Genomic screening in rare disorders: New mutations and phenotypes, highlighting ALG14 as a novel cause of severe intellectual disability.

We have investigated 20 consanguineous families with multiple children affected by rare disorders. Detailed clinical examinations, exome sequencing of affected as well as unaffected family members and further validation of likely pathogenic variants were performed. In 16/20 families, we identified pathogenic variants in autosomal recessive disease genes (ALMS1, PIGT, FLVCR2, TFG, CYP7B1, ALG14, EXOSC3, MEGF10, ASAH1, WDR62, ASPM, PNPO, ERCC5, KIAA1109, RIPK4, MAN1B1). A number of these genes have only rarely been reported previously and our findings thus confirm them as disease genes, further delineate the associated phenotypes and expand the mutation spectrum with reports of novel variants. We highlight the findings in two affected siblings with splice altering variants in ALG14 and propose a new clinical entity, which includes severe intellectual disability, epilepsy, behavioral problems and mild dysmorphic features, caused by biallelic variants in ALG14.

Deletions in the 3' part of the NFIX gene including a recurrent Alu-mediated deletion of exon 6 and 7 account for previously unexplained cases of Marshall-Smith syndrome.

Marshall-Smith syndrome (MSS) is a very rare malformation syndrome characterized by typical craniofacial anomalies, abnormal osseous maturation, developmental delay, failure to thrive, and respiratory difficulties. Mutations in the nuclear factor 1/X gene (NFIX) were recently identified as the cause of MSS. In our study cohort of 17 patients with a clinical diagnosis of MSS, conventional sequencing of NFIX revealed frameshift and splice-site mutations in 10 individuals. Using multiplex ligation-dependent probe amplification analysis, we identified a recurrent deletion of NFIX exon 6 and 7 in five individuals. We demonstrate this recurrent deletion is the product of a recombination between AluY elements located in intron 5 and 7. Two other patients had smaller deletions affecting exon 6. These findings show that MSS is a genetically homogeneous Mendelian disorder. RT-PCR experiments with newly identified NFIX mutations including the recurrent exon 6 and 7 deletion confirmed previous findings indicating that MSS-associated mutant mRNAs are not cleared by nonsense-mediated mRNA decay. Predicted MSS-associated mutant NFIX proteins consistently have a preserved DNA binding and dimerization domain, whereas they grossly vary in their C-terminal portion. This is in line with the hypothesis that MSS-associated mutations encode dysfunctional proteins that act in a dominant negative manner.

Phenotype and natural history in Marshall-Smith syndrome.

Marshall-Smith syndrome (MSS) is a distinctive entity of unknown etiology with fewer than 50 patients described in the medical literature to date. Through an International collaboration and use of an online wiki to facilitate data collection and sharing, we further delineate the phenotype and natural history of this syndrome. We present 15 new patients, the oldest being 30 years, provide an update on four previously published cases, and compare all patients with other patients reported in literature. Main clinical features are moderate to severe developmental delay with absent or limited speech, unusual behavior, dysharmonic bone maturation, respiratory compromise secondary to upper airway obstruction, short stature, and kyphoscoliosis. Facial features are characteristic with high forehead, underdeveloped midface, proptosis, anteverted nares, and everted lips. Minor abnormalities of brain morphology such as hypoplasia of the corpus callosum are common. Mortality from respiratory complications is high, but airway support increasingly allows survival into adulthood. Array-CGH was performed on 12 of the cohort and no copy number variants of clear clinical relevance were identified. The present study is the first reported use of an online wiki to aid delineation of a genetic syndrome, and illustrates its value in collecting detailed data in rare conditions.

A chromosome 10 variant with a 12 Mb inversion [inv(10)(q11.22q21.1)] identical by descent and frequent in the Swedish population.

