Association of RAD51 with homologous recombination deficiency (HRD) and clinical outcomes in untreated triple-negative breast cancer (TNBC): analysis of the GeparSixto randomized clinical trial.

BACKGROUND: Current genetic and genomic tests measuring homologous recombination deficiency (HRD) show limited predictive value. This study compares the performance of an immunohistology-based RAD51 test with genetic/genomic tests to identify patients with HRD primary triple-negative breast cancer (TNBC) and evaluates its accuracy to select patients sensitive to platinum-based neoadjuvant chemotherapy (NACT). PATIENTS AND METHODS: This is a retrospective, blinded, biomarker analysis from the GeparSixto randomized clinical trial. TNBC patients received neoadjuvant paclitaxel plus Myocet®-nonpegylated liposomal doxorubicin (PM) or PM plus carboplatin (PMCb), both arms including bevacizumab. Formalin-fixed paraffin-embedded (FFPE) tumor samples were laid on tissue microarrays. RAD51, BRCA1 and γH2AX were quantified using an immunofluorescence assay. The predictive value of RAD51 was assessed by regression models. Concordance analyses were carried out between RAD51 score and tumor BRCA (tBRCA) status or genomic HRD score (Myriad myChoice®). Associations with pathological complete response (pCR) and survival were studied. Functional HRD was predefined as a RAD51 score ≤10% (RAD51-low). RESULTS: Functional HRD by RAD51-low was evidenced in 81/133 tumors (61%). RAD51 identified 93% tBRCA-mutated tumors and 45% non-tBRCA mutant cases as functional HRD. The concordance between RAD51 and genomic HRD was 87% [95% confidence interval (CI) 79% to 93%]. In patients with RAD51-high tumors, pCR was similar between treatment arms [PMCb 31% versus PM 39%, odds ratio (OR) 0.71, 0.23-2.24, P = 0.56]. Patients with RAD51-low tumors benefited from PMCb (pCR 66% versus 33%, OR 3.96, 1.56-10.05, P = 0.004; interaction test P = 0.02). This benefit maintained statistical significance in the multivariate analysis. Carboplatin addition showed similar disease-free survival in the RAD51-high [hazard ratio (HR) 0.40, log-rank P = 0.11] and RAD51-low (0.45, P = 0.11) groups. CONCLUSIONS: The RAD51 test identifies tumors with functional HRD and is highly concordant with tBRCA mutation and genomic HRD. RAD51 independently predicts clinical benefit from adding Cb to NACT in TNBC. Our results support further development to incorporate RAD51 testing in clinical decision-making.

Neoadjuvant paclitaxel/olaparib in comparison to paclitaxel/carboplatinum in patients with HER2-negative breast cancer and homologous recombination deficiency (GeparOLA study).

BACKGROUND: The efficacy and toxicity of olaparib as combination therapy in early breast cancer (BC) patients with homologous recombinant deficiency (HRD) [score high and/or germline (g) or tumour (t) BRCA1/2 mutation] is not well described. GeparOLA (ClinicalTrials.gov, NCT02789332) investigated olaparib in combination with paclitaxel in HER2-negative early BC with HRD. PATIENTS AND METHODS: Patients with untreated primary HER2-negative cT2-cT4a-d or cT1c with either cN+ or pNSLN+ or cT1c and triple-negative breast cancer (TNBC) or cT1c and Ki-67>20% BC with HRD were randomised either to paclitaxel (P) 80 mg/m2 weekly plus olaparib (O) 100 mg twice daily for 12 weeks or P plus carboplatinum (Cb) area under the curve 2 weekly for 12 weeks, both followed by epirubicin/cyclophosphamide (EC). Stratification factors were hormone receptor (HR) status (HR+ versus HR-) and age (<40 versus ≥40 years). The primary endpoint was pathological complete response (pCR; ypT0/is ypN0). A two-sided one-group χ2-test was planned to exclude a pCR rate of ≤55% in the PO-EC arm. Secondary end points were other pCR definitions, breast conservation rate, clinical/imaging response, tolerability and safety. RESULTS: A total of 107 patients were randomised between September 2016 and July 2018; 106 (PO N = 69; PCb N = 37) started treatment. Median age was 47.0 years (range 25.0-71.0); 36.2% had cT1, 61.0% cT2, 2.9% cT3, and 31.8% cN-positive tumours; grade 3 tumours: 86.8%; Ki-67>20%: 89.6%; TNBC: 72.6%; confirmed gBRCA1/2 mutation: 56.2%. The pCR rate with PO was 55.1% [90% confidence interval (CI) 44.5% to 65.3%] versus PCb 48.6% (90% CI 34.3% to 63.2%). Analysis for the stratified subgroups showed higher pCR rates with PO in the cohorts of patients <40 years and HR+ patients. CONCLUSION: GeparOLA could not exclude a pCR rate of ≤55% in the PO arm. PO was significantly better tolerated and the combination merits further evaluation.

