described. We analysed 13 SPG4-negative families for mutations in the SPG3A gene and identified a mutation in 38% (5/13). Two of the mutations are novel, c.481G>C (p.A161P) and c.740A>C (p.H247P). One of the novel mutations was found both in a family with early onset of symptoms and in a late onset family. Furthermore, we report on numerous polymorphisms detected in the SPG3A gene.

[Genetic causes of male infertility]. / Genetische Ursachen der männlichen Infertilität.

7% of all human males suffer from infertility. In at least 10% of these males infertility is due to genetic causes. Because modern reproduction techniques like ICSI (intracytoplasmic sperm injection) can help the couples to overcome infertility, it is mandatory to analyze underlying genetic causes of male infertility. If infertility in a male is due to a genetic defect, the risk of the respective couple for abortuses or malformed children is increased. The main and relevant causes for male infertility known to day are: numerical and structural chromosomal aberrations, meiotic defects, microdeletions in the region q11.21-23 of the Y-chromosome, mutations in the gene for cystic fibrosis and genetically determined syndromes in which infertility is a symptom. The present knowledge concerning these genetic causes of male infertility is pointed out.

Genomic organisation and chromosomal assignment of ODF2 (outer dense fiber 2), encoding the main component of sperm tail outer dense fibers and a centrosomal scaffold protein.

ODF2 (outer dense fiber 2) was first described as the main protein component of the sperm tail cytoskeleton, the outer dense fibers, but was shown recently to be a component of the centrosomal scaffold in chicken. In mouse two related ODF2 cDNA clones were isolated which have been suggested to be most likely the result of alternative splicing. We show here the exon/intron organisation of mouse ODF2 and demonstrate that alternative splicing results in related cDNA sequences and most likely explains, at least partially, the highly complex protein pattern detected on Western blots. ODF2 was mapped to rat chromosome 3 and more specifically by FISH analysis at bands 3q11-->3q12. In addition, we demonstrate that ODF2 is indeed a component of the centrosome and the mitotic spindle poles in mammals.

Mutation analysis of the spastin gene (SPG4) in patients in Germany with autosomal dominant hereditary spastic paraplegia.

Hereditary spastic paraplegias (HSP) comprise a genetically and clinically heterogeneous group of neurodegenerative disorders characterized by progressive spasticity and hyperreflexia of the lower limbs. Autosomal dominant hereditary spastic paraplegia 4 linked to chromosome 2p (SPG4) is the most common form of autosomal dominant hereditary spastic paraplegia. It is caused by mutations in the SPG4 gene encoding spastin, a member of the AAA protein family of ATPases. In this study the spastin gene of HSP patients from 161 apparently unrelated families in Germany was analyzed. The authors identified mutations in 27 out of the 161 HSP families; 23 of these mutations have not been described before and only one mutation was found in two families. Among the detected mutations are 14 frameshift, four nonsense, and four missense mutations, one large deletion spanning several exons, as well as four mutations that affect splicing. Most of the novel mutations are located in the conserved AAA cassette-encoding region of the spastin gene. The relative frequency of spastin gene mutations in an unselected group of German HSP patients is approximately 17%. Frameshift mutations account for the majority of SPG4 mutations in this population. The proportion of splice mutations is considerably lower than reported elsewhere.

Identification of the human ortholog of the t-complex-encoded protein TCTE3 and evaluation as a candidate gene for primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a heterogeneous autosomal recessive disease that is caused by impaired ciliary and flagellar functions. About 50% of PCD patients show situs inversus, denoted as Kartagener syndrome. In most cases, axonemal defects in cilia and sperm tails can be demonstrated by electron microscopy, i.e. PCD patients often lack inner and/or outer dynein arms in their sperm tails and cilia, supporting the hypothesis that mutations in dynein genes may cause PCD. In order to identify novel PCD genes we have isolated the human ortholog of the murine TCTE3 gene. The human TCTE3 gene encodes a dynein light chain and shares high similarity to dynein light chains of other species. The TCTE3 gene is expressed in tissues containing cilia or flagella, it is composed of four exons and located on chromosome 6q25-->q27. To elucidate the role of TCTE3 as a candidate gene for PCD a mutational analysis of thirty-six PCD patients was performed. We detected five polymorphisms in the coding sequence and in the 5' UTR of the TCTE3 gene. In one patient a heterozygous nucleotide exchange was identified resulting in an arginine to isoleucine substitution at the amino acid level. However, this exchange was also detected in one control DNA. Our results indicate that mutations in the TCTE3 gene are not a main cause of primary ciliary dyskinesia.

