Further identification of a 140bp sequence from amid intron 9 of human FMR1 gene as a new exon.

BACKGROUND: The disease gene of fragile X syndrome, FMR1 gene, encodes fragile X mental retardation protein (FMRP). The alternative splicing (AS) of FMR1 can affect the structure and function of FMRP. However, the biological functions of alternatively spliced isoforms remain elusive. In a previous study, we identified a new 140bp exon from the intron 9 of human FMR1 gene. In this study, we further examined the biological functions of this new exon and its underlying signaling pathways. RESULTS: qRT-PCR results showed that this novel exon is commonly expressed in the peripheral blood of normal individuals. Comparative genomics showed that sequences paralogous to the 140 bp sequence only exist in the genomes of primates. To explore the biological functions of the new transcript, we constructed recombinant eukaryotic expression vectors and lentiviral overexpression vectors. Results showed that the spliced transcript encoded a truncated protein which was expressed mainly in the cell nucleus. Additionally, several genes, including the BEX1 gene involved in mGluR-LTP or mGluR-LTD signaling pathways were significantly influenced when the truncated FMRP was overexpressed. CONCLUSIONS: our work identified a new exon from amid intron 9 of human FMR1 gene with wide expression in normal healthy individuals, which emphasizes the notion that the AS of FMR1 gene is complex and may in a large part account for the multiple functions of FMRP.

Splicing of exon 9a in FMR1 transcripts results in a truncated FMRP with altered subcellular distribution.

FMRP is an RNA-binding protein, loss of which causes fragile X syndrome (FXS). FMRP has several isoforms resulted from alternative splicing (AS) of fragile X mental retardation 1 (FMR1) gene, but their biological functions are still poorly understood. In the analysis of alternatively spliced FMR1 transcripts in the blood cells from a patient with FXS-like phenotypes (normal CGG repeats and no mutation in coding sequence of FMR1), we identified three novel FMR1 transcripts that include a previously unidentified microexon (46 bp), terming the exon 9a. This microexon exists widely in unaffected individuals, inclusion of which introduces an in-frame termination codon. To address whether these exon 9a-containing transcripts could produce protein by evading nonsense-mediated decay (NMD), Western blot was used to analysis blood cell lysate from unaffected individuals and a 34 kDa protein that consistent in size with the molecular weight of the predicted truncated protein produced from mRNA with this microexon was found. Meanwhile, treatment of peripheral blood mononuclear cells with an inhibitor of NMD (Cycloheximide) did not result in significant increase in exon 9a-containing transcripts. Using confocal immunofluorescence, we found the truncated protein displayed both nuclear and cytoplasmic localization in HEK293T and HeLa cells due to lacking C-terminal domains including KH2, NES, and RGG, while the full-length FMRP protein mainly localized in the cytoplasm. Therefore, we hypothesize that the inclusion of this microexon to generate exon 9a-containing transcripts may regulate the normal functionality of FMRP, and the dysregulation of normal FMRP due to increased exon 9a-containing alternatively spliced transcripts in that patient may be associated with the manifestation of FXS phenotype.

[Phenotypic and genetic analysis of an inv dup(15) case with a BP3:BP3 rearrangement].

OBJECTIVE: To analyze a case of supernumerary marker chromosome (SMC) with combined genetic techniques and explore its correlation with the clinical phenotype. METHODS: The SMC was analyzed with G-banded karyotyping, multiplex ligation dependent probe amplification (MLPA), fluorescence in situ hybridization (FISH), and single nucleotide polymorphism array (SNP-array). RESULTS: G-banding analysis indicated that the patient has a karyotype of 47,XX,+mar. MLPA showed that there were duplications of proximal 15q. FISH assay using D15Z4 probes indicated that the SMC was a pseudodicentric chromosome derived from chromosome 15. And SNP-array revealed that there were two extra copies of 15q11-13 region spanning from locus 20 161 372 to 29 071 810. CONCLUSION: The duplication of Prader-Willi/Angelman syndrome critical region probably underlies the abnormal phenotype of the inv dup(15) case with a BP3:BP3 rearrangement.

Whole-exome sequencing identifies compound heterozygous mutations in ARSA of two siblings presented with atypical onset of metachromatic leukodystrophy from a Chinese pedigree.

