Dominant CST3 variants cause adult onset leukodystrophy without amyloid angiopathy.

Leukodystrophies are rare genetic white matter disorders that have been regarded as mainly occurring in childhood. Recent years altered this perception, as a growing number of leukodystrophies was described to have an onset at adult ages. Still, many adult patients presenting with white matter changes remain without a specific molecular diagnosis. We describe a novel adult onset leukodystrophy in 16 patients from eight families carrying one of four different stop-gain or frameshift dominant variants in the CST3 gene. Clinical and radiological features differ markedly from the previously described Icelandic Cerebral Amyloid Angiopathy that was found in patients carrying p.Leu68Asn substitution in CST3. The clinical phenotype consists of recurrent episodes of hemiplegic migraine associated with transient unilateral focal deficits and slowly progressing motor symptoms and cognitive decline in mid-old adult ages. In addition, in some cases acute onset clinical deterioration led to a prolonged episode with reduced consciousness and even early death. Radiologically, pathognomonic changes are found at typical predilection sites involving the deep cerebral white matter sparing a periventricular and directly subcortical rim, the middle blade of corpus callosum, posterior limb of the internal capsule, middle cerebellar peduncles, cerebral peduncles, and specifically the globus pallidus. Histopathologic characterization in two autopsy cases did not reveal angiopathy, but instead micro- to macrocystic degeneration of the white matter. Astrocytes were activated at early stages and later on displayed severe degeneration and loss. In addition, despite loss of myelin, elevated numbers of partly apoptotic oligodendrocytes were observed. A structural comparison of the variants in CST3 suggests that specific truncations of Cystatin C result in an abnormal function, possibly by rendering the protein more prone to aggregation. Future studies are required to confirm the assumed effect on the protein and to determine pathophysiologic downstream events at the cellular level.

Genetic counseling: development of requirements, contents, and quality management in Germany.

To carry out quality management of genetic counseling, it is important to know what genetic counseling exactly means and who the players are. The term "genetic counseling" was first defined by Reed in 1947. It describes a communication process dealing with genetic facts and psychosocial aspects and is an education process, too. It has always been understood in the context of individual and family problems, and is unrelated to eugenics. In 1975 the Ad Hoc Committee of the American Society of Human Genetics published a more detailed description. With the development of new diagnostic techniques and methods in human genetics, the requirements of genetic counseling and its contents changed. Today a genetic counselor has to apply diagnostic, predictive, susceptibility, pharmacogenetic, carrier, prenatal, and preimplantation testing, as well as genetic screening. The German Human Genetic Examination Act (Genetic Diagnosis Act - GenDG) and national and international associations recommend to embed genetic testing into genetic counseling. Based on experiences of the author, some examples of pitfalls in genetic counseling are illustrated, as there are so many individual situations and requests that it seems impossible to carry out quality management. Nevertheless, the Commission for Quality in Genetic Counseling and Clinical Genetics of the Professional Association of German Human Geneticists started a pilot ring trial in 2018 with a given counseling situation. The task was to write the human genetics comment with the help of a checklist containing all issues necessary. The evaluation was conducted with the help of a catalogue of criteria which had been established beforehand and a score adjusted to the individual situation. The first genuine pilot trial was launched in 2020. It represents a possibility for quality management in genetic counseling.

A novel strumpellin mutation and potential pitfalls in the molecular diagnosis of hereditary spastic paraplegia type SPG8.

Hereditary spastic paraplegia (HSP) is a clinically and genetically heterogeneous, neurodegenerative movement disorder. A total of eight KIAA0196/strumpellin variants have thus far been associated with SPG8, a rare dominant HSP. We present a novel strumpellin alteration in a small family with clinically pure HSP. We corroborated its causality by comparing it to rare benign variants at several levels, and, along this line, also re-considered previous genetic reports on SPG8. These analyses identified significant challenges in the interpretation of strumpellin alterations, and suggested that at least two of the few families claimed to suffer from SPG8 may have been genetically misdiagnosed.