Survival analysis of carboplatin added to an anthracycline/taxane-based neoadjuvant chemotherapy and HRD score as predictor of response-final results from GeparSixto.

Background: In the neoadjuvant GeparSixto study, adding carboplatin to taxane- and anthracycline-based chemotherapy improved pathological complete response (pCR) rates in patients with triple-negative breast cancer (TNBC). Here, we present survival data and the potential prognostic and predictive role of homologous recombination deficiency (HRD). Patients and methods: Patients were randomized to paclitaxel plus nonpegylated liposomal doxorubicin (Myocet®) (PM) or PM plus carboplatin (PMCb). The secondary study end points disease-free survival (DFS) and overall survival (OS) were analyzed. Median follow-up was 47.3 months. HRD was among the exploratory analyses in GeparSixto and was successfully measured in formalin-fixed, paraffin-embedded tumor samples of 193/315 (61.3%) participants with TNBC. Homologous recombination (HR) deficiency was defined as HRD score ≥42 and/or presence of tumor BRCA mutations (tmBRCA). Results: A significantly better DFS (hazard ratio 0.56, 95% CI 0.34-0.93; P = 0.022) was observed in patients with TNBC when treated with PMCb. The improvement of OS with PMCb was not statistically significant. Additional carboplatin did not improve DFS or OS in patients with HER2-positive tumors. HR deficiency was detected in 136 (70.5%) of 193 triple-negative tumors, of which 82 (60.3%) showed high HRD score without tmBRCA. HR deficiency independently predicted pCR (ypT0 ypN0) [odds ratio (OR) 2.60, 95% CI 1.26-5.37, P = 0.008]. Adding carboplatin to PM significantly increased the pCR rate from 33.9% to 63.5% in HR deficient tumors (P = 0.001), but only marginally in HR nondeficient tumors (from 20.0% to 29.6%, P = 0.540; test for interaction P = 0.327). pCR rates with carboplatin were also higher (63.2%) than without carboplatin (31.7%; OR 3.69, 1.46-9.37, P = 0.005) in patients with high HRD score but no tmBRCA. DFS rates were improved with addition of carboplatin, both in HR nondeficient (hazard ratio 0.44, 0.17-1.17, P = 0.086) and HR deficient tumors (hazard ratio 0.49, 0.23-1.04, P = 0.059). Conclusions: The addition of carboplatin to neoadjuvant PM improved DFS significantly in TNBC. Long-term survival analyses support the neoadjuvant use of carboplatin in TNBC. HR deficiency in TNBC and HRD score in non-tmBRCA TNBC are predictors of response. HRD does not predict for carboplatin benefit.

Dissection of mitogenic and neurodegenerative actions of cystine and glutamate in malignant gliomas.