BACKGROUND: Metachromatic leukodystrophy (MLD) is a rare inherited lysosomal storage disorder caused mainly by variants in arylsulfatase A (ARSA) gene. MLD can be divided into three major clinical forms according to the age of onset: late infantile, juvenile, and adult. We report two siblings of late infantile MLD presenting with cerebellar ataxia as the only first clinical symptom. METHODS: Because of the unspecific neurological manifestation, whole-exome sequencing (WES) was performed to find disease-causing mutations for molecular diagnosis. Then successive MRI and ARSA activity determination were performed to further confirm the diagnosis. Moreover, the prenatal diagnosis was carried out on the basis of molecular diagnosis. RESULTS: The siblings exhibited compound heterozygous variants {[c.302G>T]+[c.1344dupC]} in the ARSA gene, and both of the variants have been reported as disease-causing mutations previously. The results of MRI and low ARSA activity confirmed the diagnosis of MLD. Prenatal diagnosis showed that the fetus was a heterozygous carrier. CONCLUSIONS: It is recommended that WES be considered as a first line diagnostic procedure to discover potential disease-causing genetic variants in affected individuals with hereditary traits but without definite clinical diagnosis. However, the final diagnosis should be confirmed by comprehensive evaluations including biochemical, enzymatic or imaging investigations.

Hybrid minigene splicing assay verified the pathogenicity of a novel splice site variant in the dystrophin gene of a Chinese patient with typical Duchenne muscular dystrophy phenotype.

BACKGROUND: Duchenne muscular dystrophy (DMD) is typically caused by disrupting the reading frame of the dystrophin gene: approximately 70%-80% of mutational events are represented by deletions or duplications of one or more exons in the dystrophin gene, and the remaining cases by subtle mutations, including point mutations, small indels, small inversions, and complex small rearrangements. The dystrophin gene is the largest known gene with one of the highest known rates of new mutations. METHODS: Deletions and duplications were detected in the DMD gene of the proband by using multiple ligation-dependent probe amplification (MLPA). Targeted next-generation sequencing (NGS) was used in the subtle mutation detection, followed by Sanger sequencing confirmation. The effect of the mutation on the splicing of the DMD gene was assessed by bioinformatics prediction and hybrid minigene splicing assay (HMSA). RESULTS: Neither duplication nor deletion was found in the DMD gene of the proband. While a novel splice site mutation c.6762+1G>C was identified in the proband by NGS and Sanger sequencing, and his mother was heterozygous at the same site. Bioinformatics predicted that the 5' donor splice site of intron 46 disappeared because of the mutation, which would lead to aberrant splicing and introduce premature stop codon. The HMSA results were in agreement with the prediction. CONCLUSIONS: The novel splice site mutation caused DMD in the proband by aberrant splicing. We suggested that combined applications of MLPA, NGS, HMSA and bioinformatics are comprehensive and effective methods for diagnosis and aberrant splicing study of DMD.

[Cytogenetic and molecular genetic analysis of a case with mosaic marker chromosomes].

OBJECTIVE: To explore the source of small supernumerary marker chromosome in a case. METHODS: G-banded karyotyping, fluorescence in situ hybridization, multiple sequence tagged sites (STS) of the Y chromosome, and Illumima Human Cyto SNP-12 Beadchip analysis were carried out. RESULTS: The karyotype was mos 46,X,+mar1[21]/46,X,+mar2[78]. Y chromosome STS analysis has displayed the presence of sy84, sY86, USP9Y and DDX3Y genes from the AZFa region, and sY1227 of the AZFb region, while sY1228, sY1015, sY127, sY134 from the AZFb region, and sY254 and sY255 from the AZFc region were missing. FISH analysis has verified both of the marker chromosomes to be Y chromosome fragments. Mar1 was ish.idic(Y)(q11.2)(SRY++,DXZ1+,DYZ3++,DYZ1-), while mar2 was ish.del(Y)(q11.2)(SRY+,DXZ1+,DYZ3+,DYZ1-). Single nucleotide polymorphism (SNP) microarray analysis showed that the Yq11.2-Yq12 has lost a 10.81 Mb fragment. CONCLUSION: The marker chromosomes were verified to be aberrant Y chromosomes, with the breakage and recombination occurring in Yq11.2. Mar 1 was an isodicentric Y chromosome (idic(Y)pter to q11.2::q11.2 to pter), and mar2 was del(Y)(q11.2). The karyotype was mos 46,X,ish idic(Y)(q11.2)(DYZ3++,SRY++,DXZ1+,DYZ1-)[21]/46,X,ish del(Y)(q11.2)(DYZ3+,SRY+,DXZ1+,DYZ1-)[78]. Combined FISH, Y chromosome STS analysis, SNP microarray analysis and other technologies can facilitate determination of the nature of marker chromosomes.