We identified a paracentric inversion of chromosome 10 [inv(10)(q11.22q21.1)] in 0.20% of Swedish individuals (15/7,439) referred for cytogenetic analysis. A retrospective analysis of 8,896 karyotypes from amniocenteses in Sweden revealed a carrier frequency of 0.079% (7/8,896) for the inversion. Cloning and detailed analysis of the inversion breakpoint regions show enrichment for interspersed repeat elements and AT-stretches. The centromeric breakpoint coincides with that of a predicted inversion from HapMap data, which suggests that this region is involved in several chromosome 10 variants. No known gene or predicted transcript are disrupted by the inversion which spans approximately 12 Mb. Carriers from four non-related Swedish families have identical inversion breakpoints and haplotype analysis confirmed that the rearrangement is identical by descent. Diagnosis was retrieved in 6 out of the 15 carriers referred for cytogenetic analysis. No consistent phenotype was found to be associated with the inversion. Our study demonstrates that the inv(10)(q11.22q21.1) is a rare and inherited chromosome variant with a broad geographical distribution in Sweden.

SPG11 mutations cause Kjellin syndrome, a hereditary spastic paraplegia with thin corpus callosum and central retinal degeneration.

Autosomal recessive hereditary spastic paraplegia (ARHSP) with thin corpus callosum (TCC) is genetically heterogenous and approximately 35% of patients carry mutations in either of the SPG11 or SPG15 genes. Disease onset is during the first three decades of life with spastic paraplegia and mental impairment. Peripheral neuropathy and amyotrophy may occur. Kjellin syndrome is characterized by central retinal degeneration in addition to ARHSP-TCC and the disease is associated with mutations in the SPG15 gene. We identified five patients in four unrelated kindreds with spastic paraplegia and mental impairment. Magnetic resonance imaging revealed TCC, atrophy elsewhere in the brain and increased T2 signal intensity in the periventricular white matter. Probands from the four kindreds were screened for mutations in the SPG11 gene. All patients were found homozygous or compound heterozygous for truncating SPG11 mutations of which four are reported for the first time. Ophthalmological investigations revealed that the four index cases have central retinal degeneration consistent with Kjellin syndrome. PET examinations with N-[11C-methyl]-L-deuterodeprenyl (DED) and fluor-18 2-fluorodeoxyglucose (FDG) were performed in two patients with Kjellin syndrome. We observed a reduced glucose uptake in the thalami, anterior cingulum, and sensorimotor cortex indicating neuronal loss, and an increased DED binding in the thalami and pons which suggests astrogliosis. From our results we extend the SPG11 associated phenotype to comprise also Kjellin syndrome, previously found to be associated with mutations in the SPG15 gene. We anticipate that degeneration of the central retina is a common and previously unrecognized feature in SPG11 related disease.

Phonetograms as a tool in the voice clinic: changes across voice therapy for patients with vocal fatigue.

UNLABELLED: This study discusses phonetogram recordings as a tool in the voice clinic. It reports experiences during recordings and changes in measures across voice therapy for women with vocal fatigue. Phonetogram data are discussed along with subglottal pressure measurements and subjective evaluations of voice function and quality. Assessments were made pre-, mid-, and post behaviorally based voice therapy. MEASURES: Maximum voice range profile (VRPmax), subglottal pressure, patient's and speech and language pathologist's (SLP) ratings of voice function and quality, and voice handicap index (VHI). RESULTS: Patients and SLPs often agreed in direction of voice change across therapy. Subglottal pressure did not change systematically across therapy. VHI had decreased and VRPmax increased after therapy, although not to normal values. Increased VRPmax for individuals was mainly due to extended capacity in the low intensities; high intensities did not change noticeably. Changes tended to occur after the mid-therapy session, suggesting that the therapy should not be shortened. The results and experiences from the assessments are discussed in terms of the use of phonetograms as a tool in the voice clinic and for voice therapy outcome evaluation.