Malignant glioma represents one of the most aggressive and lethal human neoplasias. A hallmark of gliomas is their rapid proliferation and destruction of vital brain tissue, a process in which excessive glutamate release by glioma cells takes center stage. Pharmacologic antagonism with glutamate signaling through ionotropic glutamate receptors attenuates glioma progression in vivo, indicating that glutamate release by glioma cells is a prerequisite for rapid glioma growth. Glutamate has been suggested to promote glioma cell proliferation in an autocrine or paracrine manner, in particular by activation of the (RS)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrate (AMPA) subtype of glutamate receptors. Here, we dissect the effects of glutamate secretion on glioma progression. Glioma cells release glutamate through the amino-acid antiporter system X(c)(-), a process that is mechanistically linked with cystine incorporation. We show that disrupting glutamate secretion by interfering with the system X(c)(-) activity attenuates glioma cell proliferation solely cystine dependently, whereas glutamate itself does not augment glioma cell growth in vitro. Neither AMPA receptor agonism nor antagonism affects glioma growth in vitro. On a molecular level, AMPA insensitivity is concordant with a pronounced transcriptional downregulation of AMPA receptor subunits or overexpression of the fully edited GluR2 subunit, both of which block receptor activity. Strikingly, AMPA receptor inhibition in tumor-implanted brain slices resulted in markedly reduced tumor progression associated with alleviated neuronal cell death, suggesting that the ability of glutamate to promote glioma progression strictly requires the tumor microenvironment. Concerning a potential pharmacotherapy, targeting system X(c)(-) activity disrupts two major pathophysiological properties of glioma cells, that is, the induction of excitotoxic neuronal cell death and incorporation of cystine required for rapid proliferation.

Valproic acid increases the SMN2 protein level: a well-known drug as a potential therapy for spinal muscular atrophy.

Proximal spinal muscular atrophy (SMA) is a common neuromuscular disorder causing infant death in half of all patients. Homozygous absence of the survival motor neuron gene (SMN1) is the primary cause of SMA, while SMA severity is mainly determined by the number of SMN2 copies. One SMN2 copy produces only about 10% of full-length protein identical to SMN1, whereas the majority of SMN2 transcripts is aberrantly spliced due to a silent mutation within an exonic splicing enhancer in exon 7. However, correct splicing can be restored by over-expression of the SR-like splicing factor Htra2-beta 1. We show that in fibroblast cultures derived from SMA patients treated with therapeutic doses (0.5-500 microM) of valproic acid (VPA), the level of full-length SMN2 mRNA/protein increased 2- to 4-fold. Importantly, this up-regulation of SMN could be most likely attributed to increased levels of Htra2-beta 1 which facilitates the correct splicing of SMN2 RNA as well as to an SMN gene transcription activation. Especially at low VPA concentrations, the restored SMN level depended on the number of SMN2 copies. Moreover, VPA was able to increase SMN protein levels through transcription activation in organotypic hippocampal brain slices from rats. Finally, VPA also increased the expression of further SR proteins, which may have important implications for other disorders affected by alternative splicing. Since VPA is a drug highly successfully used in long-term epilepsy therapy, our findings open the exciting perspective for a first causal therapy of an inherited disease by elevating the SMN2 transcription level and restoring its correct splicing.

A novel secreted ribonuclease from Bacillus intermedius: gene structure and regulatory control.

A second secreted ribonuclease, designated binase II, has been detected in Bacillus intermedius 7P, and its structural gene was cloned and sequenced. Unlike the well-known binase I, a 109-amino acid guanyl-specific enzyme, the 292-residue binase II is closely related to the B. subtilis nuclease Bsn, in structure and in its enzymatic properties. Binase II is also insensitive to inactivation by barstar, an inhibitor protein that is specific for guanyl-specific ribonucleases. While both B. intermedius enzymes are induced upon phosphate starvation, only the gene for binase I belongs to the pho regulon system and carries pho-box elements adjacent to its promoter sequence. The gene for binase II is similar to that for Bsn in lacking such elements. The birB gene coding for binase II appears to be located next to the 3'-end of a ferric ion transport operon, with which it convergently overlaps. This would allow attenuator control over binase II expression under conditions of starvation for ferric ions.

Analysis of the SMN and NAIP genes in slovak spinal muscular atrophy patients.