A novel missense mutation in CACNA1A evaluated by in silico protein modeling is associated with non-episodic spinocerebellar ataxia with slow progression.

Spinocerebellar ataxia type 6 (SCA6), episodic ataxia type 2 (EA2) and familial hemiplegic migraine type 1 (FHM1) are allelic disorders of the gene CACNA1A encoding the P/Q subunit of a voltage gated calcium channel. While SCA6 is related to repeat expansions affecting the C-terminal part of the protein, EA2 and FHM phenotypes are usually associated with nonsense and missense mutations leading to impaired channel properties. In three unrelated families with dominant cerebellar ataxia, symptoms cosegregated with CACNA1A missense mutations of evolutionary highly conserved amino acids (exchanges p.E668K, p.R583Q and p.D302N). To evaluate pathogenic effects, in silico, protein modeling analyses were performed which indicate structural alterations of the novel mutation p.E668K within the homologous domain 2 affecting CACNA1A protein function. The phenotype is characterised by a very slowly progressive ataxia, while ataxic episodes or migraine are uncommon. These findings enlarge the phenotypic spectrum of CACNA1A mutations.

Autosomal recessive spastic ataxia of Charlevoix Saguenay (ARSACS): expanding the genetic, clinical and imaging spectrum.

BACKGROUND: Mutations in SACS, leading to autosomal-recessive spastic ataxia of Charlevoix-Saguenay (ARSACS), have been identified as a frequent cause of recessive early-onset ataxia around the world. Here we aimed to enlarge the spectrum of SACS mutations outside Quebec, to establish the pathogenicity of novel variants, and to expand the clinical and imaging phenotype. METHODS: Sequencing of SACS in 22 patients with unexplained early-onset ataxia, assessment of novel SACS variants in 3.500 European control chromosomes and extensive phenotypic investigations of all SACS carriers. RESULTS: We identified 11 index patients harbouring 17 novel SACS variants. 9/11 patients harboured two variants of at least probable pathogenicity which were not observed in controls and, in case of missense mutations, were located in highly conserved domains. These 9 patients accounted for at least 11% (9/83) in our series of unexplained early onset ataxia subjects. While most patients (7/9) showed the classical ARSACS triad, the presenting phenotype reached from pure neuropathy (leading to the initial diagnosis of Charcot-Marie-Tooth disease) in one subject to the absence of any signs of neuropathy in another. In contrast to its name "spastic ataxia", neither spasticity (absent in 2/9=22%) nor extensor plantar response (absent in 3/9=33%) nor cerebellar ataxia (absent in 1/9=11%) were obligate features. Autonomic features included urine urge incontinence and erectile dysfunction. Apart from the well-established MRI finding of pontine hypointensities, all patients (100%) showed hyperintensities of the lateral pons merging into the (thickened) middle cerebellar peduncles. In addition, 63% exhibited bilateral parietal cerebral atrophy, and 63% a short circumscribed thinning of the posterior midbody of the corpus callosum. In 2 further patients with differences in important clinical features, VUS class 3 variants (c.1373C>T [p.Thr458Ile] and c.2983 G>T [p.Val995Phe]) were identified. These variants were, however, also observed in controls, thus questioning their pathogenic relevance. CONCLUSIONS: We here demonstrate that each feature of the classical ARSACS triad (cerebellar ataxia, spasticity and peripheral neuropathy) might be missing in ARSACS. Nevertheless, characteristic MRI features - which also extend to supratentorial regions and involve the cerebral cortex - will help to establish the diagnosis in most cases.

Early onset and slow progression of SCA28, a rare dominant ataxia in a large four-generation family with a novel AFG3L2 mutation.