Alternatively spliced products lacking exon 12 dominate the expression of fragile X mental retardation 1 gene in human tissues.

Fragile X mental retardation 1 gene (FMR1) expression is associated with fragile X syndrome (FXS) and exhibits several splicing products. However, the proportion of spliced isoforms that are expressed in different tissues remains unclear. In the present study, long-chain reverse transcription-polymerase chain reaction with a T cloning-sequencing method was conducted in order to analyze the entire coding region of the FMR1 gene in human tissues. In particular, FXS-associated tissues were analyzed, including the brain and testis. Twenty alternatively spliced isoforms were observed among 271 recombinants, including six novel ones. The isoform that consisted of the entire FMR1 coding region (ISO1) accounted for a small proportion of all isoforms. Isoforms lacking exon 12 were the most abundant. In particular, spliced isoforms ISO7 and ISO17 were the most abundant. However, their relative abundance varied between the peripheral blood cells, and the testis and brain tissues. Bioinformatic analyses suggested that exon 12 may be the sole exon undergoing positive selection. The results of the present study suggested that the mechanisms underlying alternative splicing (AS) of the FMR1 gene may be more complex. Furthermore, the functions of alternatively spliced products lacking exon 12 require further investigation. The results of the present study provide novel insights into the association between AS and the structure and function of the FMR1 gene.

[Analysis of APC gene mutation in a familial adenomatous polyposis pedigree].

OBJECTIVE: To analyze mutation of adenomatous polyposis coli (APC) gene in a family affected with familial adenomatous polyposis. METHODS: The diagnosis was made based on clinical manifestations, family history, presence of numerous polyps in the colon as well as pathological examination. Peripheral blood samples were collected, and genomic DNA was extracted. Potential mutation of the APC gene was detected by polymerase chain reaction (PCR) and DNA sequencing. After finding the mutation in the proband, the same mutation was screened among other family members. The mutation was also confirmed with PCR-restriction fragment length polymorphism (RFLP), with which 100 unrelated healthy controls were examined. RESULTS: A novel heterozygous nonsense mutation c.2891T>G (L964X) of the APC gene was identified in this pedigree. The mutation has led to premature termination of translation. The same mutation was not detected among the 100 healthy controls. CONCLUSION: The c.2891T>G (L964X) of the APC gene probably underlies the familial adenomatous polyposis in this pedigree. The combined DNA sequencing and PCR-RFLP method is efficient and accurate for the diagnosis.

[Genetic diagnosis and analysis of related genes for a pedigree with 2p25 and 12p13 cryptic rearrangements].

OBJECTIVE: To analyze chromosome aberration in a child with mental retardation and abnormalities and its parents. METHODS: Chromosome G banding, multiplex ligation-dependent probe amplification, fluorescence in situ hybridization and single nucleotide polymorphisms array were employed for analysis. RESULTS: Karyotype analysis revealed that the child was 46,XX and the father was 46,XY, while the mother was 46,XX, add (12)(p13). Subtelomeric region analysis with MLPA displayed that the child has reduced ACP1 gene copy number in 2p25 region and increased SLC6A12,KDM5A gene copy numbers in 12p11 region. SNP-array has fine mapped the duplication to 12p13.33-p12.3, a 15.142 Mb region, and a deletion to 2p25.3 for 3.194 Mb, which resulted in duplication of 9 genes including SLC6A12 as well as deletion of 11 genes including SNTG2, respectively. FISH analysis revealed that the child was 46,XX,ish,der(2),t(2;12)(p25;p13)mat, or partial monosomy 2p25 and partial trisomy 12p13. In addition,the mother was a carrier with cryptic balanced translocation chromosome, 46,XX,isht(2;12) (p25;p13). Mental abnormalities and retardation of the child may be attributed to heterozygous deletion of SNTG2, MYT1L genes and duplication of SLC6A12 gene. CONCLUSION: Combined use of MLPA, FISH and SNP-array can facilitate accurate diagnosis of cryptic rearrangement at genomic level.