We identified homozygous absence of exon 7 of the telomeric copy of the survival motor neuron gene (telSMN) in 88.4% (38/43) of spinal muscular atrophy (SMA) patients from Slovakia. Additional deletions within the neuronal apoptosis inhibitory protein (NAIP) gene were found in 38.5% of type I, 12.5% of type II and never in type III SMA patients. Neither the SMN nor the NAIP gene was deleted in 81 healthy relatives and 25 controls tested. In one family, pseudodominant inheritance was identified. Both the type III SMA father and type II SMA son carried the homozygous deletion of the telSMN gene. One SMA I patient showed an SMN hybrid gene, probably created by intrachromosomal deletion. In two haploidentical type II SMA sibs, the telSMN exon 7 was absent on one chromosome, while the other carried an A-->G transition 96 bp upstream of exon 7 of the telSMN gene, a potential disease-causing mutation in these patients.

A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy.

SMN1 and SMN2 (survival motor neuron) encode identical proteins. A critical question is why only the homozygous loss of SMN1, and not SMN2, results in spinal muscular atrophy (SMA). Analysis of transcripts from SMN1/SMN2 hybrid genes and a new SMN1 mutation showed a direct relationship between presence of disease and exon 7 skipping. We have reported previously that the exon-skipped product SMNDelta7 is partially defective for self-association and SMN self-oligomerization correlated with clinical severity. To evaluate systematically which of the five nucleotides that differ between SMN1 and SMN2 effect alternative splicing of exon 7, a series of SMN minigenes was engineered and transfected into cultured cells, and their transcripts were characterized. Of these nucleotide differences, the exon 7 C-to-T transition at codon 280, a translationally silent variance, was necessary and sufficient to dictate exon 7 alternative splicing. Thus, the failure of SMN2 to fully compensate for SMN1 and protect from SMA is due to a nucleotide exchange (C/T) that attenuates activity of an exonic enhancer. These findings demonstrate the molecular genetic basis for the nature and pathogenesis of SMA and illustrate a novel disease mechanism. Because individuals with SMA retain the SMN2 allele, therapy targeted at preventing exon 7 skipping could modify clinical outcome.

Quantitative analysis of survival motor neuron copies: identification of subtle SMN1 mutations in patients with spinal muscular atrophy, genotype-phenotype correlation, and implications for genetic counseling.

Problems with diagnosis and genetic counseling occur for patients with autosomal recessive proximal spinal muscular atrophy (SMA) who do not show the most common mutation: homozygous absence of at least exon 7 of the telomeric survival motor neuron gene (SMN1). Here we present molecular genetic data for 42 independent nondeleted SMA patients. A nonradioactive quantitative PCR test showed one SMN1 copy in 19 patients (45%). By sequencing cloned reverse-transcription (RT) PCR products or genomic fragments of SMN1, we identified nine different mutations in 18 of the 19 patients, six described for the first time: three missense mutations (Y272C, T274I, S262I), three frameshift mutations in exons 2a, 2b, and 4 (124insT, 241-242ins4, 591delA), one nonsense mutation in exon 1 (Q15X), one Alu-mediated deletion from intron 4 to intron 6, and one donor splice site mutation in intron 7 (c.922+6T-->G). The most frequent mutation, Y272C, was found in 6 (33%) of 18 patients. Each intragenic mutation found in at least two patients occurred on the same haplotype background, indicating founder mutations. Genotype-phenotype correlation allowed inference of the effect of each mutation on the function of the SMN1 protein and the role of the SMN2 copy number in modulating the SMA phenotype. In 14 of 23 SMA patients with two SMN1 copies, at least one intact SMN1 copy was sequenced, which excludes a 5q-SMA and suggests the existence of further gene(s) responsible for approximately 4%-5% of phenotypes indistinguishable from SMA. We determined the validity of the test, and we discuss its practical implications and limitations.

SMN oligomerization defect correlates with spinal muscular atrophy severity.