Autosomal dominantly inherited spinocerebellar ataxias (SCAs) are a heterogeneous group of neurodegenerative disorders primarily affecting the cerebellum. Genetically, 26 different loci have been identified so far, although the corresponding gene has not yet been determined for 10 of them. Recently, mutations in the ATPase family gene 3-like 2 gene were presented to cause SCA type 28. To define the frequency of SCA28 mutations, we performed molecular genetic analyses in 140 unrelated familial cases with ataxia. Among other variations, we found a novel missense mutation at an evolutionarily conserved amino-acid position using a single-strand conformation polymorphism approach, followed by DNA sequencing. This amino-acid exchange p.E700K was detected in a four-generation German family and was not observed in a survey of 400 chromosomes from healthy control individuals.

Exon deletions and intragenic insertions are not rare in ataxia with oculomotor apraxia 2.

BACKGROUND: The autosomal recessively inherited ataxia with oculomotor apraxia 2 (AOA2) is a neurodegenerative disorder characterized by juvenile or adolescent age of onset, gait ataxia, cerebellar atrophy, axonal sensorimotor neuropathy, oculomotor apraxia, and elevated serum AFP levels. AOA2 is caused by mutations within the senataxin gene (SETX). The majority of known mutations are nonsense, missense, and splice site mutations, as well as small deletions and insertions. METHODS: To detect mutations in patients showing a clinical phenotype consistent with AOA2, the coding region including splice sites of the SETX gene was sequenced and dosage analyses for all exons were performed on genomic DNA. The sequence of cDNA fragments of alternative transcripts isolated after RT-PCR was determined. RESULTS: Sequence analyses of the SETX gene in four patients revealed a heterozygous nonsense mutation or a 4 bp deletion in three cases. In another patient, PCR amplification of exon 11 to 15 dropped out. Dosage analyses and breakpoint localisation yielded a 1.3 kb LINE1 insertion in exon 12 (patient P1) and a 6.1 kb deletion between intron 11 and intron 14 (patient P2) in addition to the heterozygous nonsense mutation R1606X. Patient P3 was compound heterozygous for a 4 bp deletion in exon 10 and a 20.7 kb deletion between intron 10 and 15. This deletion was present in a homozygous state in patient P4. CONCLUSION: Our findings indicate that gross mutations seem to be a frequent cause of AOA2 and reveal the importance of additional copy number analysis for routine diagnostics.

Genetic analysis of candidate genes modifying the age-at-onset in Huntington's disease.

The expansion of a polymorphic CAG repeat in the HD gene encoding huntingtin has been identified as the major cause of Huntington's disease (HD) and determines 42-73% of the variance in the age-at-onset of the disease. Polymorphisms in huntingtin interacting or associated genes are thought to modify the course of the disease. To identify genetic modifiers influencing the age at disease onset, we searched for polymorphic markers in the GRIK2, TBP, BDNF, HIP1 and ZDHHC17 genes and analysed seven of them by association studies in 980 independent European HD patients. Screening for unknown sequence variations we found besides several silent variations three polymorphisms in the ZDHHC17 gene. These and polymorphisms in the GRIK2, TBP and BDNF genes were analysed with respect to their association with the HD age-at-onset. Although some of the factors have been defined as genetic modifier factors in previous studies, none of the genes encoding GRIK2, TBP, BDNF and ZDHHC17 could be identified as a genetic modifier for HD.

Mutations in TITF1 are not relevant to sporadic and familial chorea of unknown cause.

Benign hereditary chorea (BHC; OMIM 118700) is an autosomal dominant movement disorder. Mutations in the thyroid transcription factor 1 (TITF1) gene have been linked with BHC. The phenotype for BHC is highly variable and may include atypical features such as dystonia, slow saccades, and even cognitive deficits. Although BHC is commonly transmitted in a dominant manner, assessment of TITF1 mutations in familial or sporadic patients with late-onset nonprogressive or early-onset progressive chorea is of practical relevance in order to evaluate diagnostic strategies in single patients. In this study, 18 patients with chorea of unknown cause including index patients of three families with autosomal dominantly inherited nonprogressive chorea have been screened for TITF1 mutations by means of denaturating high-pressure liquid chromatography (dHPLC). No sequence variations were detected for the complete open reading frame, suggesting that TITF1 mutations are not a common cause of sporadic or familial chorea of unknown cause. Additionally, linkage analysis excluded TITF1 mutations in a large family with benign hereditary chorea.