Multiplex ligation-dependent probe amplification improves diagnostics in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive disease caused by decreased levels of survival motor neuron protein (SMN). In the majority of cases, this decrease is due to absence of the SMN1 gene. Multiplex ligation-dependent probe amplification (MLPA) is a modern quantitative molecular method. Applied in SMA cases, it improves diagnostics by simultaneously identifying the number of copies of several target sequences in the SMN1 gene and in nearby genes. Using MLPA in clinical diagnostics, we have identified a previously unreported, partial deletion of SMN1 (exons 1-6) in two apparently unrelated Swedish families. This mutation would not have been detected by conventional diagnostic methods. This paper illustrates the broad clinical and genetic spectrum of SMA and includes reports of MLPA results and clinical descriptions of a patient with homozygous absence of SMN1 and only one SMN2 (prenatal onset SMA type 1), an asymptomatic woman with five SMN2 (lacking SMN1) and representative patients with SMA types 1, 2 and 3.

Genome-wide screening using array-CGH does not reveal microdeletions/microduplications in children with Kabuki syndrome.

Kabuki syndrome (KS) is a rare multiple congenital anomaly/mental retardation syndrome. It is characterized by a distinct facial appearance, mental retardation, postnatal growth retardation, skeletal anomalies, unusual dermatoglyphics and fetal fingertip pads. It has previously been speculated that KS is caused by a microdeletion or duplication. In a recent report, an interstitial microduplication of 8p22-23.1 was presented in several cases with this disorder. We investigated 10 Caucasian patients diagnosed with KS by fluorescence in situ hybridization and microsatellite markers located on 8p22-23.1. Using the same clones that were previously reported to be duplicated on chromosome 8p, we could exclude the duplication in all our patients. In addition, we performed a genome-wide screening on this group of patients using array-based comparative genomic hybridization containing BAC clones spaced at approximately 1 Mb intervals across the genome and could not find any evidence for gene dose alterations. The characteristics of KS are variable, a fact that complicates the diagnosis of this disorder. It is possible that we will find genetic heterogeneity among Kabuki patients, and therefore it is unlikely that all patients have an interstitial 8p duplication. We conclude that the etiology of KS remains to be solved and further genetic studies are necessary to delineate its genetic cause.

Identification of genomic deletions of the APC gene in familial adenomatous polyposis by two independent quantitative techniques.

Large deletions in the APC (adenomatous polyposis coli) gene, causing familial adenomatous polyposis (FAP), cannot easily be detected by conventional mutation-detection techniques. Therefore, we have developed two independent quantitative methods for the detection of large deletions, encompassing one or more exons, of APC. Multiplex ligation-dependent probe amplification (MLPA) is performed in one reaction for the initial quantification of all APC exon copy numbers. Subsequently, quantitative real-time PCR (QRT-PCR) is used to verify the results obtained in the MLPA reaction. The identification of a deletion of the whole APC gene in a patient with classical FAP is described. The mutation was detected with the two quantitative methods and further verified on chromosomal level by the use of FISH (fluorescence in situ hybridization) on metaphase spreads. Furthermore, a large deletion covering exons 11-13 of the APC gene was detected in two apparently unrelated families. This deletion was further verified and characterized with long-range PCR. The MLPA test ensures a sensitive high-throughput screening for large deletions of the APC gene and can easily be implemented in the diagnostic testing for FAP.

Aerodynamic and acoustic voice measurements of patients with vocal nodules: variation in baseline and changes across voice therapy.

An important clinical issue concerns the efficacy of current voice therapy approaches in treating voice disorders, such as vocal nodules. Much research focuses on finding reliable methods for documentation of treatment results. In this second treatment study of ten patients with vocal nodules, who participated in a behaviorally based voice therapy program, 11 aerodynamic (transglottal air pressure and glottal waveform) and acoustic (spl, f0, and spectrum slope) measures were used. Three pretherapy baseline assessments were carried out, followed by one assessment after each of five therapy phases. Measurements were made of two types of speech materials: Strings of repeated /pae/ syllables and sustained /ae/ phonations in two loudness conditions: comfortable loudness and loud voice. The data were normalized using z-scores, which were based on data from 22 normal subjects. The results showed that the aerodynamic measures reflected the presence of vocal pathology to a higher degree than did the acoustic spectral measures, and they should be useful in studies comparing nodule and normal voice production. Large individual session-to-session variation was found for all measures across pretherapy baseline recordings, which contributed to nonsignificant differences between baseline and therapy data.