Spinal muscular atrophy (SMA) is a motor-neuron disorder resulting from anterior-horn-cell death. The autosomal recessive form has a carrier frequency of 1 in 50 and is the most common genetic cause of infant death. SMA is categorized as types I-III, ranging from severe to mild, based upon age of onset and clinical course. Two closely flanking copies of the survival motor neuron (SMN) gene are on chromosome 5q13 (ref. 1). The telomeric SMN (SMN1) copy is homozygously deleted or converted in >95% of SMA patients, while a small number of SMA disease alleles contain missense mutations within the carboxy terminus. We have identified a modular oligomerization domain within exon 6 of SMN1. All previously identified missense mutations map within or immediately adjacent to this domain. Comparison of wild-type to mutant SMN proteins of type I, II and III SMA patients showed a direct correlation between oligomerization and clinical type. Moreover, the most abundant centromeric SMN product, which encodes exons 1-6 but not 7, demonstrated reduced self-association. These findings identify decreased SMN self-association as a biochemical defect in SMA, and imply that disease severity is proportional to the intracellular concentration of oligomerization-competent SMN proteins.

De novo rearrangements found in 2% of index patients with spinal muscular atrophy: mutational mechanisms, parental origin, mutation rate, and implications for genetic counseling.

Spinal muscular atrophy (SMA) is a relatively common autosomal recessive neuromuscular disorder. We have identified de novo rearrangements in 7 (approximately 2%) index patients from 340 informative SMA families. In each, the rearrangements resulted in the absence of the telomeric copy of the survival motor neuron (SMN) gene (telSMN), in two cases accompanied by the loss of the neuronal apoptosis-inhibitory protein gene . Haplotype analysis revealed unequal recombination in four cases, with loss of markers Ag1-CA and C212, which are near the 5' ends of the SMN genes. In one case, an interchromosomal rearrangement involving both the SMN genes and a regrouping of Ag1-CA and C212 alleles must have occurred, suggesting either interchromosomal gene conversion or double recombination. In two cases, no such rearrangement was observed, but loss of telSMN plus Ag1-CA and C212 alleles in one case suggested intrachromosomal deletion or gene conversion. In six of the seven cases, the de novo rearrangement had occurred during paternal meiosis. Direct detection of de novo SMA mutations by molecular genetic means has allowed us to estimate for the first time the mutation rate for a recessive disorder in humans. The sex-averaged rate of 1.1 x 10(-4), arrived at in a proband-based approach, compares well with the rate of 0.9 x 10(-4) expected under a mutation-selection equilibrium for SMA. These findings have important implications for genetic counseling and prenatal diagnosis in that they emphasize the relevance of indirect genotype analysis in combination with direct SMN-gene deletion testing in SMA families.

Different entities of proximal spinal muscular atrophy within one family.

The molecular analysis of the survival motor neuron (SMN) gene and several closely flanking polymorphic markers in an atypical pedigree with four patients suffering from spinal muscular atrophy (SMA) over two generations has raised new aspects concerning the etiology and the molecular spectrum of autosomal recessive SMA. Three patients in two generations show homozygous deletions of exons 7 and 8 of the telomeric copy of SMN (telSMN), thus confirming the presence of autosomal recessive SMA, with localisation on chromosome 5q12. The fourth SMA patient with mild neurogenic atrophy (confirmed by muscle biopsy and electromyography) shows no homozygous deletion of telSMN but carries a heterozygous deletion of telSMN, as can be deduced from her two affected homozygously deleted children. No intragenic mutation has been identified in the remaining telSMN. In addition, she shares only one SMA chromosome with her affected brother, is haploidentical with two healthy brothers, and has a 31-year-old healthy son, who has inherited an SMN-deleted paternal chromosome and the SMN non-deleted maternal chromosome. These results suggest that this patient either has a neurogenic atrophy of a different origin or exhibits an unusual heterozygous manifestation of SMA 5q12. Interestingly, the two haploidentical telSMN-deleted affected sibs in the second generation show a strikingly discordant clinical picture indicating that, in addition to telSMN mutations, other factors influence the phenotype of SMA in the reported pedigree.

Missense mutations in exon 6 of the survival motor neuron gene in patients with spinal muscular atrophy (SMA).