The S18Y polymorphism in the UCHL1 gene is a genetic modifier in Huntington's disease.

An expanded polyglutamine stretch in the huntingtin protein has been identified as the pathogenetic cause of Huntington's disease (HD). Although the length of the expanded polyglutamine repeat is inversely correlated with the age-at-onset, additional genetic factors are thought to modify the variance in the disease onset. As linkage analysis suggested a modifier locus on chromosome 4p, we investigated the functional relevance of S18Y polymorphism of the ubiquitin carboxy-terminal hydrolase L1 in 946 Caucasian HD patients. In this group, the allelic variation on locus S18Y is responsible for 1.1% of the variance in the HD age-at-onset, and the rare Y allele is associated with younger-aged cases.

Mutation analysis in the fibroblast growth factor 14 gene: frameshift mutation and polymorphisms in patients with inherited ataxias.

The spinocerebellar ataxias (SCAs) with autosomal dominant inheritance are a group of neurodegenerative disorders with overlapping as well as highly variable phenotypes. Genetically, at least 25 different loci have been identified. Seven SCAs are caused by CAG trinucleotide repeat expansions, for 13 the chromosomal localization is known solely. Recently, a missense mutation in the fibroblast growth factor 14 gene (FGF14) has been reported in a Dutch family with a new dominantly inherited form of SCA. To evaluate the frequency of mutations in the FGF14 gene, we performed molecular genetic analyses for the five exons in 208 nonrelated familial ataxia cases and 208 control samples. In one patient, we detected a novel single base pair deletion in exon 4 (c.487delA) creating a frameshift mutation. In addition, we found DNA polymorphisms in exon 1a, 4, and 5, an amino-acid exchange at position 124, as well as a single-nucleotide polymorphism in the 3'-untranslated region of exon 5.

Ten novel MSH2 and MLH1 germline mutations in families with HNPCC.

Hereditary nonpolyposis colorectal cancer (HNPCC) is one of the most common hereditary cancer-susceptibility syndromes. Germline mutations in mismatch repair genes are associated with the clinical phenotype of HNPCC. We report ten novel germline mutations, three in MSH2 and seven in MLH1. All but one mutation have been found in families fulfilling criteria of the Bethesda guidelines; four of them additionally fulfilled the Amsterdam criteria I or II. Eight mutations were considered pathogenic and predictive diagnostics in healthy family members at risk shall be undertaken; these include five frameshift mutations leading to premature stop codons, in MSH2: c.1672delT (p.S558Xfs) and c.2466_2467delTG (p.C822X) and in MLH1: c.1023delG (p.R341Xfs), c.1127_1128dupAT (p.K377Xfs) and c.1310delC (p.P437Xfs); three mutations leading to splice aberrations, in MSH2: c.1661G>C (r.1511_1661del) and in MLH1: c.677+3A>C (r.589_677del) and c.1990-2A>G predicted to result in a splice site defect. The remaining two mutations are unclassified variants with assumed pathogenicity: one missense mutation in the highly conserved ATPase domain of MLH1 (c.122A>G [p.D41G]) and one in-frame insertion of twelve nucleotides in MLH1 (c.2155_2156insATGTGTTCCACA [p.I719delinsNVFHI]). These two mutations were not found in 102 alleles of healthy control individuals. The corresponding tumors from all patients showed a high level of microsatellite instability (MSI-H). Immunohistochemistry (IHC) revealed complete loss of expression of the affected protein in the tumor cells from all but three patients. The tumors from the patients with the mutations c.1127_1128dupAT and c.1990-2A>G showed a reduction of expression of the MLH1-protein, rather than complete loss. In the tumor from the patient with the missense mutation c.122A>G [p.D41G] a normal expression of the proteins coded by MLH1 and MSH2 was noticed.