Human cyritestin genes (CYRN1 and CYRN2) are non-functional.

The mouse cyritestin gene is a member of the ADAM (a disintegrin and metalloprotease) gene family and codes for a membrane-anchored sperm protein. Recently, it was shown that cyritestin is critical for male fertility in the mouse. Spermatozoa of cyritestin-deficient mice are not able to bind to the zona pellucida of the oocyte and therefore unable to fertilize the egg. However, zona-free oocytes can be fertilized and the resulting embryos show normal development. In contrast to the mouse, where only one gene for cyritestin (Cyrn) is reported, two cyritestin genes (CYRN1 and CYRN2) are known in humans. The human CYRN1 and CYRN2 genes are located on chromosomes 8 and 16, respectively. We report that 27% of fertile men are deficient for the CYRN1 gene but that all have a CYRN2 gene, suggesting that the CYRN2 gene is the orthologous mouse cyritestin gene in humans and might be involved in sperm-egg interactions. However, the characterization of CYRN2 transcripts from testicular RNA of CYRN1-deficient men demonstrated many termination codons in the synthesized cyritestin cDNA. Furthermore, Western-blot analysis with human testicular protein extracts using an anti-cyritestin antibody failed to detect any cyritestin protein. These results demonstrate clearly that both cyritestin genes are non-functional in humans.

Disruption of an inner arm dynein heavy chain gene results in asthenozoospermia and reduced ciliary beat frequency.

Impaired ciliary and flagellar functions resulting in male infertility and recurrent respiratory tract infections are found in patients suffering from primary ciliary dyskinesia (PCD). In most cases, axonemal defects are present, i.e. PCD patients often lack inner and/or outer dynein arms in their sperm tails and cilia, supporting the hypothesis that mutations in dynein genes may cause PCD. However, to date it is unclear whether mutations in dynein heavy chain genes are responsible for impaired flagellar and ciliary motility in mammals. To elucidate the role of the mouse dynein heavy chain 7 (MDHC7) gene, which encodes a component of the inner dynein arm, we have generated mice lacking this dynein heavy chain isoform. Both MDHC7(+/-) and MDHC7(-/-) mice are viable and show no malformations; however, homozygous males produce no offspring. In comparison to MDHC7(+/-) and wild-type mice the spermatozoa of MDHC7(-/-) mice revealed a dramatic reduced straight line velocity and progressive movement, resulting in the inability of MDHC7-deficient sperm to move from the uterus into the oviduct. Additionally, we measured the beat frequency of tracheal cilia and observed a decrease in the beat frequency of approximately 50% in MDHC7(-/-) mice. The reduction in both ciliary and flagellar motility is not correlated with any gross defects in the axonemal structure. The phenotype of MDHC7(-/-) mice is similar to that observed in some patients suffering from PCD, and our data strongly suggest that in some patients this disease could be due to mutations in the homologous human gene DNAH1 (HDHC7).

Homozygous factor V Leiden mutation in a woman with multiple adverse pregnancy outcomes.

We report a case with one intrauterine fetal death (IUFD) at 32 weeks of gestation, one premature delivery at the same week, and one abortion of unknown etiology at 12 weeks of gestation. We discuss that the presence of homozygosity for Factor V Leiden may be associated with placental insufficiency in this woman. Application of anticoagulant therapy may have been beneficial in her current pregnancy.

Identification of a testis-specific gene (C15orf2) in the Prader-Willi syndrome region on chromosome 15.