[Analysis of small supernumerary marker chromosome 15q11 in four infertile males].

OBJECTIVE: To delineate the origins of small supernumerary marker chromosomes (sSMCs) identified in 4 infertile males. METHODS: The sSMCs were analyzed with combined G-banding, N-banding, multiplex ligation-dependent probe amplification (MLPA), fluorescence in situ hybridization (FISH) and single nucleotide polymorphisms array (SNP-array) techniques. RESULTS: G-banding analysis has suggested a 46,X,-Y,+mar karyotype in all of the 4 cases. N-banding revealed that all of the sSMCs have possessed two satellites located on both sides. By MLPA, 1 patient showed copy number gains for 15q11.2 region. SNP-array analysis suggested that all had duplication for 15q11.1-q11.2 region, spanning 3.06 Mb, 0.9118 Mb, 1.728 Mb and 0.287 Mb, respectively. By FISH analysis, all of the sSMCs showed two hybridization signals, indicating that they were dicentric chromosomes. CONCLUSION: In all of the four cases, the marker chromosomes have derived from chromosome 15 and were bisatellited and dicentric, which gave rise to a karyotype of 47,XY,+ish,inv dup(15)(q11)(D15Z4++). sSMC 15q11 therefore may be a major cause for male infertility.

[Delineation of three structural Y chromosome aberrations combined molecular techniques].

OBJECTIVE: To delineate the structure of Y chromosome aberrations and recombinant mechanisms for three patients. METHODS: Karyotype analysis, multiplex ligation dependent probe amplification (MLPA), fluorescence in situ hybridization (FISH), Y chromosome sequence tagged sites (STS) analysis, human whole genome-wide SNP array were used. RESULTS: The karyotypes of the three patients were 46, X, +mar. As suggested by MLPA analysis, case 1 has increased copy numbers of SRY, ZFY and UTY genes, case 2 had increased copies of SRY and ZFY genes, and deletion of UTY gene, and case 3 had decreased copies for subtelomeric regions of X/Yp and X/Yq. By STSs analysis, case 1 has retained SRY, sY84 and sY86 in the AZFa region, sY1227 in the AZFb region, whilst lost sY1228 in the AZFb region and other STSs in the AZFc region. Its breakpoint was thereby mapped between sY1227 and sY1228. Case 2 has retained SRY and sY1200 in the centromeric region, whilst has deletion of other STSs. Case 3 has retained SRY and STSs in the AZF regions. By SNP array, case 1 had duplicated Yp11.31-p11.2 and deletion of Yq11.22-q11.23 (approximately 5.18 Mb). Case 2 had duplicated Yp11.31-p11.2 and deletion of Yq11.21-q11.23 (approximately 14.644 Mb). Case 3 had single copy number deletion of p22.33 and q28 in the subtelomeric region of X/Yp and X/Yq. By FISH, cases 1 and 2 showed two signals for SRY and DYZ3 but no signal for DYZ1 on their marker chromosomes. Combining above results, the karyotypes of cases 1, 2 and 3 were determined as 46, X, idic(Y) (q11.23), 46, X, idic(Y) (q10) and 46, X, r(Y) (p11q12), respectively. CONCLUSION: Y chromosome aberrations are variable. Combined use of MLPA, STSs, FISH and SNP array is effective for revealing the breakpoints and recombinant mechanisms.

Familial skewed x chromosome inactivation in adrenoleukodystrophy manifesting heterozygotes from a Chinese pedigree.

BACKGROUND: X-linked adrenoleukodystrophy (X-ALD) is an inherited neurodegenerative disorder caused by mutations in the ABCD1 gene. Approximately 20% of X-ALD female carriers may develop neurological symptoms. Skewed X chromosome inactivation (XCI) has been proposed to influence the manifestation of symptoms in X-ALD carriers, but data remain conflicting so far. We identified a three generation kindred, with five heterozygous females, including two manifesting carriers. XCI pattern and the ABCD1 allele expression were assessed in order to determine if symptoms in X-ALD carriers could be related to skewed XCI and whether skewing within this family is more consistent with genetically influenced or completely random XCI. RESULTS: We found a high frequency of skewing in this family. Four of five females had skewed XCI, including two manifesting carriers favoring the mutant allele, one asymptomatic carrier favoring the normal allele, and one female who was not an X-ALD carrier. Known causes of skewing, such as chromosomal abnormalities, selection against deleterious alleles, XIST promoter mutations, were not consistent with our results. CONCLUSIONS: Our data support that skewed XCI in favor of the mutant ABCD1 allele would be associated with the manifestation of heterozygous symptoms. Furthermore, XCI skewing in this family is genetically influenced. However, the underlying mechanism remains to be substantiated by further experiments.

