Background
Methods
Cytogenetic and FISH analysis
DNA methylation study
Expression studies by RT-PCR and quantitative RT-PCR
RT-PCR | ||||
---|---|---|---|---|
Gene/Exon | fwd (5'-3') | rev (5'-3'); complement strand | size | T ann. |
ZNF127 | GGG TTG CGG TTT TGC TAT TA | TTT CTC GTG TGC TTC AAT GC | 168 bp | 59C |
MAGEL2 | CTG AAG CCT GGG ACT TTC TG | GGA CCT TGG CCA CAA ACT TA | 225 bp | 59C |
Necdin | GAA GAA GCA CTC CAC CTT CG | CCA TGA TTT GCA TCT TGG TG | 164 bp | 59C |
SNRPN Ex 1–3 | ATG GAG CGG GCA AGG GAT CGC | GGT ACA ACT GAC ACT CTT GG | 124 bp | 53C |
SNRPN Ex 14/16 | CTG CAA ACA TAG GAG ATG ATA GTT CC | CTT ATG AAA GCA CTG AGA TGA AGC C | 459 bp | 53C |
SNRPN Ex 16/17 | GAA AGT GAC CTA AAG AGT GTC ATT G | CTT GCA GTT GGA CAG CCG ACT C | 515 bp | 53C |
SNRPN Ex 17/18 | AGA TAT CTT TAA AAT TGA GTC TTC TGT CCA | TGA AGA TGC AGC ACT TTT GAA GAA | 218 bp | 53C |
SNRPN Ex 19/20a | CAT TGT GCT TAT TTA CTA TTT TTG TAG ACG | CTG CAG GTG GTG ACC ATG TG | 150 bp | 53C |
AK094315 | TCT TCT CTA CCC TCA TTC CCA GC | TCG CTA CAC CCC TTT GCT TAT G | 222 bp | 53C |
AB061718 | AGG AGG GGT TCA AAG ATG C | CTG GTA AAC AAA CTG GTA AAG GTG | 204 bp | 50C |
HBII-85/PWCR1 | CGA TGA TGA GTC CCC CAT AAA AAC | CAG TTC CGA TGA GAA CGA CG | 79 bp | 53C |
HBII-13 | GGA TTT GTG ATG AGC TGT GTT TAC | GGA CTT CAG AGT AAT CAC GTT G | 67 bp | 54C |
HBII-438A/B | GGA TCG ATG ATG AGA ATA ATT ATT G | GGA CCT CAG ATT GAC ATC TG | 67 bp | 53C |
GABRB3 | TCA GGC GGC ATT GGC GAT ACC | ATA AAA ACT TGA CAG GCA GAG | 352 bp | 52C |
GABRA5 | AAT ATT GCC TTA ATG TTT CTA | GCC TAT TCT ATT TCT TCG TGT | 425 bp | 48C |
GABRG3 | GCG TAT TCA CAT AGA CAT CTT G | GAT TGG TCA CTA CTG GTC TGG | 188 bp | 52C |
GAPDH | TGG GCT ACA CTG AGC ACC AG | GGG TGT CGC TGT TGA AGT CA | 50 bp | 53C |
Quantitative RT-PCR
| ||||
Gene/Exon
|
fwd (5'-3')
|
rev (5'-3');complement strand
|
size
|
T ann.
|
SNURF Ex2 | ACG AAC TAC AGA ACA GCA CGT ACC | CTG CGT TTG ACT TGG ACT TCC | 50 bp | 60C |
SNURF Ex3 | TTC TCA GCA GCA GCA AGT ACC T | TGC CTC AGT TCA GCC TGG A | 50 bp | 60C |
HBII-437 | ATC ATT ATT TCT TGA ATT GG | CCC TCA CGC TCC CTT TGC | 50 bp | 60C |
SNRPN Ex 14/15 | CTG CAA ACA TAG GAG ATG ATA GTT CC | CAA AGA CGA TAA AAT GTT CCT TCT TG | 50 bp | 60C |
SNRPN Ex 19/20a | GGA ACC ACC ATT TGT CTA TGA TCC | CTG CAG GTG GTG ACC ATG TG | 50 bp | 60C |
HBII-438 | ATA ATT GTC TGA GGA TGC T | GAT TGA CAT CTG GAA TGA GTC | 50 bp | 60C |
HBII-85/PWCR1 | TCG ATG ATG AGT CCC CCA TAA | CAT TTT GTT CAG CTT TTC CAA GG | 50 bp | 60C |
PCR to generate Southern probes in intron 17
| ||||
Gene/Exon
|
fwd (5'-3')
|
rev (5'-3');complement strand
|
size
|
T ann.