Prader-Willi syndrome (PWS) results from the loss of paternal contributions for a 2-Mb imprinted region on the proximal long arm of human chromosome 15. Hitherto, five paternally active genes have been identified in this region (ZNF127, NDN, MAGEL2, SNURF-SNRPN, and IPW). Here we report the identification of a novel gene in the PWS critical region, which has been designated "chromosome 15 open reading frame 2" (C15orf2). C15orf2 is an intronless gene located between MAGEL2 and SNURF-SNRPN. It is associated with a CpG island, which is methylated in all tissues tested except for germ cells. C15orf2 is transcribed as a 7.5-kb mRNA and contains an open reading frame encoding a predicted 1156-amino-acid protein of unknown function. Transcription of C15orf2 occurs exclusively in the testis, and in adult testis samples, we observed biallelic expression. By zoo-blot analysis, we found related sequences in DNA from other primates, but not in nonprimate DNA. We conclude that C15orf2 may play a role in primate spermatogenesis.

Alternative splicing, chromosome assignment and subcellular localization of the testicular haploid expressed gene (THEG).

We have previously isolated and characterized the mouse Testicular Haploid Expressed Gene (Theg) that is specifically expressed in haploid germ cells. We now describe the molecular cloning and characterization of the human homologue (THEG) of mouse Theg. Expression studies by using both dot blot and Northern blot techniques revealed that human THEG is expressed specifically in the testis. Additionally, we found two alternatively spliced transcripts (THEG major and THEG minor) for THEG by using reverse transcription-polymerase chain reaction on human testicular RNA. Sequence analysis of these PCR products demonstrated that the smaller transcript (THEG minor) lacks 72 bp which was also observed for the mouse Theg. We have isolated the cDNAs of human THEG major and THEG minor, containing the complete open reading frames, which encode putative nuclear proteins of 379 amino acids and 355 amino acids, respectively. Database searches identified two genomic clones on chromosome 19 harboring the human THEG gene, which is approximately 14 kb pairs in size, contains eight exons, and comparison of the two cDNA sequences with the genomic sequence indicated that the smaller transcript lacks exon 3. Furthermore, we assigned the human THEG gene (THEG) to human chromosome 19ptel--> p13 by fluorescence in situ hybridization. Moreover, we detected mouse THEG protein prominently in the nucleus of round spermatids by using an antibody against THEG on both testicular sections and cellular suspensions. Additionally, the subcellular localization of mouse THEG was confirmed by a green fluorescent protein (GFP) fusion protein of mouse THEG which was found mainly in the nucleus of transfected NIH3T3 cells. These data suggest that both human and mouse THEG are specifically expressed in the nucleus of haploid male germ cells and are involved in the regulation of nuclear functions.

Proteins with tandemly arranged repeats of a highly charged 16-amino-acid motif encoded by the Dhmst101 gene family are structural components of the outer sheath of the extremely elongated sperm tails of Drosophila hydei.

Fruit fly species of the genus Drosophila show a remarkable variation in sperm length. Some of them produce gigantic sperm several times the total male body length. Sperm of Drosophila hydei, for example, are more than 20 mm long. Little is known about the advantage of such elongated sperm or about the proteins that stabilize their thin flagellar tails. Recently, two members of a novel gene family Dhmst101(1) and Dhmst101(2), whose gene products are associated with the sperm tail, were isolated and characterized. Here a third member of this gene family, Dhmst101(3), is described. It was previously demonstrated that all three genes are located in a single small cluster on chromosome 5 of D. hydei. They are located within 15 kb of genomic DNA, oriented in the same direction and transcribed testis-specifically. The encoded sperm tail-specific proteins are mainly composed of tandemly arranged repeats of a highly charged, cysteine-containing motif of 16 amino acids with the consensus sequence KKKCA/EEAAKKEKEAAE. Experiments with synthetic repeat monomers and dimers have demonstrated a tendency for alpha-helical rod formation, which increased strongly with an increase in repeat number. Therefore, Dhmst101 proteins with 7-60 repeats with regularly spaced cystein-residues are thus expected to form long alpha-helical rods cross-linked by numerous Cys-Cys bridges. Here we apply immunoelectron microscopy and monospecific antibodies, alpha-mst101, raised against the KKKCAEAAKKEKEAAE-motif to investigate the distribution of Dhmst101 proteins within the sperm tail of D. hydei. We show that Dhmst101 proteins are part of the outer sheath of the sperm tail where they presumably help to provide a tight but elastic envelope for the extremely extended spermatozoa of D. hydei.