Spinal muscular atrophy (SMA) is a frequent autosomal recessive neurodegenerative disorder leading to weakness and atrophy of voluntary muscles. The survival motor neuron gene (SMN) is a strong candidate for SMA and present in two highly homologous copies (telSMN and cenSMN) within the SMA region (5q11.2-q13.3). More than 90% of SMA patients show homozygous deletions of at least exon 7 of telSMN, whereas absence of cenSMN seems to have no clinical consequences. In 23 non-deleted SMA patients, we searched for intragenic mutations of the SMN genes in exons 1-7 and the promotor region by single strand conformation analysis. We identified two different missense mutations, S2621 and T2741, in exon 6 of telSMN in three independent SMA families, providing further evidence for the telSMN gene as a SMA determining gene. Both mutations, as well as two previously described mutations (Y272C and G279V) are located within a highly conserved interval from codon 258 to codon 279 which seems to be an important functional domain of the telSMN protein. Recently, this region has been shown to contain a tyrosine/glycine-rich motif, which is also present in various RNA binding proteins, suggesting a potential role of SMN in RNA metabolism. Missense mutations might be useful for in vivo and transgenic experiments and further investigations on understanding the function of the telSMN protein.

Hybrid survival motor neuron genes in patients with autosomal recessive spinal muscular atrophy: new insights into molecular mechanisms responsible for the disease.

Spinal muscular atrophy (SMA) is a frequent autosomal recessive neurodegenerative disorder leading to weakness and atrophy of voluntary muscles. The survival motor-neuron gene (SMN), a strong candidate for SMA, is present in two highly homologous copies (telSMN and cenSMN) within the SMA region. Only five nucleotide differences within the region between intron 6 and exon 8 distinguish these homologues. Independent of the severity of the disease, 90%-98% of all SMA patients carry homozygous deletions in telSMN, affecting either exon 7 or both exons 7 and 8. We present the molecular analysis of 42 SMA patients who carry homozygous deletions of telSMN exon 7 but not of exon 8. The question arises whether in these cases the telSMN is truncated upstream of exon 8 or whether hybrid SMN genes exist that are composed of centromeric and telomeric sequences. By a simple PCR-based assay we demonstrate that in each case the remaining telSMN exon 8 is part of a hybrid SMN gene. Sequencing of cloned hybrid SMN genes from seven patients, as well as direct sequencing and single-strand conformation analysis of all patients, revealed the same composition in all but two patients: the base-pair differences in introns 6 and 7 and exon 7 are of centromeric origin whereas exon 8 is of telomeric origin. Nonetheless, haplotype analysis with polymorphic multicopy markers, Ag1-CA and C212, localized at the 5' end of the SMN genes suggests different mechanisms of occurrence, unequal rearrangements, and gene conversion involving both copies of the SMN genes. In approximately half of all patients, we identified a consensus haplotype, suggesting a common origin. Interestingly, we identified a putative recombination hot spot represented by recombination-stimulating elements (TGGGG and TGAGGT) in exon 8 that is homologous to the human deletion-hot spot consensus sequence in the immunoglobulin switch region, the alpha-globin cluster, and the polymerase alpha arrest sites. This may explain why independent hybrid SMN genes show identical sequences.

Frequent DNA variant in exon 2a of the survival motor neuron gene (SMN): a further possibility for distinguishing the two copies of the gene.

An intragenic single-strand conformation polymorphism (SSCP) variant in exon 2a of the survival motor neuron gene (SMN) has been identified. The SSCP band shift is caused by a silent mutation (AGC-->AGT) at codon 28, which is the first codon of exon 2a. Five exchanges of base pairs at the 3'-end of the gene have been described that allow the two copies of SMN (telSMN and cenSMN) to be distinguished, whereas no DNA variant has been found at the 5'-end. The new DNA variant belongs to cenSMN and may be important for the assignment of point mutations to one of the two copies of SMN in spinal muscular atrophy (SMA) patients. The frequency of this variant is lower in SMA patients (10%) than in controls (24%).

Clinical spectrum and diagnostic criteria of infantile spinal muscular atrophy: further delineation on the basis of SMN gene deletion findings.