|
SB 1 | ACC ATC AGT GAA TGA CCT GTT GC | CCC AGC CTC TTT CCT ATG TCT TG | 565 bp | 53C |
SB 3 | TGG TAA ACT GAT GAG AGC ACA GCC | GCC TGG GAG ACA GAA TGA GAA AC | 416 bp | 53C |
Southern blot analysis
Breakpoint cloning with a PCR-based method
Results
Clinical case report
Cytogenetic analysis
DNA methylation analysis
Mapping of the translocation breakpoint by FISH
Fine mapping of the breakpoint by SNRPN expression and Southern blot analysis
Amplification product | PWS | Normal control | t-PWS (4;15) | PWS triplication | t-PWS intron 2 | t-PWS (4;15) |
---|---|---|---|---|---|---|
LCL | LCL | LCL | LCL | FB | FB | |
SNURF Ex 2 | 0.0001 | 0.53 | 0.54 | 2 | 0.002 | 0.56 |
SNURF Ex 3 | 0.0002 | 1.1 | 1.43 | 6.4 | 0.0004 | 0.56 |
HBII-437 | 0.00003 | 0.86 | 1.13 | 4.9 | 0.00008 | 0.1 |
SNRPN 14/15 | 0.0013 | 4.7 | 4.91 | 17.23 | - | - |
SNRPN 19/20a | 0.005 | 1.34 | 0.003 | 4.7 | 0.007 | 0.007 |
HBII-438 | 0.03 | 1.5 | 0.02 | 6.2 | 0.07 | 0.07 |
PWCR1/HBII-85 | 0.03 | 3.7 | 0.02 | 16.8 | 0.04 | 0.06 |
Breakpoint mapping at the nucleotide level
Expression of upstream genes MKRN3, MAGEL2, and NDN
Expression of C/D box snoRNAs and intron-encoded ESTs
Discussion
Breakpoint mapping and mechanism of the translocation event
Breakpoint in SNRPN Intron 2 | Breakpoint in SNRPN Exon 20/ Intron 20 | Breakpoint in SNRPN Intron 17 | ||||
---|---|---|---|---|---|---|
Sun et al. 1996 | Kuslich et al. 1999 | Schulze et al. 1996 | Conroy et al. 1997 | Wirth et al. 2001 | Present case | |
Karyotype designations | 46, XY, t(15;19) (q12;q13.41) | 46, XY, t(4;15) (q27;q11.2) | 46, XY, t(9;15) (q21;q12–q13) | 46, XY, t(2;15) (q37.2;q11.2) | 46, X, t(X;15) (q28;q12) | 46 XY, t(4;15)(q27;q11.2) |
Age of examination | 3.5 years | 3 years 3 months | 29 years | 4.5 years | 18 years | 22 years |
Major criteria (each scores one point) from [1] as revised in [2]. | ||||||
1. Neonatal central hypotonia | Floppy and lethargic in the first 6 months with poor suck (1pt.) | Hypotonicity, poor sucking reflex during infancy (1pt.) | Neonatal hypotonia (weak cry, poor suck) (1 pt.) | Neonatal hypotonia, lethargy, poor suck (1 pt.) | - | Reduced tone with poor head control, poor suck (1 pt.) |
2. Infantile feeding problems/ failure to thrive | Failure to thrive (1pt.) | Feeding problems in infancy, failure to thrive (1 pt.) | Special feeding techniques, but no failure to thrive | - | Feeding problems, but no failure to thrive (1pt.) | |
3. Rapid weight gain between 1–6 years | Obesity starting at 6 months, hyperphagia (1 pt.) | Eating behavior leading to increased weight gain at age 2 yr (1 pt.) | Periodic excessive weight gain from age 7 yr | Onset of obesity at 1.5–2 yr with excessive appetite and food foraging (1 pt.) | Obesity began at 4–5 yr with hyperphagia and food foraging (1 pt.) | Late onset obesity (at approx. 8 yr) |
4. Characteristic facial features | Narrow bifrontal diameter, almond-shaped eyes, down-turned mouth (1pt.) | Narrow bifrontal diameter, almond-shaped eyes, upslanted palpebral fissures (1 pt.) | Narrow bifrontal diameter, narrow face, small mouth, poor facial mimic (1pt.) | Narrow bifrontal diameter, squared nasal tip, downturned mouth (1 pt.) | - | - |