Speech and voice range profiles of adults with untrained normal voices: methodological implications.

Automatic recordings were made of speech and voice range profiles for 63 vocally healthy Australian men and women without voice training (30 males and 33 females aged 21 to 65 years). Test-retest reliability, evaluated for a subgroup, was high. Speech range profile results were consistent with results reported by others. However, voice range profiles were larger than shown in several previous studies. Nevertheless, voice range profiles were consistent with results reported for a recent study that used a similar elicitation and recording protocol and similar equipment. Results are discussed with reference to methodological issues important for reliable phonetogram recordings. The data may also be clinically useful for comparisons between disordered and healthy voices if similar equipment and elicitation and recording protocols are used.

Leiomodin-3 dysfunction results in thin filament disorganization and nemaline myopathy.

Nemaline myopathy (NM) is a genetic muscle disorder characterized by muscle dysfunction and electron-dense protein accumulations (nemaline bodies) in myofibers. Pathogenic mutations have been described in 9 genes to date, but the genetic basis remains unknown in many cases. Here, using an approach that combined whole-exome sequencing (WES) and Sanger sequencing, we identified homozygous or compound heterozygous variants in LMOD3 in 21 patients from 14 families with severe, usually lethal, NM. LMOD3 encodes leiomodin-3 (LMOD3), a 65-kDa protein expressed in skeletal and cardiac muscle. LMOD3 was expressed from early stages of muscle differentiation; localized to actin thin filaments, with enrichment near the pointed ends; and had strong actin filament-nucleating activity. Loss of LMOD3 in patient muscle resulted in shortening and disorganization of thin filaments. Knockdown of lmod3 in zebrafish replicated NM-associated functional and pathological phenotypes. Together, these findings indicate that mutations in the gene encoding LMOD3 underlie congenital myopathy and demonstrate that LMOD3 is essential for the organization of sarcomeric thin filaments in skeletal muscle.

Helical mutations in type I collagen that affect the processing of the amino-propeptide result in an Osteogenesis Imperfecta/Ehlers-Danlos Syndrome overlap syndrome.

BACKGROUND: Whereas mutations affecting the helical domain of type I procollagen classically cause Osteogenesis Imperfecta (OI), helical mutations near the amino (N)-proteinase cleavage site have been suggested to result in a mixed OI/Ehlers-Danlos syndrome (EDS)-phenotype. METHODS: We performed biochemical and molecular analysis of type I (pro-) collagen in a cohort of seven patients referred with a clinical diagnosis of EDS and showing only subtle signs of OI. Transmission electron microscopy of the dermis was available for one patient. RESULTS: All of these patients harboured a COL1A1 / COL1A2 mutation residing within the most N-terminal part of the type I collagen helix. These mutations affect the rate of type I collagen N-propeptide cleavage and disturb normal collagen fibrillogenesis. Importantly, patients with this type of mutation do not show a typical OI phenotype but mainly present as EDS patients displaying severe joint hyperlaxity, soft and hyperextensible skin, abnormal wound healing, easy bruising, and sometimes signs of arterial fragility. In addition, they show subtle signs of OI including blue sclerae, relatively short stature and osteopenia or fractures. CONCLUSION: Recognition of this distinct phenotype is important for accurate genetic counselling, clinical management and surveillance, particularly in relation to the potential risk for vascular rupture associated with these mutations. Because these patients present clinical overlap with other EDS subtypes, biochemical collagen analysis is necessary to establish the correct diagnosis.

Voice and speech range profiles and Voice Handicap Index for males--methodological issues and data.