Loss of MSH3 protein expression is frequent in MLH1-deficient colorectal cancer and is associated with disease progression.

Mononucleotide repeat sequences are particularly prone to frameshift mutations in tumors with biallelic inactivation of the mismatch repair (MMR) genes MLH1 or MSH2. In these tumors, several genes harboring mononucleotide repeats in their coding region have been proposed as targets involved in tumor progression, among which are also the MMR genes MSH3 and MSH6. We have analyzed the expression of the MSH3 and MSH6 proteins by immunohistochemistry in 31 colorectal carcinomas in which MLH1 was inactivated. Loss of MSH3 expression was identified in 15 tumors (48.5%), whereas all tumors expressed MSH6. Frameshift mutations at coding microsatellites were more frequent in MSH3 (16 of 31) than in MSH6 (3 of 31; Fisher's exact test, P < 0.001). Frameshift mutations and allelic losses of MSH3 were more frequent in MSH3-negative tumors compared with those with normal expression (22 mutations in 30 alleles versus 8 mutations in 28 alleles; chi(2), P = 0.001). Biallelic inactivation was evident or inferred for 60% of MSH3-negative tumors but none of the tumors with normal MSH3 expression. In contrast, we did not identify frameshift mutations in the (A)8 tract of MSH3 in a control group of 18 colorectal carcinomas in which the MMR deficiency was based on the inactivation of MSH2. As it has been suggested that mutations of MSH3 might play a role in tumor progression, we studied the association between MSH3 expression and disease stage assessed by lymph node and distant metastases status. Dukes stages C and D were more frequent in primary tumors with loss of MSH3 expression (9 of 13), compared with tumors with retained expression (1 of 14; Fisher's exact test, P = 0.001), suggesting that MSH3 abrogation may be a predictor of metastatic disease or even favor tumor cell spread in MLH1-deficient colorectal cancers.

Mutations in NSD1 are responsible for Sotos syndrome, but are not a frequent finding in other overgrowth phenotypes.

Recently, deletions encompassing the nuclear receptor binding SET-Domain 1 (NSD1) gene have been described as the major cause of Japanese patients with the Sotos syndrome, whereas point mutations have been identified in the majority of European Sotos syndrome patients. In order to investigate a possible phenotype-genotype correlation and to further define the predictive value of NSD1 mutations, we performed mutational analysis of the NSD1 gene in 20 patients and one familial case with Sotos syndrome, five patients with Weaver syndrome, six patients with unclassified overgrowth/mental retardation, and six patients with macrocephaly/mental retardation. We were able to identify mutations within the NSD1 gene in 18 patients and the familial case with Sotos syndrome (90%). The mutations (six nonsense, eight frame shifts, three splice site, one missense, one in-frame deletion) are expected to result in an impairment of NSD1 function. The best correlation between clinical assessment and molecular results was obtained for the Sotos facial gestalt in conjunction with overgrowth, macrocephaly, and developmental delay. In contrast to the high mutation detection rate in Sotos syndrome, none of the patients with Weaver syndrome, unclassified overgrowth/mental retardation and macrocephaly/mental retardation, harbored NSD1 mutations. We tested for large deletions by FISH analysis but were not able to identify any deletion cases. The results indicate that the great majority of patients with Sotos syndrome are caused by mutations in NSD1. Deletions covering the NSD1 locus were not found in the patients analyzed here.

Identification of six novel MSH2 and MLH1 germline mutations in HNPCC.