5. Hypogonadism: genital hypoplasia, pubertal deficiency | Undescended testes (1 pt.) | Undescended small testes, hypogonadism (1 pt.) | Hypoplastic genitalia, incomplete gonadal maturation with delayed pubertal signs after age 16 yr (1 pt.) | Scrotum normal, penile length at 10th %ile | Primary amenorrhea, hypoplastic uterus (1 pt.) | Undescended small testes, hypogonadism, delayed pubertal signs (1 pt.) |
6. Mental retardation, developmental delay | Developmental delay (1 pt.) | Developmental delay (1 pt.) | Mental retardation, developmental delay/ learning problems (1 pt.) | Developmental delay, special school setting (1 pt.) | Slight developmental delay, school for mentally retarded children (1 pt.) | Developmental delay, special school setting (1 pt.) |
Score | 5 points | 6 points | 5 points | 4 points | 3 points | 4 points |
Sun et al. 1996 | Kuslich et al. 1999 | Schulze et al. 1996 | Conroy et al. 1997 | Wirth et al. 2001 | Present case | |
---|---|---|---|---|---|---|
Minor criteria (1/2 point each) | ||||||
1. Decreased fetal movement and infantile lethargy | Decreased fetal activity (0.5 pt.) | Decreased fetal movements (0.5pt.) | - | - | Slightly reduced fetal movements (0.5pt.) | |
2. Typical behaviour problems | Behavior problems (0.5pt.) | Temper tantrums, violent outbursts, obsessive-compulsive (0.5 pt.) | Aggressive outbursts, rigid personality, perseveration (0.5pt.) | Behavior problems with temper tantrums and severe aggressiveness (0.5 pt.) | Temper tantrums, violent outbursts after food restrictions (0.5pt.) | Temper tantrums, abnormal social behavior (0.5pt.) |
3. Sleep disturbance, sleep apnea | Sleep disturbance, sleep apnea (0.5pt.) | Sleep disturbance (0.5pt.) | Sleep disturbance, amphetamine treatment from age 9 ys. (0.5pt.) | |||
4. Short stature for the family by age 15 years | Short stature at the age of 15 (0.5pt.) | 50–75th percentile (0.5pt.) | 151 cm (3rd%tile) (0.5pt.) | Height 155.7 cm at 16 years < 3rd %tile (0.5 pt.) | ||
5. Hypopigmentation | - | - | Hypopigmentation (0.5 pt.) | - | - | |
6. Small hands and /or feet for height age | Hand length 25th percentile, finger length 10th%ile (0.5pt.) | - | Normal hands, but small feet (< 10th%tile) (0.5 pt.) | Short 3rd finger bilaterally | Hands 20th %ile, feet 5th %ile (0.5pt.) | |
7. Narrow hands with straight ulnar border | - | - | ||||
8. Eye abnormalities: esotropia, myopia | - | Esotropia (0.5 pt.) | Alternating esotropia in infancy (0.5 pt.) | Left esotropia (0.5 pt.) | Esotropia (0.5pt.) | |
9. Thick viscous saliva | Viscous saliva (0.5pt.) | Thick viscous saliva (0.5 pt.) | - | |||
10. Speech articulation defect | Articulation difficulty (0.5 pt.) | Poor articulation (0.5pt.) | - | |||
11. Skin picking | Skin picking (0.5pt.) | Skin picking (0.5pt.) | Skin picking (0.5pt.) | |||
Score (minor only) | 1.5 points | 3 points | 3.5 points | 2.5 points | 1.5 points | 3.5 points |
Total Score | 6.5 points | 9 points | 8.5 points | 8.5 points | 4.5 points | 7.5 points |