Identification of dynein heavy chain genes expressed in human and mouse testis: chromosomal localization of an axonemal dynein gene.

Dynein heavy chains are involved in microtubule-dependent transport processes. While cytoplasmic dyneins are involved in chromosome or vesicle movement, axonemal dyneins are essential for motility of cilia and flagella. Here we report the isolation of dynein heavy chain (DHC)-like sequences in man and mouse. Using polymerase chain reaction and reverse-transcribed human and mouse testis RNA cDNA fragments encoding the conserved ATP binding region of dynein heavy chains were amplified. We identified 11 different mouse and eight human dynein-like sequences in testis which show high similarity to known dyneins of different species such as rat, sea urchin or green algae. Sequence similarities suggest that two of the mouse clones and one human clone encode putative cytoplasmic dynein heavy chains, whereas the other sequences show higher similarity to axonemal dyneins. Two of nine axonemal dynein isoforms identified in the mouse testis are more closely related to known outer arm dyneins, while seven clones seem to belong to the inner arm dynein group. Of the isolated human isoforms three clones were classified as outer arm and four clones as inner arm dynein heavy chains. Each of the DHC cDNAs corresponds to an individual gene as determined by Southern blot experiments. The alignment of the deduced protein sequences between human (HDHC) and mouse (MDHC) dynein fragments reveals higher similarity between single human and mouse sequences than between two sequences of the same species. Human and mouse cDNA fragments were used to isolate genomic clones. Two of these clones, gHDHC7 and gMDHC7, are homologous genes encoding axonemal inner arm dyneins. While the human clone is assigned to 3p21, the mouse gene maps to chromosome 14.

Tandemly arranged repeats of a novel highly charged 16-amino-acid motif representing the major component of the sperm-tail-specific axoneme-associated protein family Dhmst101 form extended alpha-helical rods within the extremely elongated spermatozoa of Drosophila hydei.

We have previously reported that the sperm-tail-specific proteins in Drosophila hydei, encoded by the small Dhmst101 gene family, contained several tandem repeats of a novel highly charged, well-conserved cysteine-containing motif of 16 amino acids, KKKCAEAAKKEKEAAE [Neesen, J., Bünemann, H. & Heinlein, U. A. O. (1994) Dev. Biol. 162, 414-425] and suggested that this motif might be important in the structural and functional integrity of the sperm tail. We tested this suggestion by examining structure formation by model synthetic peptides containing the 16-residue sequence and corresponding peptides with one and two repeats of the sequence with Cys being replaced by Ala. We find that all these peptides form monomeric alpha-helices and that the helix content is considerably enhanced as the number of tandem repeats increases. These results are consistent with tandemly arranged 16-amino-acid repeats in Dhmst101 proteins forming extended alpha-helical rods, with the highly conserved Cys present in each 16-amino-acid motif being involved in regular interhelical cross-linking, thus providing a rigid, stable framework within the extremely elongated spermatozoa of Drosophila hydei.

The Drosophila hydei gene Dhmst101(1) encodes a testis-specific, repetitive, axoneme-associated protein with differential abundance in Y chromosomal deletion mutant flies.

To understand the effect of the megabase-sized, Y chromosomal fertility genes on different stages of spermatogenesis in Drosophila hydei, an immunoscreening was performed to search for testis-specific protein-encoding cDNAs. The array of isolated clones contained cDNA sequences derived from a gene on chromosome 5 at 101BC. The gene, Dhmst101(1), is a member of a small gene family and is specifically expressed in adult testis tissue. The mRNA encodes a protein of 344 amino acids with a deduced apparent molecular weight of 37,793 Da. The main portion of the protein sequence comprises repetitive, highly charged amino acid units and shows repeat number variations among several D. hydei laboratory stocks. Immunocytochemistry with antibodies raised against synthetic peptides localized the protein product in elongated spermatids. This pattern of expression and the evaluation of biophysical considerations on the protein sequence data suggest that the Dhmst101(1) gene product may have some importance for the structural integrity of the sperm tail. Moreover, Y chromosomal deletions affecting correct spermiogenesis lead to degradation of the Dhmst101(1) gene product.