Germline mutations in mismatch repair genes are responsible for hereditary nonpolyposis colorectal cancer (HNPCC), the most common hereditary cancer-susceptibility syndrome. We report six novel germline mutations, three in MSH2 and three in MLH1. All but one mutation have been found in families fulfilling the criteria of the Bethesda guidelines; two of them additionally fulfilled the Amsterdam criteria. We identified two nonsense mutations in MSH2 (c.1764T>G [p.Y588X], c.2579C>A [p.S860X]), one duplication of four nucleotides causing premature stop codon (MLH1: c.821_824dupAAGC [p.A275fsX307]), one splice site mutation resulting in skipping of exon 8 from the MLH1 transcript (c.677+3A>G), one duplication of 18 nucleotides leading to duplication of six amino acids in the mismatch-binding domain of MSH2 (c.4_21dup [p.A2_E7dup) and one missense mutation in the PMS2 interaction domain of MLH1 (c.1756G>C [p.A586P]). The three latter mutations were not found in 73, 90 and 94 healthy control individuals, respectively. The corresponding tumors from all patients showed a high level of microsatellite instability (MSI-H). Immunohistochemistry (IH) revealed complete loss of expression of the affected protein in the tumor cells from the patients with the nonsense, splice-site and missense mutation. The tumor from the patient with the c.821_824dupAAGC mutation showed a reduced, rather than lost, expression of the MLH1-protein.

Cardiac energetics correlates to myocardial hypertrophy in Friedreich's ataxia.

Friedreich's ataxia is a neurodegenerative disease frequently associated with hypertrophic cardiomyopathy. We have determined mitochondrial ATP, phosphocreatine, and intracellular inorganic phosphate levels by 31P nuclear magnetic resonance spectroscopy in the heart of 11 Friedreich's ataxia patients and 11 healthy controls. For the first time, to our knowledge, we showed a significant correlation between the extent of myocardial energy deficiency and the degree of myocardial hypertrophy. When combining our results with previous works on Friedreich's ataxia, these novel findings suggest that energy metabolism is most likely the cause and hypertrophy the effect in Friedreich's ataxia.

Apparent genotype-phenotype correlation in childhood, adolescent, and adult Chediak-Higashi syndrome.

Chediak-Higashi syndrome (CHS) is a rare autosomal recessive disorder characterized by severe immunologic defects, reduced pigmentation, bleeding tendency, and progressive neurological dysfunction. Most patients present in early childhood and die unless treated by bone marrow transplantation. About 10-15% of patients exhibit a much milder clinical phenotype and survive to adulthood, but develop progressive and often fatal neurological dysfunction. Very rare patients exhibit an intermediate adolescent CHS phenotype, presenting with severe infections in early childhood, but a milder course by adolescence, with no accelerated phase. Here, we describe the organization and genomic DNA sequence of the CHS1 gene and mutation analysis of 21 unrelated patients with the childhood, adolescent, and adult forms of CHS. In patients with severe childhood CHS, we found only functionally null mutant CHS1 alleles, whereas in patients with the adolescent and adult forms of CHS we also found missense mutant alleles that likely encode CHS1 polypeptides with partial function. Together, these results suggest an allelic genotype-phenotype relationship among the various clinical forms of CHS.

Seven novel MLH1 and MSH2 germline mutations in hereditary nonpolyposis colorectal cancer.

Hereditary nonpolyposis colorectal cancer (HNPCC) is the most frequent hereditary form of colorectal cancer and is caused by germline mutations in mismatch repair (MMR) genes. The majority of mutations occur in MLH1 and MSH2. We report hereby seven novel germline mutations in these two genes (five in MLH1 and two in MSH2). All mutations have been found in families fulfilling criteria of the Bethesda guidelines and four of which also fulfilled the Amsterdam criteria. We identified three insertions or deletions of 1 bp leading to premature stop codons (MLH1: c.341delC, c.1413-1414insA; MSH2: c.1119delG) and three nonsense mutations (MLH1: c.67G>T [E23X], c.436C>T [Q146X]; MSH2: c.1857T>G [Y619X]). The corresponding tumors showed a high level of microsatellite instability (MSI-H) and a complete loss of expression of the affected protein. In addition, a missense mutation in MLH1 was identified (c.1984A>C [T662P]). The respective tumor also showed a high level of microsatellite instability but a reduced, rather then lost, expression of the MLH1-protein. This missense mutation was not found in 107 healthy control individuals and in 54 HNPCC patients.