Establishment of a molecular diagnostic system for spinal muscular atrophy experience from a clinical laboratory in china.

Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder characterized by degeneration of the anterior horn of the spinal cord. The disease gene survival motor neuron 1 (SMN1) is homozygously absent in approximately 95% of patients, and approximately 5% of patients are believed to have subtle mutations. Although methods for molecular diagnosis of SMA have been reported singly, no diagnostic methodological system to tackle different SMA cases has been reported. Thirty-two families affected by SMA enrolled into this study. Our system comprised PCR-restriction fragment length polymorphism and allele-specific PCR for homozygous deletion analysis of SMN1, multiplex ligation-dependent probe amplification analysis for the determination of the copy number of SMN1, and SMN1 subtle mutation analysis at both the transcript and genomic levels. In 23 families, 21 patients had a homozygous deletion of SMN1. The remaining two patients without a deletion had a single SMN1 copy containing the subtle mutations S230L and L228X, respectively. In nine families in whom samples from the index patients were unavailable, parents from eight families showed one SMN1 copy, and one parent in the remaining family showed two SMN1 copies, one being normal and the other carrying the subtle mutation 22_23insA. To our knowledge, our methodological system for the molecular diagnosis of SMA offers the most complete evaluation of family members affected by SMA at this time.

[Partial AZfc region deletions of the Y chromosome in spermatogenic dysfunction patients].

OBJECTIVE: To investigate the influence of partial deletions in the AZFc region of the Y chromosome on spermatogenesis. METHODS: We selected 9 sequence tagged sites (sY1258, sY1291, sY254, sY255, sY1201, sY1206, sY1161, sY1197 and sY1191) in the AZFc region of the Y chromosome, with ZFX/ZFY and SRY (sY14) as the interior control. We amplified by multiplex PCR the DNA of 160 patients with azoospermia or severe oligozoospermia that showed no microdeletion of the Y chromosome (the case group) and another 76 males with normal fertility (the control group). For the individuals suspected of DAZ gene deletion, we detected the single nucleotide polymorphism sites (SNPs) in the four copies of the DAZ gene by single nucleotide variation (SNV) analysis to determine the types of DAZ copy deletion. RESULTS: In the case group, there were 10 cases of gr/gr (sY1291) deletion (6.3%), 14 b2/b3 (sY1191) deletion (8.8%), 1 sY1291,sY1197 deletion (0.6%), 1 b1/b2 deletion (0.6%) and 1 b1/b3 deletion (0.6%), while in the control group, there were 4 cases of gr/gr deletion (5.3%) and 4 b2/b3 deletion (5.3%). SNV analysis showed DAZ1/DAZ2 deletion in all those with gr/gr and those with b1/b3 deletion, DAZ3/DAZ4 deletion in those with b2/b3 deletion, and DAZ-SNV sY587 deletion in 1 case of sY1291, sY1197 deletion, but no DAZ deletion was found in 1 case of b1/b2 deletion. CONCLUSION: B2/b3 (sY1191) and gr/gr (sY1291) deletions are genomic polymorphisms and quite common in the normal Chinese population; while b1/b2, b1/b3, and sY1291, sY1197 deletions may be high risk factors of dyszoospermia.

Pre-symptomatic molecular diagnosis of X-linked adrenoleukodystrophy in Chinese families.