Reference data for speech range profiles (SRP), voice range profiles (VRP), and Voice Handicap Index (VHI) are presented for Swedish males (n = 30). For comparisons, individual data for four male contact granuloma patients are also reported. For the vocally healthy group mean values were: speaking fundamental frequency 123 Hz (SD 12.1), speaking equivalent level, Leq, 72.2 dB (SD 2.1), SRP area 142 ST*dB (SD 24.1), and VRP area 1,706 ST*dB (SD 340). Mean VHI was 5 (SD 4.8). Test-retest recordings of VRP and SRP for three subjects suggested good reliability. SRP and VRP values for three of the patients fell more than 2 SD outside the reference values. Protocols and results are discussed and standardized recording and analyses procedures are suggested.

Phonetograms, aerodynamic measurements, self-evaluations, and auditory perceptual ratings of male-to-female transsexual voice.

OBJECTIVES: This exploratory study reports instrumental and subjective data for 25 male-to-female transsexual (M-F TS) individuals using their attempted female voice. The aim was to examine the usefulness of phonetograms and aerodynamic measures for voice assessment of this client group. STUDY DESIGN: Descriptive and correlational. METHODS: Phonetogram speech-range profiles (SRPs) were recorded for the M-F TS participants' attempted female voice. Transglottal air pressure and airflow were estimated from oral recordings. All recordings were made in typical- and loud-voice conditions. Relationships among acoustical and aerodynamic measurements, background data, self-evaluations, and auditory perceptual ratings were examined. M-F TS data were compared with male and female normative data. RESULTS: Agreement between naive and voice-expert listeners as well as intra- and interlistener reliability was good. Fundamental frequency (F(0)) accounted for 41-49% of variation in gender ratings for the group, but individual exceptions were found. Background data did not account for female voice success. Perceptual ratings of strain and breathiness were low. No data indicated hyperfunctional vocal behavior. The aerodynamic data agreed with normative male high-pitch data. The speech sound pressure level (SPL) was higher than the female norms. Phonetogram speech-range data fell between male and female data. CONCLUSIONS: The importance of speaking fundamental frequency (SFF) in perception of gender was confirmed. Instrumental and subjective data suggested that the use of low speech intensities and avoidance of vocal fry could help contribute to a successful female voice. Phonetograms were suggested to be useful for visual feedback and documentation of changes in voice therapy for M-F TS clients.

Mutations in CCBE1 cause generalized lymph vessel dysplasia in humans.

Lymphedema, lymphangiectasias, mental retardation and unusual facial characteristics define the autosomal recessive Hennekam syndrome. Homozygosity mapping identified a critical chromosomal region containing CCBE1, the human ortholog of a gene essential for lymphangiogenesis in zebrafish. Homozygous and compound heterozygous mutations in seven subjects paired with functional analysis in a zebrafish model identify CCBE1 as one of few genes causing primary generalized lymph-vessel dysplasia in humans.

A family with pyruvate dehydrogenase complex deficiency due to a novel C>T substitution at nucleotide position 407 in exon 4 of the X-linked Epsilon1alpha gene.

UNLABELLED: The pyruvate dehydrogenase complex (PDHc; McKusick 312170), localised in the mitochondrial matrix, is a multienzyme complex which converts pyruvate to acetyl-CoA. A deficiency of PDHc leads to inadequate removal of pyruvate and lactate resulting in lactic acidaemia and insufficient energy production. The major cause of PDHc deficiency is a defect in the E1alpha component. The gene of this component is localised to Xp22.1. We describe two brothers with a relatively mild clinical phenotype of PDHc deficiency. Onset of disease was associated with muscle weakness and swallowing difficulties in both. At follow-up, the older brother developed encephalopathic features consistent with Leigh syndrome. Lactate to pyruvate ratios were low, consistent with a PDHc deficiency which was confirmed by measurements of PDHc activity in thrombocytes. A 407C>T change in exon 4 of the E1alpha gene was found in both brothers and their mother. This substitution predicts a replacement of a conserved alanine at position 136 by valine. CONCLUSION: Due to the X-linked inheritance pattern combined with the overall results of clinical investigations, molecular genetic findings and a corresponding functional deficiency of the gene product we believe that this substitution in the pyruvate dehydrogenase E1alpha gene is a mutation leading to pyruvate dehydrogenase complex deficiency in this family.