With the evidence of deletions in the region responsible for autosomal recessive spinal muscular atrophy (SMA) on chromosome 5, it is now possible to further clarify the clinical and diagnostic findings in proximal SMA. Homozygous deletions of the survival motor neuron (SMN) gene can be detected in about 95% of patients with early onset SMA. In a series of more than 200 patients, we tested 31 patients with atypical features of SMA who fulfilled at least one exclusion criterion according to the diagnostic criteria of the International SMA Consortium for the presence of SMN gene deletions. The patients were subdivided into two groups: 1. Seven index patients being not deleted for the SMN gene who belonged to a well-defined SMA plus variant that has already been shown to be unlinked with chromosome 5q markers: diaphragmatic SMA, SMA plus olivopontocerebellar hypoplasia, SMA with congenital arthrogryposis and bone fractures. 2. Twenty-four patients with clinical signs of SMA and neurogenic findings in EMG/muscle biopsy who had unusual features or other organ involvement. In order to structure this heterogeneous group, each patient was assigned to a subgroup according to the leading atypical feature. In 5 out of 8 unrelated patients with a history of preterm birth and/or perinatal asphyxia leading to a picture of severe SMA in combination with respiratory distress and/or cerebral palsy, no deletion of the SMN gene could be detected. There were five unrelated patients with extended central nervous system involvement (cerebral atrophy, EEG abnormalities, pyramidal tract signs, evidence of cerebellar involvement). Most of these patients (4/5) proved to belong to SMA 5q on the basis of SMN gene deletion findings. The same applied to a group of three patients with classical SMA in association with congenital malformations (mainly heart defect). A fourth group of three patients was characterized mainly by an unusual improvement of the condition; in these patients no SMN gene deletions were present. In three index patients a more complex syndrome of the CNS and other organs was suggested, but the detection of SMN gene deletions in two of them made a coincidence of features more likely. In addition, SMN gene deletions were found in two patients with evidence of congenital fibre type dysproportion in one and extremely raised CK activity ( > 10fold) in the other. While the confirmation of SMN gene deletions is very useful in cases with diagnostic doubts, caution is required when offering prenatal prediction with regard to SMA 5q in families with atypical features. There is strong evidence that there are clinical entities resembling SMA which most likely have another pathogenetic background.

Molecular analysis of candidate genes on chromosome 5q13 in autosomal recessive spinal muscular atrophy: evidence of homozygous deletions of the SMN gene in unaffected individuals.

Proximal spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder characterized by degeneration of anterior horn cells in the spinal cord leading to weakness and wasting of voluntary muscles. Here we present the molecular analysis of both SMA candidate genes, the survival motor neuron gene (SMN; exons 7 and 8) and the neuronal apoptosis inhibitory protein gene (NAIP; exons 5, 6 and 13), in 195 patients and 348 parents of SMA families mainly of German origin. The SMN gene is homozygously deleted for both exons 7 and 8 or exon 7 only in 96% of type I SMA, 94% of type II SMA and 82% of type III SMA as well as in 0.3% of SMA parents. The NAIP gene is homozygously deleted in 46% of type I SMA, 17% of type II SMA, 7% of type III SMA and 2% of SMA parents. The frequencies of deletions in patients for both genes, SMN and NAIP, correspond to those for the NAIP gene only. SMA patients of this series who did not show deletions were clinically indistinguishable from deleted patients. In addition to one unaffected mother of a type II SMA patient, we found homozygous deletions of the SMN gene exons 7 and 8 in six further unaffected individuals, all sibs of type II and III patients. These belonged to four families with affected and unaffected sibs who showed identical haplotypes for all SMA flanking markers; therefore, we had regarded these families as chromosome 5 unlinked. All seven unaffected individuals in whom we detected SMA deletions do not show any signs of muscle weakness and are physically inconspicuous. The largest divergence between age at onset of an affected subject and the present age of unaffected deleted sibs is four decades now. The occurrence of SMN deletions in unaffected individuals suggests that other genes or mechanisms may be necessary to produce the SMA phenotype.