OBJECTIVE: To identify asymptomatic males with X-linked adrenoleukodystrophy (X-ALD) from Chinese pedigrees by molecular genetic testing. METHODS: Genomic DNA was extracted from peripheral blood of the asymptomatic individuals from X-ALD families, and fragments spanning the proband's mutation were amplified. PCR-RFLP, direct sequencing and denaturing high performance liquid chromatography (DHPLC) were used to detect the PCR products. RESULTS: Four asymptomatic male subjects from three Chinese X-ALD pedigrees were found to carry the same mutation with the probands. In Pedigree 1, by restriction analysis with endonuclease Eco47 I, the digestion pattern of the proband's elder brother (Subject 1) was same with the proband, which indicated that both carried the same mutation. In Pedigree 2 and Pedigree 3, the PCR products were analysed by DHPLC, and the patterns of elution peaks of the Subjects 2-4 and the heterozygous mothers were similar, which indicated the presence of sequence alterations in the ABCD1 gene. DNA sequencing of the corresponding PCR products confirmed the mutations. CONCLUSIONS: Molecular testing was an effective way to determine the genotype of family members of X-ALD before they develop any symptoms. Early and preferable pre-symptomatic identification of hemizygotes is of great benefit to affected individuals and their families.

[Mutation analysis of SMN gene in a patient and his family with spinal muscular atrophy].

OBJECTIVE: To perform mutation analysis and describe the genotype of the SMN gene in a patient with spinal muscular atrophy (SMA) and his family. METHODS: Deletion analysis of the SMN1 exon 7 by conventional PCR-restriction fragment length polymorphism (RFLP) and allele-specific PCR, and gene dosage of SMN1 and SMN2 by multiplex ligation-dependent probe amplification (MLPA) were performed for the patient and his parents; reverse transcriptase (RT)-PCR and sequencing were performed for the patient. To determine whether the SMN variant was exclusive to transcripts derived from SMN1, the RT-PCR product of the patient was subcloned and multiple clones were sequenced directly; PCR of SMN exon 5 from the genomic DNA of the parents and direct sequencing were performed to confirm the mutation. RESULTS: In SMN1 exon 7 deletion analysis, no homozygous deletion of the SMN1 was observed in the family; the gene dosage analysis by MLPA showed that the patient had 1 copy of SMN1 and 1 copy of SMN2 his father had 2 copies of SMN1 and 2 copies of SMN2, and his mother had 1 copy of SMN1 and no SMN2. A previously unreported missense mutation of S230L was identified from the patient and this mutation was also found in his father. CONCLUSION: A novel missense mutation of S230L was identified in the SMA family and the genotype of the family members were investigated.

[Molecular diagnosis of a Chinese pedigree with osteogenesis imperfecta type I].

OBJECTIVE: To perform molecular diagnosis for a Chinese pedigree with osteogenesis imperfecta type I. METHODS: Thirty pairs of primers were designed to amplify all the 52 exons, exon boundaries and promoter region of the COL1A1 gene from genomic DNA of peripheral blood cells of the family members. The PCR products were purified and directly sequenced. To check the mutation in normal controls, the genomic DNA from peripheral blood cells of the index patient, his mother and 60 normal controls were analyzed by amplification refractory mutation system. RESULTS: A missense mutation of GAT>CAT was identified at codon 1441 of the COL1A1 gene from the family, which resulted in the replacement of aspartic acid by histidine (D1441H). This mutation was not found in a group of 60 normal controls. CONCLUSION: The method for molecular diagnosis of osteogenesis imperfecta was established and a novel COL1A1 gene mutation, D1441H, was identified in the Chinese pedigree with osteogenesis imperfecta type I.

Identification of two de novo mutations in Chinese patients with X-linked adrenoleukodystrophy.

BACKGROUND: Mutations in the ABCD1 gene lead to X-linked adrenoleukodystrophy, a neurodegenerative disorder. Hundreds of hereditary mutations of the gene have been reported in patients with X-linked adrenoleukodystrophy, but there have been no reports of de novo mutations. METHODS: The coding region of ABCD1 cDNA of two patients was amplified and sequenced. To confirm the mutations in the ABCD1 gene of the patients and screen for mutations in their family members, the genomic DNA was analyzed by direct sequencing and denaturing high performance liquid chromatography. RESULTS: Two missense mutations (C631Y and G512S) were identified in the probands, but the mutations were not found in their parents. Tests for paternity identification excluded the possibility of misparentage. CONCLUSIONS: The mutations identified in the two male patients were de novo mutations. Mutation analysis of parents of the proband may be helpful for pregnancy planning and evaluation of the recurrence risk to siblings of the proband.